TRANSMI SS IBLE DRUG RESISTANCE, PLASMID 
CIIARACTERI ZATION IN SHIGELLA AND 
SALMONELLA, AND VIRDLENCE OF SHIGELLA 
ISOLATED FROM DIARRHOEIC HUMANS AND PIGLETS\
BY
ISAAC ADEYEMI ADELEYE
B.Sc. (Hons.) Lagos,
M.Sc. (Microbiology) TFE.
A TH$S4$ 
l^THE
DEPARTMENT OF Vjj^ERINARY MICROBIOLOGY 
AND PARASITOLOGY,
/^JWBMITTED TO THE
TY OF VETERINARY MEDICINE
C^IN PARTIAL FULFILMENT OF THE 
REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPY (Ph.D) 
OF THE
UNIVERSITY OF IBADAN
MARCH, 1990
i
ABSTRACT
A total of 1U,200 faecal samples »ncluding 10,000 from
diarrhoeic human beings and 200 from diarrhoeic ^i&iets 
were collected. The human faecal samples were collected 
from the three government hospitals in Ibadan, naraely: T^e
University College Hospital, State Hospitallse 2 t Adeoyo and
Ring Road, while the piglets were <-»n the Teaching and 
Research Farm, of the University oi lbauau.
.................  - S T
Thirty Shigella and twenty-two Salmone11a spp. were 
isolated from the human fae cat s amples while one Shigella 
isolate was obtained from the faecal samples of piglets.
The Shigellae were sero1ogica 11y identified as £. y 1exner i 
(23 from humans and 1 from a piglet), £. d.vsenter iae 
(4 isolates from human) and S . bo.vdi i (3, all were isolated 
from human). Of the Salmone11a isolates, 9 were identified 
as S .typhi while the remaining 13 were classified as 
Salmone11a species. One microgram per ml potassium 
tellurite in MacConkey agar was used to enhance the 
isolation of Shigellae.
Antimicrobial susceptibi1ity testing to eight
antibiotics were peformed on the fifty -three bacterial 
isolates. Twenty-one antibiotic resistance patterns
were identified.
ii
The highest pattern T-CT-F-A-S-C-Te (Septemdrip1e) was found 
in eleven isolates, sextiple resistant pattern in thirteen; 
quintiple pattern in eight isolates; quadriple in four 
isolates; triple in eight isolates; double in four isolates 
while resistance to single antibiotic (Te and CT) was found 
in five bacterial isolates.
The Minimal Inhibitory Concentrations &(MIC) of 
4 antibiotics (ampicillin, ch1oramphenico1, Streptomycin 
and tetracyc1ine) were deterrained for the bacterial 
isolates. Two of the Shigella isolates were sensitive 
to ampicillin, the MIC of ampicillin was 8 ug per ml 
for them, 1 to chloramphenicol; 4 to Streptomycin while 
all isolates were resistant to tetracyc1ine. The MIC of
I
ampicillin for 14 of the Salmonel1a isolates was 8 ug per ml 
while 11 and 9 isolates of the same organism: were resistant 
to chloramphenicol and Streptomycin respective1y .
The following reistance patterns were observed: A-C-S-T, 
C-S-T, A-C-T and A-S-T.
All the forty-two isolates screened transferred 
ampicillin resistance (100 percent). Twenty-one isolates 
(50.0 percent) transfered two determinants, either A-T or 
A-S or A-C. aeyen v16.6 percent) transfered three 
determinants either A-T-S or A-S-C.
111
None of the isoiates transfered four determinants (A-T-S-C).
Among the Shigel1a isoiates the ch1oramphenico1 determinant
was transfered at a low frequency (only 3 i
screened) whereas the Salmonella isoiates
higher frequency ^6 of the 14 screened).
The R plasmids ränge in size between I 1.
The chloramphenicol R plasmid has a molecular »,eight of
4.00 Mdal., Streptomycin
Mdal .
The plasmid profile
investigated using agaros w . The
Shigella isoiates exhibited a large number of small cryptic
plasmids. In contras the SalmoneI1a isoiates exhibited
fewer number of plasmids. All the plasmids ränge between
0.8 and
The virulence of the Shige 11a isoiates was investigated 
using Sereny Test and Rabbit ligated ileal loop test. Four 
of the fourteen Shigella isoiates including £.f1eyp<?ri (2), 
£. bovdi i (1), S .dvsenter i ae (1) produced Keraconjuc- 
tivities in guinea pigs.
Six of the Shigella isoiates including four f 1 gxne.rjL»
one S.dvsenteriae and one S . bo.vd i i dilated ligated rabbit 
ileal loop with accumulatiön of fluid. Histological 
ulterations found in the ileal loops exposed to these
iv
enterotoxins included inflamation, general degeneration, 
subraucosal oedema and neutrophilic infi1 trations.
Four of the Shige 11a isolates coroprising of two 
£• f 1 exneri . one £. dysent er tag and one 5.. bo.vdi i dilated 
the ligated intestinal loop of rabbit with accumulation
of fluid when enterotoxin heated at 65 C for 30 min. wer e
used. It was observed for the first time thalt the S . bo.vd i i 
produced heat labile and heat stable enterotoxin in ligated 
rabbi t i11eal 1oop.
Oral inoculation of the invaOsive' Shigella isolates 
into pretreated (starved and calcium carbonate treated) 
mice and guinea pigs failed to produce clinical 
manifestation of dysentery-1ike diarrhoea and febrile 
condi t ion.
V
ACKNOWLEDGEMENTS
It is ray pleasure to acknowledge my sincerest thanks 
to all persons who through various ways rendered valuable 
assistance towards making it possible for me to accomplish
this work.
First and foremost, I am most grateful to my able 
Supervisor, Dr. A.I. Adetosoye for his criticisms and 
suggestions, as well as his generous Cooperation, 
encouragement and advice.
My profound gratiiuue also goes to Dr. D.O. Olukoy
Head, Department of Genetic Research and Oncology, Nigeria 
I ns t i t u t e Medical Research, Yaba for placing his
laboratory facilities at my disposal for the plasmid 
characterization of my isolates.
I would also like to place on record my gratitude 
to Mr. I.0. Rotilu of Department of Medical Mftgfcrobi o 1 ogy 
U.C.H. for his wonderful cooperation and generous gift 
of many ^eagents and Dr. Dupe Akintola for his efforts in 
minimising the delays before viva-voce.
Finally, I am greatly indebted to the authors of the 
papers cited and publishers of relevant books and journals
vi
iPEDLCAT.l.OM
vn
CERTIF1CAT1QN
I certify that this work was done by 
Isaac Adeyemi Adeleye in the Department of 
Veterinary Microbiology and Parasitology 
University of Ibadan.
DR.•A . I. ADETOSOYE 
SUPERVISOR.
DVM (ABU), Dr. Med. Vet. (VIENNA), 
Cert. in Animal Management & 
Reproductive Biology (VIENNA)
PROF. M. OLA. OJO, 
SUPERVISOR.
KSG, BVM&S, MRCVS, (EDIN.), 
Dip. Bact. (LOND.),
Ph.D. (IBADAN).
Vlll
! A m iE_jQIl_mNTMX£
EA£E
v 
v i 
vi i 
v i i i 
x i v
r XV
!
1
5
Prevention of contact with the infecting agent....... 7
Replacement of sodium, bicarbonate and potassiura.... . 8
Interference with mechanism of diarrhoea............. 9
Use of antibiotics...................................... 10
Problem of plasmid mediated transmissib1e drug
res i stance.............................................. 11
Previous work on Salmomel1a and Shigella in Nigeria.. .14
Chemotherapeutics and Bacterial resistance in Nigeria .15
Objectives of study.................................... 18
ix
CHARTER 2
LITERATURE REVIEW......................................... .
Description of Shigpl1«.......... .20 
Sh i ere 1 I a infections.............. .23 
Clinical manifestation of disease . 25 
........................
Pathogenes i s............... ...... .26 
Role of Shigel1a toxin .......... £ ............. ,28 
Bacterial invasion........... V 29 
Pathology of Shigel1a infectio .......... 31
Treatment therapy in Shigellosis.........................31
Fluid and electrolytc..................................... 33
Salmonel la,................................................ 35
Salmone 1 1 a infections................ -.................. 36
Transmission.............................................. «37
Enterotoxin production by Salmone 1 1 a .................... 39
Treatment of Sa 1 mone 1 1 os .................................. 41
IsolatioAn arnd  characterizati, on of,  Shigeltllae
and Salmonel lad........................................... 44
Differentiation and Character i zat ion of Salmonellaß.
and Shigellae.............................................  49
Ser o 1 ogy..................................    50
Antibiotics................................................ 52
Mode of action of antibiotics............................ 53
X
Ant irai crobi al resistance in man......................... 53
Ant imi crobi al resistance in animal...................... 56
Properties of infectious drug resistance............... 60
Chromosomal or plasmid mediated resistance..............63
Methods of plasmid transfer ............................. 64
Conditions governing the frequency of transfer.........65
Incompat ibi 1 l ty behaviour................................68
Mechanism of plasmid mediated resistance................ 70
The levels of drug resistance confered by R-factors....75 
Non-plasmid mediated resistance in enteric bacteria ....78
Transposons. s y  ..... 80
Plasmids and virulence in Salmone11a andS higella....... 81
Immuni^y Salmone11a and Salmonel1a vaccines...... 84
Immunlt« to Shigel1a infection & Shige11a vaccines ..87
CHARTER 3 _
INTRODUCTION............................................... 90
MATERIALS AND METHODS..................................... 96
Collection of samples and isolation of organisms...... 96
Cultivation techniques.................................... 96
Purification of Isolates..................................97
Biochemical char ac t er i za t i on of isolates............... 97
Serological Identification of isolates.................102
xi
RESULTS.............................*.....................104
DI SCUSS ION............................................... 108
CHAPTER 4
INTRODUCT ION................................ O W ' .......116
MATERIALS AND METHODS....................#A / . ............................ 119
Antimicrobial Susceptibi 1ity tests ............................
Disc diffusion method............ ......................119
Minimal inhibitory Concentration (MIC) determination.120 
Conjugation (interrupted raating) experiment..........122
Selection Method........<..y.N.............................122
RESULTS.............. I..................................125
DISCUSSION 129
I NTRODUCT ION............................................142
MATERIALS AND METHODS..................................146
Plasmid DNA Isolation procedures ..................... 146
Bacterial Isolates.....................'............... 146
Agarose gel e 1 ec t r ophores i s of DNA.................. 148
RESULTS................................................. 15°
DISCUSSION 152
xii
CHARTER 6
INTRODUCTION...................................... 166
MATERIALS AND METHODS..................................... 170
Test for invass i veness (Sereny Test)....................170
Experimental Shigellosis in Guinea pigsa nd mice.........170
Pretreatment of animals for experiment................. 171
Assay for enterotoxin......................  ............. 172
Bacterial isolates....................................... .172
Preparation of crude bacterial enterotoxin..............172
Surgical procedure. . . . ................... . ... i...........173
Assay for heat labile (LT) Oenvte rotoxin.................'. 174
Fluid accumulation evaluation............................ 174
Assay for heat stable (ST) enterotoxin.................. 175
Fluid accumulation evaluation............................ 175
Histopathologicale valuation............................... 175
RESULTS. . U . ............................................... 176
i V /
Ileal loop ligation tests................................. 177
Histopathogica 1 changos in rabbit gut caused
by cholerae ........................................... 178
Histopathogical changes in rabbit gut caused by
£. f 1 exner i 6 .............................................*78
Histopathological’ changes in rabbit gut caused by
dvsent er i ..............................................
Histopaythological** changes in rabbit gut caused by 
£. bovdi ......................................  178
M S T  OF TABLES
TABLE PAGE
3.1 Biochemical characteristics of isolates........ 252
4.1 Selection plate showing concentrations of
antibiotics in MacConkey plate...................123
4.2a Result of antibiotic susceptibi 1ity testing
of isolate (Disc diffusion)........✓..v..T... 264
4.2b Incidence of antibiotics resistance in
"hJjLeJLLa and S,alroQneU a  isolates in faec&l
samplesof diarrhoeic patients and 
piglets 135
4.3a In Vitro activity of antibiotics against
30 Shige 1 1 a and C22 rSalmonel 1 a . ......... 1364.3b In Vitro activity of antibiotics' against 
30 Shigel 1 a isolates............................ 137
4.3c In vitro activity of antibiotics against 
22 Salmonel 1 a isolates.........................  138
4.4a Conjugation* Determinants transfered on
rnating each donor with E_. coli K 12 Na r.....  139
5.1 Number and sizes of different plasmidoj in
Shigel 1 a and Sa lmone I 1 a isolates............  158
5.2 Plasmid profile of transconjugants...........  165
6.1 Invasiness of Shigella isolates from clinical
diarrhoeal cases..............................  185
6.2 Enterotoxigenicity of Shigella isolates as 
demonstrated by the illeal loop (ligation)
test (test labile)...........................  186
6.3 Enterotoxigencity of Shigella isolates as 
demonstrated by the ileal loop ligation
test (heat stable enterotoxin)..............  189
x iv
CHAPTKR 7.
SUMMARY AND CONCLUSION 193
REFERENCES............ . . . . 202
APPENDIX I.......... 250
APPENDIX II.......... 251 
APPENDIX III.......... .............. 251b
APPENDIX IV.......... 252 
........••••
APPENDIX V ........... . ......................... 264
XV ,
FIGURE PAGE
5.1 Agarose gel e1 ectrophoresis of R Plasmids..... 181
6.1 Keratoconjunctivitis in guinea pig caused
by isolates 3. f 1 exner i )................
6.2 Ileal loop ligation test.................... .
6.3 Ileal loop ligation test........................ 192
CHAPTER owe
INTRQDUCTIQN
Infectious enteric diseases, including acu arrhoeal
disease are of great public health importance i eloping
and underdeve1oped countries of the worl
Diarrhoea has long been recognised as the greatest 
killer of infants and young children in the developing
countries (WHO Scientific Worki oup, 1983). Well over
five hundred million episode iarrhoea in children under
five are estimated to occur annually in Asia, Africa and 
Latin America with abo ve million deaths recorded in
1980 (Cutting and Elliot, 1980). Diarrhoea is often lethal
among the very young. It is also a major cause of i11 —
health and death among children and adult of all ages when 
badly managed.
In the investigations of bacterial etiology of 
di arrhoeal infections, Shigella has been recognised as a 
pathogen of man and primates (Tiwary and Prasad, 1972). 
However, £. f 1 exner i. £• sonnei and £. bo.vdi i have been 
isolated from diarrhoea and faecal materials of dogs 
(Doem and MortelmanA» 1954; Floyd, 1955 and Carwadine,
1982) .
2
Nyaga Ai Ai* (1985) reported the isolation of £. dvsent er i ae 
3 organism, a human pathogen, from poultry in Kenya.
Ibu Ai Ai* (1987) also reported a case of fatal Shigellosis
caused by £. flexner i in piglets in Jos (Nigerilaai);.
Many enteric gram negative organisms e _S_a lmonel 1 a
spp and some strains of Campy1obacter infect man as well 
as animals (Coker and Dosunmu-Ogunbi, 1984)'. äThe larger 
proportions of Salmonella infections in man are derived
from eating contaminated meat and therefore, indirectly 
from animal sources. Domesticated animals like dogs and 
cats have been known to be carriers of Salmone11a (Khan, 
1970) while frogs, lizard and snakes are actual reservois 
of this organism (Sarvamangala and Shivanda, 1983). Other 
wellknown causes of diarrhoea are V. cholerae and certain 
strains of j . coli. •
In the last ten years owing to active research in 
different parts of the world, various agents including 
rotavirus, Campy1obacter, Yersinia , Aeromonas, . aeruginosa
JP1 esiomonas, C. perfiring es have been associated with 
diarrhoea (Candy, 1984; Reunels et al., 1985 and Williams 
et al., 1985).
Rotavirus which was first reported in children in 1973 
is known to cause perhaps fifty percent of childhood 
diarrhoea, the incidence increases to eighty percent in 
temperate climates (Banatvala, 1979; Malta et al., 1983
3
In addition to bacteria and viruses, parasites have 
also been implicated in the outbreak of diarrhoea. The 
■ost important protozoan parasities known to cause dli^arirhoea
are Gi.ftLsU.ft Iftmbl ift» Entamoeba histol.vt i ca and
Cr.YPt9SP9riflim g (Rhode, 1984 ). Among helminths . S t£ rong.v 1 o i de 
and occasiona11y , Tr i churi s have been known to be
causative agents of di. arrhoea. The mecha,nsiss?m of amoebic 
dysentery is related to adherence through mierofi1 ament 
invasion and microabscess formation (Radvin and Guerrant, 
1981). •
Shigellosis (Bacillary dysOenyter y). has attracted 
attention throughout the course of history by its dramatic 
clinical manifes tations. It was described in the Bible
in association with rectal proplase ("Emrods").Hippocrates 
described it as the bloody flux and Thomas Willis as a 
"dysentery" in the course of a "putrid fever" (Schwartz and 
Bishop 1958). It has been associated with major mobidity 
and often high mortality wherever it has struck.
The Shigellae are thin, non-motile, non-sporulating 
gram negative rods closely related to Escherichia coli in 
the family Enterobacteriaceae. There are four related
Shigella species namely S. dysenteriae. S . f 1 exneri , S . bQy_d.i_j
4
tad sonnei d i f f erent i a t ed by biocheraical and serological 
reactions. Within each species a number of distinct 
sabspecies are grouped together, all sharing major :a  
somatic) antigenic determinants, classified as A,B,C and 
group D respectively. They all ferment glucose but do 
not produce gas (except for a few isolates of £. f1exner i1 
and they neither ferment lactose (except Sonnei which 
turns positive after sometime in broth culture) nor produce
H^S (Burrows et al., 1968)
These few properties are t he basis of microbio1ogica 1 
diagnosis of the infection-» which relies upon differential
inhibitory agar media to suppr ess growth of gram-positive
organisms and coliforms and to reveal the capacity of the 
latter to rapidly ferment lactose. •
Shigella d.vsenteriae is the classical agent of 
invasive diarrhoea. The organism elaborates a variety 
of toxins (neurotoxin, cytotoxin and exterotoxin) many 
of which are plasmid encoded, and may facilitate penetration 
of the mucosa of the colon (Keusch, 1979). Shige 1 1 a 
dvsent er i ae is cytotoxic and moves laterally to invade 
contiguous cells. Microabscesses and denuded ulcers
lead to bloody dysentery.
5
Salmonellosis is one of the zoonotic di seases of public 
health importance. The natural habitat of members of the 
genus Salmonel la is the intestinal, tract of warm-blooded and
many cold-blooded vertebrates from where the 
and even multiply.
In general, members of the genus can be 
three categories on the basis of their host
1. Man adapted Salmonellae;
Those primarily adapted to man i.e. the enteric
fever organisms, ß. t.vphi and ;Sy. parat.vphi A,B,C belong
to this category. S. t.vphi and S. parat.vphi A and C have 
no known secondary hosts and hence are rarely isolated 
from animals other than man. ß. parat.vphi ß, while 
primarily adapted to man« is occasionally isolated from 
seondary hosts including cattle, swine, dogs and fowls 
(USDA Committee on Salmonella, 1969; Anon, 1971).
2. Animal adapted Salmonel laeJ *
Those primarily adapted to particular animal 
hosts, for example, ß. cho1era-sui s (pigs); ß. d u M  in 
cattle; ß. p u Iloruro and ß. gal 1inarium (chickens); 
ß. arbor tus-eoui (horses) and ß. ftbor t US-ttV-i-S. (sheep).
6
These are all capapble of causing clinical illness in 
man, although only £. cholera-suis and £. dublin are of 
any significant consequence in this context.
3 . UnadftPted. Saimonel lae;
The remainder of the more than two thousand
serotypes of the genus have no apparent host 
preference and belong to this category. They 
are all potential agents of human infection, but, 
in practice, the real problem of human Saimonel1osis
is limited to a relative 1 serotypes (CDC
Salmonella Survillance, 1976).
The epidemiology of Salmonellae of categories 2 and 3 
with respect to infection in man mostly involves the animal 
reservoir and transmission through food in which multi­
plication may take place. The most common clinical 
manifestation of Saimonel1a infection is gastro- 
enteroco1itis . After incubation period of 6 to 48 hours
(usually 12 to 24 hours) headache, malaise, nausea, 
vomitting and diarrhoea appear abruptly. Abdominal pain 
is frequent and may cause mild to severe discomfort.
The patient may have fever and shivering (Yoshikawa et al., 
1980). In complicated cases, the acute stage usually
terminates within forty-eight hours, although it may be
7
prolonged and the patient may not feel well for several more 
days. The diarrhoea may also be severe with frequent 
passage of watery, greenish, offensive stools with mucus; 
blood may also be present. Abdominal cramps and diarrhoea 
may persist after nauseaa and vomitting have ceased
(Mandal; 1979)
Diarrhoea, whatever the etiology, leads to acute 
fluid and electrolyte loss (Rohde and Northrup, 1976). ■
This loss of fluid and electrolytes leads to dehydration 
and> i» S e e  ca.es „eath ,f
In the treatment of diarrhoea the following steps 
are usually considered (Mandal, 1979):
(i) Prevention of contact with the infecting agent;
(ii) replacement of sodium, bicarbonate, potassium,
Substrate and water loss
(iii) interference with the mechanism of diarrhoea;
(iv) prevention of infection through the use of 
vaccine; and
(v) use of antibiotics.
(1) Prevention of contact with the infecting aeent-L
Bacterial diarrhoea (Shigellosis and 
Salmonellosis) is known to spread through contact
8
with faeces, food, foul water and by "fingers" 
(Blaser and Newman, 1982). Thus interruption of 
the faecal-oral t ranstni ss i on of diarrhoea is
accomp1ished through widespread introduction o jA  
clean water supplies, personal and food hygiene
and sanitary waste disposal (Rhode, 1984 ) Q -  
Inadequate refrigeration of stored foods encourages 
growth of organisms causing acute food poisoning. Salmonella
organisms are spread through the mlaasss pProo«cessing of poultry. 
Control of this and other factors is part of the complex 
health regulations of the food chain necessary to interrupt 
transmission of diarrhoea causing pathogens;
Ren 1 acement of sodium. bi carbonat e. .pg.tft8.si.wn 
substrate and water loss:
The development of oral fluid therapy for diarrhoea, 
which was the outcome of research on Vibrio cho1erae 
demonstrated the high efficacy of replacement of water and 
electrolytes with an appropriate, absorbable substrate, 
thereby off-setting the effects of dehydration and 
substantia11y reducing the number of deaths. Extensive 
reviews have documented both scientific basis and the 
efficacy of oral rehydration therapy (ORT) in acute diarr­
hoea (Rohde, 1984; Candy, 1984). This is being extensively
9
practiced in Bangladesh as well as other third world 
countries including Nigeria. The principle involved 
is that of co-transport of sodium equimolar with active 
transport of sugar (Keusch, 1979).
3. Interf erence with the mechanism of Diarrhoi-  
A number of antisecretory drugs (aspirin, 
indomethacin, and ch1orpromazine) working through 
a variety of mechanisms in the mucosal cells have 
been shown to diminish or reverse the secretory process 
and, therefore, effectively reduce loss of fluid and 
electrolyte (Rhode* 1984). For example Aspirin and 
Indomethacin have been shown to stimulate Sodium and 
Chlorine absorption in normal tissue and it may be 
this activity which is responsible for their effect
on cholera toxin (Turnberg, 1978). Chloropromazine 
prevents intestinal secretion in the mouse by 
inhibiting adeny1cyc1ase activity (Lonroth et al., 
197'
4. Prevention through the use of vaccinesi.
For oral vaccines against Shigella, five doses 
and booster doses were required (Levine et al, 1976).
10
In the case of Salmonella, the Ty21a oral typhoid 
-accine appears to offer some protection, even in single 
doses (Wahdan et al., 1982) but there are still no vaccines
for the over two thousand or more serotypes of common A
>almone11a . Recombinant DNA technologies make production
of a wide variety of specific antigens possible Cm open
the way for eventual polyvalent vaccines aimed- against a 
host of organisms. For example Shigella-Salmonella hybrid 
has been created (Formal et al., 1981) by transfering 
plasmids responsible for form 1 antigen synthesis in 
Shigel la to established gal E. t.vphi strain. At 
present research is still going on in various laboratories 
in the world to find a suitable vaccine to combat the 
menace of Salmonella and Shigella infections.
use 9t Antibiotics; V
Several a uthors have advanced three congent reasons 
for reconsid the use of anti-microbials in the
therapy o gellosis (Weissraan et al., 1973): the
disease is usually se1f-1imiting and may be mild 
(especially that due to £. sonnei)? post-infection 
carriage is usually less than three to four weeks and 
with increasing frequency, the organism possesses plasmids
11
coding for transferable drug resistance (Anderson, 1968
and Weissman et al., 1974). In cases in which the
invasive organism causes a dys entery-1ike syndrome, the
specific drug of choice is based on knowledge of sensitivity
patterns of the pathogen when cultured in t'he same 
envi ronment.
In case of enteric fever caused by Salmone11a t.yphi. 
chloramphenico1 has been the most successful drug. The 
clinical response to the drug has been fairly uniform 
throughout the world (Mandal, 1979). A rapid improvement 
in the patient's general condition followed be defervescence 
within two to five days has beeh^recognised. However, the 
wide-scale emergence of R-factor mediated resistance to 
chl or ampheni co 1 (as well as other antibiotics) in _S_. typhi 
(Datta, 1965) has introduced a new sense of urgency in the 
search for suitable antibiotics.
Problem of P lasmid mediated tranmissible drug resistance.;.
K
Many of the antibiotics used in treating human 
infections are also used as chemotherapeutic and 
prophylactic agents in animals as well as feed additives 
at low concentrations to increase their growth rate and 
reduce economic losses of animals (Walton, 1966a; Ojo and 
Adetosoye, 1977).
12
J
Soon after the introduction of antibiotics into medical 
practice and as feed additives, antibiotics resistance 
amongst bacteria emerged as a significant problem. A  
Studies on the nature of antibiotic resistance in 
bacteria reveal that several biochemical mechanisms may 
be responsible. These include:
(a) alteration of target site in the cell that
eliminate or decrease the binding of the drug;
(b) decreased drug accumulation into the cell; and
(c) antibiotic inactivation by enzymes, etc. 
Resistance of type a, which arise by mutational
alteration of a cellul. ar comp4onent are very common in 
laboratory derived resistant strains but clinical isolates 
of this have also been reported (Benveniste and pävies
1973). .<£>
Resistance of types b and c are generally found only 
in clinical isolates of resistant . bacteria, and are
normal 1y associated with extrachromosomal element or plasmid 
(O'Brien, 1987). The plasmids carry "resistance factors" 
or R-factors. Transfer is effected by contact resulting 
in conjugation between drug resistant donor cells and 
senstive recipient cells.
13
Apart from transmissible drug resistance, plasmids also 
play an important role in the pathogenicity of Shige11a and 
Salmonella. Takabashi et al. (1988) demonstrated that 
large plasmid (150 megadalton) found in £. bo.vd i i was 
responsible for the epithelial cell Penetration by the 
organism. There is also a correlation betwei
presence of a fifty megadalton plasmid in Salmonel1a
dublin and its virulence for mice (■Tarakado et al . , 1983).
Central to the studies on Shigella and Salmonel1a is 
the question of the virulence. Levine £_L (1973)
established that the primary virulence determinants of 
Shigellae is the invasive capacity of the organism as 
strains which cannot penetrate and multiply within the 
colonic epithelial cells do not cause disease in humans.
It is also know that &. f 1 exner i adhere very well to the 
colonic mucosa of guinea pig in the presence of adhesin 
(binding protein) found in the intestinal cells (Izhar et
al., 1982).
S h i gc11a dvsent er i ae is known to elaborate a toxin 
with cytotoxic, enterotoxic and neurotoxic properties.
This heat labile toxin is enterotoxic when injected into 
rabbit illeal loops (Cavanagh et al., 1956; Keusch et al.,
1972a) and lethal to mice and rabbits (Van Heyningen and 
Glads tone, 1953). Moreover, it is cytotoxic for
14
#*
eukaryotic cells such as HeLa cells (Keusch et al., 1972b), 
KB (human epidermal carcinoma), normal human liver and 
monkey kidney (Vicari et al., 1960). Furthermore, purified 
Shigella toxin inhibits protein synthesis in cell-free 
translation System (Oienick and Wolfe, 1980).
Likewise cell-free extract of Salmonel1a has been found 
to inhibit protein synthesis and cause cytotoxicity in 
eukaryotic cells (Koo and Peterson, 1982)
PreviQus_H-Qrk gii-Salmonei l.s^aiid Shigella in Nigeria;
Salmonel1a spp. have been isolated from various 
animals in Nigeria: cattle (Collard and Sen, 1956). 
fowls (Sen and Collard, 1957a) pig (Sen and Co 11 ard, 1957b, ) 
agama lizard (Collard and Montefiore, 1956), goats (Falade, 
1976) and captive animals (Falade and Durojaiye, 1977).
Other workers, Oduye and Olayemi (1977) and Britt et al.
(1978) have also reported the isolation of Salmonellae
.............................................................
from dogs.
The isolation of Salmonella serntypes from man 
was first reported by Collard and Sen, (1957). The picture 
of Salmone11osis in Nigeria is one of wide diversity of 
types in both man and animals. Many of the Salmonella 
types obtained from man had also widespread distribution in 
other sources, whereas other types were limited to man only.
15
Perhaps at one end of the scale would be £. tvphi which has 
not been isolated from other sources other than man and at
the other end would be £. agama which has been found in
several sources.
In contrast, the distribution of
is less widespread. It has been isol
few domestic animals. Collard and Sen
strains of Shigella over a five-year period (1956-61).
Of these TI. 335 were .£• f 1 exner i . 6.135 £. bo vd i i while £.
sonnei was least common. Odugbemi et al. (1982) recorded a
than Salmonella (0.935)
infections in children undervf^ve years of age in Lagos.
The most common specie was found to be £,. f1exner i.
Shigella has also been isolated from diarrhoeic piglets and 
fowls (Ibu et al., 1987).
Chcmotherapeiiti.cg_.?n.d Bac t gr j a )  r g sis.Un.ee in N iger  U?.
Diarrhoea caused by Salmonella and Shigella is 
very common in Nigeria (Odugbemi et al., 1982). The 
drugs used in treating this condition include ampicillin, 
Streptomycin, tetracycline and chloramphenicol (Adetosoye 
and Rotilu, 1986). The laws Controlling the sales of
these drugs are not enforced in Nigeria. As a result
16
antimicrobial agents are easily purchased by individuals 
without prescription; they are sold in motor parks and 
market places. Thus the indi scr irninate use of these agents
in Veterinary and human medicine has resulted into energence 
of drug resistance (Ojo, 1973; Osoba, 1979 ; Ogunnlaranwo,
1987).
The first case of ch1oramphenico1 reCsisvtant Salmonella
in Nigeria was reported by Njoku-Obi and Njoku-Obi (1965). 
Ojo (1973) isolated twenty-four strains of Salmone11a 
from animals in Ibadan and tested them for the sensitivity 
to eleven chemotherapeutic agents. Two of the isolates 
were found to show multiple resistance to chlora- 
mphenicol, sulphonami de, st reptomycin, tetracyc1ine, 
ampicillin and septrin. Ibe et al. (1987) isolated S . 
f1exner i which weH  t ound to be resistant to septrin,
ampicillin, penicillin, Streptomycin, neomycin and 
erythromycin from diarrhoeic piglets.
Some of the resistant strains have been found to 
harbour R-factor. Adetosoye and Rotilu (1986) reported 
the presence of drug resistance plasmids in twenty-four 
S. typhimurium which were isolated from calves with 
diarrhoea and fifteen other Salmone11a spp. as well as
17
twenty-four Shigella isolates which were isolated from
diarrhoeric children in Ibadan.
From these studies it is apparent that drug resistance
18
OBJECTIVES QF STUDY
Diarrhoeal diseases of various causes including
bacteria, virus and parasitic agents are common and of
great public health importance in Nigeria. O^hey are 
also of great economic importance to the livestock industry. 
There is some evidence that a Proportion of this disease 
is caused by Shige 1la and Salmone11a (Akinkugbe et al.,
1968; Alonge and Oyekole, 1982; Odugbemi et al., 1982; 
Adetosoye and Rotil
This study is
(i) Isolate Salmone11a and Shigella from diarrhoeic 
humans and piglets.
(ii) Characterise such isolates biochemica 1 ly and 
serologically.
(iii) Determine whether there is transmissib1e drug 
resistance among the isolates.
(iv) Isolate and characterise the plasmids harboured 
by the isolates.
(v) Study the pathogenicity of Shigella isolates using
various animal models.
19
CHAPTER TWO
LITERATURE REVIEW A
The first dysentery bacillus was isolated and 
identified by Kiyoshi Shiga in Japan in 1898 during a severe 
bacillary dysentery epidemic. Morpho1ogica 11y ,it was
described as short, plump and aniline stainable bacillus. 
Kruse in 1900 isolated bacteria that resembled those of
Shiga from ,d ysentery pati, ents ,i n Wes^t Ph al„i a. Fljexner in 
the same year reported the isolation of mieroorganism he 
thought looked like Shiga's bacillus from American soldier 
in the Philipines. Strong and Mustgrave (1900) confirmed 
the work of Flexner and produced dysentery with the bacteria 
in a monkey and in a condemned criminal. Castellani (1904) 
described an acute case of bacillary dysentery which 1ed to 
death. He found at autopsy that the mucosa of the colon and 
rectum were thickened, covered with mucus and studded with a 
large number of very small superficial roundish ulcers.
These reports were followed in rapid succession by 
a large number of publications from virtually all parts of
the world which recorded the isolation of these and similar 
microogranisms from acute and chronic cases of dysentery
20
(Edward and Ewing, 1972). However, important advances in 
the knowledge of this disease was not made until the 1940s. 
This was largely through the work of Boyd (1940) who A  
described a number of Shige11a paradysenteriae from India; 
Sachs (1943) isolated non-mannito1-fermenting Shigella from 
Middle-East; Christensen and Gowen (1944) characterized two 
strains in North-Africa and Ewing (1946) did similar work on 
strains isolated in Algeria. - «
DescriPtion of Shigella;
Shigella is Gram negative, non-motile bacillus 
belonging to the family Enterobacteriaceae (Cowan and Steel,
1974). It produces acid from a number of carbohydrates 
except lactose; it is methyl-red positive and 
Voges-Proskauer negative. It does not grow in Koser's 
citrate medium, does not hydrolyse urea; deaminate 
Phenylalanine; blackens kliglers hydrogen sulphide 
medium.lt grows in Moller's cyanide medium; utilizes 
malonate; oxidizes gluconate and liquefies gelatine; 
however does not fort lysine decarboxylase.
There are four species of dysentery bacilli.
They are designated A,B,C,D (Topley and Wilson, 1983)
21
These are described as follows:
(1) Slligella dysenteriae (Group A): The dysentery 
bacteria making up this group are set apart by 
their inabi1ity to ferment mannitol.
S .d.vsent er iae is inramnolotri cal l.v heterotn
made up of ten sharply separated serotyp&es 
arbitrarily designated by numbers. The serotypes 
appears to be unrelated antigentically except for 
unilateral cross reaction between some strains of 
types 2 and 6, and some sttrati;ns of types 3 and 5.
dvsenteriae type 1 was the first of the 
dysentery bacilli to be described and it was 
referred to as Sh.ga's bacillus (Shiga, 1898). 
It has marked toxicity for man and experimental 
animals due to the formation of toxin. This has 
an effect upon the nervous System of experimental 
animals and has as an affinity for the gastro­
intestinal tract (Keusch el al., 197i2a).
£. dvsenter i ae type 2 differs in that it produces 
indole and ferments sorbitol and rhamnose. Other 
serotypes which are culturally identical with &. 
d.vsenteriae but immunologi cal ly unrelated are known 
as the Sachs group of dysentery bacilli and are
22
des ignated £. d.vsent er i ae types 3,4,5,6 to 10 
(Sachs, 1943).
(ii) Shigella f1exner i (Group B). This is composed of
those microorganisms formerly referred to as Shigella 
par adysent er i ae Flexner, and is now naraed 5.. f 1 exner i 
serotypes 1-6 (Edward and Ewing, 1972). Members of 
this Group are related one to another through the 
possession of common group antigens but each serotype 
contains a type of main antigen by which it may be 
identified. Mannitol is usually fermented by members 
of subgroup B. They may be differentiated frora 
member of subgroups A and C by means of some 
biochemical reactions as well as serological analysis
(iii) Shigel1a boyd i i (Group C). These are also mannitol 
fermenting dysentery bacilli closely resembling 
£. f1exner i in biochemical characteristics but 
differ in several important aspects and are unrelated 
serologically. There are fifteen serotypes of 
Shigella boydi i recognized (Edward and Ewing, 1972).
(iv) Shigella sonnei (Group D): These are late lactose
fermenting dysentery bacilli. The Sonnei bacillus 
ferments mannitol and does not produce indole; it 
is sero1ogica 11y distinctive and homogenous.
23
£. soiinei can occur in two phases, namely; Phase I 
and II or S and R forms. The serological properties 
of ant igens of R forms differed from those of S forms 
and an R or form II antiserum will not react with the
S or form I (Edward and Ewing, 
Shigella infections:
The Shigellae were previously thought to be highly
host adapted, naturally infecting only humans and certain
subprimates like chimpanzeez and monkeys (Keusch, 1979).However, cases of Shigello ave also been reported indogs (Doems and Mortelmans, 1955); swine and calf (Ueda 
et al., 1963); poultry (Nyaga et al., 1985) and pigs 
(Ibu et al., 1987). In most instances the epidemiology 01
Shigellosis is traceable to a human host, whether 
symptomatic or asymptomatic apd the route describe a 
circle from the anus to the mouth and back to the anus 
once again (Christie, 1968; Grady and Keush, 1971). In 
the case of dvsenteriae 1, as few as 10 to 100 bacteria 
suffice to produce symptomatic disease in 10 to 40 percent 
of adult volunteers respectively (Levine et al., 1973).
As a result of this miniscule inoculum it is rather simple
24
for Shigeallae to spread by contact without interposition 
of a vehicle such as food, water or drink to amplify 
the infection doses (Keusch, 1979). A thin veener 
of infected faeces on the fingers may be all that is
required and indeed viable Shigellae can regu
cultured from the fingers hours after experi
inoculation of bacteria on the skin (Christi ek,? 1968).
Transmission is enhanced however, when sanitation is poor 
or there is opportunity for the organism to reach food or 
water. Nonetheless, in more developed countries, epidemic
shigellosis can be traced to faulI t.y .water supply or sewage
(Hardy and Watt, 1948) or contamination of food as varied 
as milk, cheese and fruits (Christie, 1968). In those 
countries, Shigellosis is often an endemic contact-spread 
disease affecting children under the age of ten years
Epidemiologically, there has been a noticeable 
change in species prevalence over the past hundred years. 
The outbreaks which occured in Japan early in this 
Century was due to J5_. dysenter i ae 1 (Kostrewski and 
S typulkowska-Mi siurewicz, 1968). In the periods between
WorldWars (1920-1939) this picture gradually changed and
25
sonnei emerged as the most common isolate in Europe 
and Japan (Reller et al., 1969). Of late 5.. dysenter i ae 
has once again caused epidemic disease in many part s of 
the world such as Bangladesh (Rahaman et al., 1975
Clinical manifestation of the disease;
Oral infection with Shige11a generally results in 
the onset of fever and watery diarrhoea within twenty-four 
and seventy-two hours (Dupont et al., 1969). In many 
instances this is followed by dysie ntery syndrome, including
abdominal cramps, tenesmus and bloody mucoid stools
(Dupont et al., 1969; Mata et al, .1, 1970). Whenever 
diarrhoea is detected in experimental Shigellosis; the 
jejunum is in a secretory state (Keusch, 1979); whenever 
there is dysentery, the colon is markedly inflammed and many 
invading bacteria are present (Takeuchi et al., 1968).
Thus shigellosis appear to be a two-organ disease - a 
proximal bowel secretory diarrhoea and an acute bacterial 
colitis resulting in dysentery (Keusch, 1978).
26
Pathogenesis;
Enteric pathogens are known to cause disease by 
one or two distinct mechanisms: elaborat ion of enterotoxin 
or epithelial penetration (Sprinz, 1969). A
Enterotoxin of Shigella d.vsenteriae was first described 
in 1903 and called Shiga neurotoxin (Keusch and Jacemicz,
1975). Parenteral injection of bacterial lysate or cell-free 
media supernatant into susceptible animal^caused 
characteristic 1imb paralysis which led to the •
Classification of Shigella toxin as neurotoxin (Olitsky 
and Kligler, 1920). Thiee Ssym[p toms prioduced by neurotoxin
were unlike the disease induWced b y intact b*acteria in man
(described above). Thus the concept that neutrotoxin 
participated in the pathogenesis of Shigella feil into 
disfavour.
It was discovered later that £. dysenter iae produced a 
protein toxin capable of causing intestinal fluid secretion 
(Keusch et al., 1972). This discovery opened the question 
of the role of toxins in the pathogenesis of Shigella 
diarrhoea. Eiklid and Olsnes (1983) compared the 
biological activities of the enterotoxin of d.vsenter i ae
with that of a well studied 20-year-old partially purified
27
preparation of neurotoxin from same organism. . They found 
Tr ;r». were:
i) intestinal fluid accumulation
ii) limb paralysis in mice and
iii) cell death in both preparations
When the toxin was further subjected to Sephadex ge 1 
filteration, chromatogprahy, isoelectric focusing and poly 
acrilamide gel e1 ectrophoresis, two fractions which were 
cytotoxic to heLa cells were obtained. One of the fractions 
was also associated with enterotoxigenicity and 
neurotoxicity. These data suggest that Shigella
enterotoxin and neurotoxin are closely related proteins 
and may indeed by indentical (Koo and Peterson, 1982, Eiklid
t
and Olsnes, 1983). Other properties of Shigella toxin 
include its heat-1iabi1ity, destruction by proteolytic 
enzymes; isoelectric pH 7.2 and molecular weight of 72,000 
daltons (Keusch and Jacewizs, 1975; Mclver et at., 1975)
Two other species of Shigella (flexneri and sonnei) have 
been shown to produce indentical enterotoxin (Keusch and
Jacewicz 1977a).
28
Role of Shigella toxini
Opinions seem divided on the actual role played 
by Shigella toxins in the pathogenesis of diarrhoea.
The toxin is lethal to mice and rabbits (Van Heyningen 
and Gladstone, 1953). Moreover it is cytotoxic for 
eukaryotic cells, such as HeLa cells (Keusch et al., 1972b) 
KB (human epidermoid carcinoma), normal human liver and 
monkey (Vicari et al., 1966). Fürthermore, purified
Shigella toxin inhibit protein synethesis in intact HeLa 
cells (Brown et al., 1980) as well as in cell free
translation System ,( Thompson eOt ayl., 1976; Oienick and 
Wolfe, 1980). It has been shown that the most provocative 
biologic activity of the toxin is its ability to cause 
transudation of fluid in rabbit illeum (Keusch et al., 
1972a). Since this model has proved so us.eful for 
definition of pathogenicity of diarrhoea producing strains 
of V. cho1 erae and £. co1i (De and Chatterjee, 1953; Moon et 
al., 1970), it was logical to suggest that Shiga toxin is an 
enterotoxin capable of causing human diarrhoea in a manner 
analogous to cholera and Jjj. co1i toxins. However 
this view was contrary to the results of studies in 
animals and volunteers. Levin et al. (1973) demonstrated
29
that large number of non-invasive but enterotoxigenic 
Shige11a strains were well tolerated by human models when 
fed orally. In contrast, the same workers showed that an
invasive but non-toxigenic strain caused Shigellosis in 
monkeys and human volunteers. Also Flores et al. (1974) 
and Steinberg et al. (1975) demonstrated that Shigella 
enterotoxin applied in vivo and vitro into intestinal 
preparations did not produce any marked increase in 
adenylate cyclase or cyclic AMP as noted after the
application of Y« cho1 erae entesrrotoxin. The Consensus 
of opinion was that the ability of Shigella organism to 
penerate the intestine was the primary determinant of 
virulence. A
Bacterial invasio
Many investigators have argued in favour of bacterial 
invasion of small intestinal wall as an essential process 
in the pathogenesis of Shigel la diarthoea (Formal £_L 
al.. 1972; Levine et al., 1973). It is known that 
piliated strains of Shigel1a flexneri adhere to human 
intesitinal epithelial cells better than non-piliated 
strains (Duguid and Gillies, 1957). Also the presence of a 
mucosal adhesin has been implicated in the adherence of £. 
flexneri to guinea pig intestinal cells (Nuchamowitz and
30
Mirelman, 1982). In experimental Shigella and Salmonella 
infection, electron microscop^ has shown that the brush 
border was destroyed and the bacteria were engulfed, by an 
invagination of the cell membrane of the host cells. A  
Fron the vacuoles in the epithelial cells the organisms 
invade and destroy adjacent cells producing ulceration and 
local inf1ammation (Takeuchi et al., 1965 and Takeuchi, 
1967).
Evidence reviewed by Formal and Hornick (1978) 
suggested that invasion, at least in Shigella, is determined 
by multiple chromosonal genes.
A major problem in the stOudyy of invasive organisms is 
the lack of a practical assay System. In the Standard 
in vitro test (Sereny) organisms are instilled into the 
conjunctival sac of guinea pigs. Invasion of the corneal 
epithelial cells produce keratoconjunctivits which 
correlates with intestinal invasion (LaBrec £.t al ., 1964). 
Invasion of HeLa monolayer has been used as a more 
practical assay, but organism adhering to the cells make 
Interpretation of results difficult and the assay has 
fallen into disuse (Keusch, 1979).
Human volunteer experiments and challenge of laboratory 
animals are alternative techniques but of limited value 
in screening large number of isolates.
31
Patholog.v of Shigella infections:
Bacillary dysentery is an infection localized in
the alimentary tract (Burrows et al., 1968). Histological
examination of infected guinea pig illeum revealed the
following changes: markedly altered villi, severe acute
inflamation of the mucosa with haemorrhage, discharge of
goblet cells and denudation of surface epithelium
(Keusch et al., 1972a; Steinberg et al., 1972; Levine et
at., 1973). Protoscopic examination of the rectal mucosa
demonstrates significant colitis manifested by inflammed
oedematous mucosa with patechial haemorrhages and mucus
(Levine et al., 1973; Mathan et al., 1986). Rectal biospy 
revealed po1ymorphonuc1ear infi1teration, goblet cell
depletion, microulcers, haemorrhage and oedema
(Levine et al., 1973; Annond et al., 1986).
Treatment thera.P.y in Shigellosis;
In the early 1940s the Sulfonamide were used for the 
treatment of bacillary dysentery. However, as early as 
the mid 1940s several countries began to note that a large 
Proportion of isolates of Shigellae were resistant to 
Sulfonamides (Cheever, 1952). As a result of the eaergence 
of drug resistance the sulphonamides became ineffective in 
the treatment of bacillary dysentery in Japan by 1959
32
(Watanabe and Fukasawa, 1960; Anderson, 1965; Datta, 1965; 
Weissman et al., 1973). A study of almost 1,500 cases in 
Korea in 1953 showed that almost all strains of Shigella
were resistant to Sulfonamide thus indicating that the 
emergence of drug resistance is world wide (Garfinkei et
al., 1953).
Other antibiotics including the tetracyclines and
ampicillins were introduced for the treatment of bacillary 
dysentary (Maltalin et al., 1967). Ampicillin therapy 
resulted in bacterio1ogica 1 clearance of stool in over
90 percent of patients within ours. Multiple drug
resistance to ampicillin was recognised in the early 50s in 
Japan and in vitro transfer of R-factors fromj^. coli to 
Shigella was demonstrated (Watanabe, 1963; and Farrar and 
Eidson, 1971). r V
As result of this development, the hitherto reliable 
ampicillin feil out of choice for Shigellosis in many areas 
of the world. When isolates were increasingly resistant 
to ampicillin, triraethoprim-sulphamethoxazo1e was prescribed 
(Keusch, 1979). Nalidixic acid is also effective in vitro 
and in general in vivo but less so than the above drugs 
(Keusch, 1979). Again reistance to nalidixic acid has been 
reported (Panhotra et al., 1985).
33
Generally, antimicrobia1 therapy follows recognized 
guideliness for all infectious diseases: the orgnni sm 
should be sensitive in vitro, the infection should be 
responsive in vivo and the risk of side effects should be 
minimal compared to the expected benefits and certainly no 
greater risk if the disease was not treated (Keusch, 1979). 
An additional criterion is that the drug must be absorbable 
in order to reach the population of organism within the 
lamina propria of the gut (Haltalin et al., 1967). The ■ 
guidelines ruled out the use of oral neomycin or 
furazolidone (non-absorbable) (Haltalin and Nelson, 1972), 
chloramphenicol (potentially too toxic), suphadiazine or 
or other sulpha drugs (resistant in vivo) (Haltalin et al.,
1976). A
Flmid-and eleclroüytg.;.
Rahaman et al**(l975)observed that Shigella diarrhoea 
is not usually accompanied by massive loss of fluid leading 
to severe dehydration, but, however, there is significant 
fluid losses, requiring attention to the state of 
dehydration. Mild to moderate dehydration should be 
easily managed with oral rehydration Solutions, consisting 
of electrolytes and an actively transported sugar such as
34
giucose (Keusch, 1979). The principle involved is that 
of co-transport of sodium equimolar with active transport 
of sugar (Field, 1977). Controversy still reigns regarding 
the specific nature and concentration of salts and sugar to
be chosen. Keusch ( 1 979) based on considerable exppeerniie nce 
in the field recommended a single ad libitum oral
rehydration solution (in mmol/1: Na 81 * CI-71, HCO 28,
3
Glucose 139) for treatment of all dehydrating infectious 
diarrhoea, regardless of stool sodium concentration, so 
long as physiological regulatory mechanism (thirst, renal
response) remain intact, unless giucose intolerance is
present. The electrolyte composition 
suggested for oral rehydration can be achieved 
in the household by mixing hald a teaspoonful of table salt, 
half a teaspoonful of bicarbonate of soda, a quarter of 
teaspoon of po’assium Chloride and four teaspoonful of table 
sugar (Glucose) in a a litre of water (WHO Diarrhoeal 
Disease Control (CDD) Programme, 1980). For some time, the 
WHO Programme for the control of diarrhoeal disease 
(CDD) has been researching into a more stable oral 
rehydration salts (ORS) formula. Laboratory tests have 
shown that this was achievable by replacing the sodium
bicabonate in the formular with trisodium citrate hydrate
35
(WHO Diarrhoeal Disease Control (CDD) Programme, 1984).
SALMQNEILAE
The Salmonel1a group was originally created by medical
bacterialogists to include organisms that gave rise to a
certain type of illness in man and animals and were related
antigentica 1 ly. It is now customary to lay emphasis on
biochemical activity (Topley and Wilson, 1983). Generally'
they are motile, produce acid and gas from glucose and
mannitol, and usually from sorbitol. They rarely ferment
sucrose or adonitol, and rarely form indole.
Acety-methy1 carbin ol is not formed, do not hydrolyse urea
or deaminate pheny1a1 anine, produce H S actively and grows
2
in citrate medium and form lysine decarboxyläse. The many 
serotypes in the group are closely related to each other by 
somatic and flagella antigens and most strains show diphasic 
Variation on flagella antigens. They are pathogenic for 
man and many species of animals giving rise to enteritis and 
typhoid-like disease (Edward and Ewing, 1972).
36
Salmonellae are divided into four subgenera. The 
first of these includes the typhoid and parathyphoid bacilli 
and other animal types while the remaining three sub: A  
genera coraprise of organisms that are in the main parasites 
of cold blooded animals. (Topley and Wilson, 1983). 
Sub-genera I: Most ferments dulcitol but not 
Lactose or salicin, KCN sensitive and do not 
1iquef y gela t in,
Sub-genera II: Also ferment dulcitol but liquefy 
gelat in.
Sub-genera III:(Arizona gfp)4:.  Acidi fy lactose but not 
dulcitol and liquefy gelatin.
Sub-general IV:Members acidify salicin but not lactose 
or dulcitol. Liquefy gelatin and resistant to KCN. 
Salmonel.l.a. .I.nfections;: < j y
The epidemiology of Salmonellae other than those 
causing typhoid and paratyphoid fever with respect to 
infection in man mostly involves the animal reservoir 
and transmission through food in which mu1tip1ication may 
take place (Edel et al., 1981). Large scale or intensified 
farming practices confine animals ori fowls in close
quarters. Frequently the animals have to live in contact
37
with their own excreta, which also may contaminate their 
feed or drinkin? arrangement. Salmonellae, inadvertenly 
introduced into a herd of flock through contaminated feed, 
water, pasture, sub-c1 inica 11y infected new stock or 
sometimes by rodents, wild birds or even man himself rapidly 
spread by cross infection (Edel et al., 1981). The 
excrement through effluents and through being used as 
fertilizer carries the contaminant to the environment and 
surface waters while at the abattoir their infecting
.SalJDQUe11a can be transfered t destined for human
comsumption (Richardson, 1975; Turnbull, 1979).
Typhoid and paratypoid fever occur throughout the 
world (Bockemuehl, 1976). Endemie disease is prevalent 
in many countries of the Far East, Middle East, Central and 
South America and Africa. This is largely a reflection of 
the Standard of water supply and sanitation. ln the rest 
of Europe and North America the incidence of enteric fever 
has declined steadily-in the last half Century, and the 
disease has largely become an imported one (Mandal, 1979). 
Transmission: £.. typhi and £. paratvphi are strict human
pathogens and the ultimate source is almost invariably man 
(Burrows et al., 1968). Tramsmission is through direct or
38
indirect contact with faeces or urine of a patient or a
carrier (Bennet, Jr. and Hook, 1959; Yoshikawa et al.f
1980). The principal vehicle of spread are conittaaim ni nat te e<d
water and food. Water has always been a dominant vehii c 1 e
of typhoid spread and many large out-brea_k_s _h_a„vje  bbeeeenn  \water- 
j Q
borne; these tend to be explosive because of the simiunlutlitaneous 
inolvement of a large number of people drinking from the 
same source (Mandal, 1979).
Paratyphoid outbreaks on the othseVr !hand are much 
less likely to be water-borne. #  s is because typhoid 
bacilli can initiate infection in small numbers (Blaser 
and Newman, 1982) whereas paratyphoid infection needs a 
higher infecting dose which is unlikely to be found in 
drinking water unless there is heavy polution (Mandal,
1979). Foods are common vehicles for both typhoid and 
paratyphoid infections. Raw fruits and vegetables are
impor tan t vehVi d e s  in some tropical countries where use 
of human faeces for manuring vegetable crops is a common 
practice (Bennett, Jr. and Hook, 1959; Mandal, 1979). 
Unpasteurized milk and milk products such as diary cream and 
ice-cream have been involved in many outbreaks 
(Blasser and Newman, 1982). In tropical countries,
39
carriers among the public food handlers dealing with 
high-risk food and beverage such as cold dishes,
fresh fruits and co>l1d drii:n ks play an important role 
in maintaining the endemicity of infection (Cvj etanovic,
1976; Onile and Odugbemi, 1987). Incidence of 
contamination of "fast food" has also been documented in 
Nigeria (Alonge and Oyekole, 1982).
The classical Symptoms of Salmes *1 1 1 a infection has been 
described (see Introduct ion) . Ho.wever, Symptoms are also
related to the severity of infect n, the sites of action of
> X ?
Salmonellosis has been associated are malignances, liver 
disease, malaria, bar tone 11osis, Schistosomiasis and 
haemog1obinopathies (Bennet, Jr. and Hook, 1959). The 
incidence of complication of Salmonella infection include 
arthritis and bacteremia (Torrey et al., 1985); 
Osteomyelitis (Wu et al, 1985); pneumonia (Canney, 1985); 
and endophthalamitis in infants (Appel, 1986). Other 
comp 1ications of enteric fever are intestinal haemorrhage 
and Perforation (Mandal, 1979).
Enterotoxin Production by Salmonella;
Despite the progress made in the study of enteric 
infections, the mechanism by which Salmonella causes
40
Gastroenteritis still remains an enigma to many 
investigators. Koupal and Deible (1975) demonstrated
the presence of enterotoxin in culture filterates o t A
S. enteritidis. In subseauent vears the entero to3xin ?
was demonstrated in other species of Sa lmone 1 1 
(Sandefur and Peterson, 1976; Thapliyal and Singh, 1978). 
Salmonella toxin has been shown to cause cell detachment 
and inhibited protein synthesis in intact Vero cells 
(Koo and Peterson, 1982). Maximum toxin was produced 
at pH 7.3 after 18h aerated incubation at 37 C in £. 
enteritidis (Sobeh and Vadel >ra, , 983), and evoked fluid 
secretion in the rabbit illeum (Giannella et al., 1973).
Pathology of Salmonella infections;
Rectal biopsies of patients with Salmonella 
infections showed abnorma1ities ranging from mucosal 
oedema and hyperaemia with or without petechial 
haemorranges to mucosal friability with slough formation 
and spontaneous haemorrhages (Day et al., 1978).
There was dilation and congestion of capillaries in 
the mucosa and submucosa and focal collections of 
polymorphonuclear leucocytes were present in the lamina 
propria and in the lumen of mucosal capillaries.
41
In most of the biopsies, crypt abscesses and variable 
destruction of the crypts associated with mucus deple- 
tion were seen (Mandat, 1979).
Jj-SAtment pf SftlrwnelIpqis ; A
For the past thirty years ch1oramphenico1 has been 
the most successful drug in the treatment of enteric 
fever. The recommended dose in adult is 500mg every 
four hours tili defervescence, then 500 mg six-hourly, 
the total lasting for two weeks (Mandat, 1979). The 
drug is generally given orally but in the initial stage, 
when the patient is frequently anorexic and perhaps having 
diarhoea, the drug should be given intravenously (Synder et 
al. , 1976). However, ch1oramphenico1 is not the ideal drug 
because of the risk of marrow toxicity and the high relapse 
rate (Kamat, 1970)C. Av  reover, recent wide-scale emergenceof £. typhi strains with R factor-mediated resistänce to 
Ch1oramphenico1 has brought about a new sense of urgency in 
the search for a suitable alternative (Mandal, 1979).
Such alternatives are:
(i) Ampi ci11 in: Düring the sixties, several
studies (Robertson et al., 1968; Herzog, 1976) 
showed the drug to be distinctly inferior to 
ch1orampheni co1.
42
The response was slow and the failure rate was as high 
as 30 percent. This drug is populär in Nigeria and has 
been known to induce resistance (Adetosoye and Rotilu,
1986).
(ii) Amox.vci 11 in; This drug was introduced into Nigeria 
in 1973 and its use has given more promising 
results. When amoxycillin was used Pillay et al., 
(1975) found that fever in these patients lasted 
6.8 days whii1e it lasted V7.3 day s inch1oramphenico1 group in a randomized comparative 
trial. Four of the ch1oramphenico1 group relapsed 
but none on t(»e amoxycillin group.
(iii) Co-trimoxazolet This drug has emerged as the most 
successful challenger to chloramphenico1 as the 
drug of choice in enteric fever. After the 
original report by Akinkugbe et al.f (1968) of 
more rapid defervescence with co-trimoxazo1e than 
with chloramphenico1, the drug has been used 
successfully in the treatment of typhoid fever in 
well over thousands of cases. It was later found 
that "toxic crisis" which may sometimes
complicate ch1oramphenico1 treatment did not occur 
with co-trimoxazole (Kamat, 1970).
43
(iv) Furazolidone: This nitrofurantoin derivative has 
been used mainly in the Indian subcontinent 
(Mandat, 1979). It has the advantage Of being
cheap but appears to be somewhat less effective 
than chloramphenicol in man (Herzog, 1976).
It has however been used to control experimental 
Salmonella gallingfium infect n chicken
(Ojo and Adetosoye, 1977).
.
44
Isolation and characterizat ion of Shitrellae and
Salmonellae
A nuraber of suitabie cultural techniques have been 
developed over the years for isolation of Salmonel1a 
and Shigella. For instance there is a wide acceptance 
of the value of elevated temperature enrichment for 
isolating Salmonella (Georgala and Boothroyd, 1964; 
Spino, 1966). Banfer (1971) found 43 C to be Optimum 
incubation temperature and selenite F broth a suitabie 
enrichment medium. Livings tUne (1965) found that 
incubation of Salmonella-Shigella plates (SS agar Difco 
No. 0074) at 40 C allowed Salmonellas and Shigellas to 
form colonies but controlled the growth of unwanted 
organ i sms.
It is well known that Salmonella and Shigella are 
not uniformly distributed through contaminated material 
and invariably in enrichment cultures and that several 
subcultures, all made at the same time, may be 
necessary to demonstrate them (Cowan and Steel, 1974). 
Harvey and Price (1979) subcultured, after 24 hours 
incubation, a Selenite broth inoculated with faeces 
onto eight separate plates of Brillant green and
MacConkey agar
45
Only two out of the eight plates were found
positive for Salmonella after incubation The
importance of subculture timing from fluid media has
been increasingly recognized f ound
that it was no advantage to use enrichment periods 
longer than 24 hrs. At 24h incubation 6035 of his 
samples were positive however , subculturing at 48h and 
96h decreased the isolations rate to 23% and 11% 
respectively. * •
The problem of infection with multiple serotypes 
in man has been recognized (Harvey and Price, 1979). 
Multiple serotype infection is also of interest to
Veter inarry bacterio1ogists (Edward and Brunner, 1946)
Although multiple infections are not common in 
Shigellosis as in Salmonella, a number of techniques 
have been found to be useful in dealing with this
oblem. These include the use of agg1utinating serum. 
iley and Laidley (1955) used the method for the 
Separation of single Salmonella serotype from a 
mixture by use of 0 and H agglutinating sera in plates 
of semi-solid agar. The usual method of obtaining
pure cultures by dilution of a mixed culture may 
also be applied to material containing multiple
46
serotypes (Cowan and Steel, 1974). If a sample is 
divided into many subsamp1 es, it is proibbaable that 
the serotypes isolated from the subsamp]>1 es will not 
be identical. Identical result can be achieve d by
multiple subculture from enrichment media.
The earliest reference to an enrichment method 
was found in the work of Vincent (1890) who used 
five drops of 5% (w/v) phenol in 10ml of broth.
This was inoculated with 5-10 drops of sewage-polluted 
water and incubated at Ä f  The point was made by 
Thomson (1955) who recorded that ordinary nutrient 
broth was generally able to act as enrichment medium 
for the isolation of pathogen in faeces. Selenite F 
broth did not come into general use until Leifson 
(1936) developed several fuild media containing 
different concentration of sodium hydrogen selenite.
It was found that 0.8% Sodium Selenite yielded Optimum 
result for the isolation of Salmonella tvohi. Sodium 
tetrathionate broth (containing a mixture of sodium 
thiosuplhate and iodine) is also very populär as 
an enrichment medium (Edward and Ewing, 1972). Knox 
et al., (1943) ha<\ expressed the formulae they studied
47
in terms of ml of 0.5 mol/1 iodine and 1 mol/1 
thiosulphate to 100ml of medium. Between 1923 and 
1957, there was a series of media used with a 
concentration ränge of sodium tetrathionate varying 
from 0.015 - 0.039 mol/1. To increase the complexity 
of the Situation, other workers have further modified 
tetrathionate media by addition of novobiocii^ and
sodium laurylsulphate (Harvey and Price, 1979). '
Over the years, a series of enrichment broths
have been described. V This include magnesium Chloride 
malachite green (Rappaport et al., 1956); Strontium 
Chloride and Strontium selenite (Chau and Huang, 1971) 
Bismuth sulphite (Gell et al., 1945); Gram negative 
(GN) broth (Hajna, 1955) e.t.c. Obviously the choice
of enrichment media to be used will be determined by 
the specialist interest of the bacteriologist.
< seelenite F and GN broth were found to be essential for 5 the diagnosis of typhoid fever and shigellosis (Harvey 
and Price, 1979).
For selective plating a number of selective agar 
are in current use. Among them are Brilliant green 
agar; bile salt agar. Bismuth sulphate agar,
Salmonella - Shigella (SS) agar, MacConkey agar,
48
Hektoen enteric agar, Xylose-lysine deoxycholate agar, 
and Deoxycholate citrate agar. A comparative study 
carried out by Goyal et al., (1981) demonstrated the
superiority of Xy1 ose-1ysine deoxycho ilate agar aind
MacConkey agar in the isolation of Salmone11a and 
Shigella. Devenish et al. (1980) d«eveloped a 
new medium called nov<robiocin-bri11iant green-ig-lgil ucose 
(NBG) agar for the isolation of Salmone11. spp.
According to them, the distinct advantage of NBG 
over the conventional media is the "consistent
differential reaction 11 Salmonella subgroups
including biochemically atypical strains".
In the final analysis the choice of Optimum 
selective agar is made on common sense grounds. If
it is important to have a medium capable of growing 
Shigella, deoxycholate citrate (DCA), Salmonella­
Shigella (S-S), Xylose-lysine deoxycholate, and 
Hektoen enteric agar must be used. If the bacterio-
log ist is only interested in Salmonel1a tvohi  ̂Bismuth
ulphite agar would be mandatory (Harvey and Price, 
1979). Also addition of 1 ug/ml potassium tellurite to 
MacConkey agar is known to serve as secondary enrich- 
ment medium and facilitate the isolation of Shigellae.
49
Differentiation and Characterization of Salmonellae
and Shieellae
Generally, the differ entiation and characteri- 
zation of enteric bacilli is based upon a variety of 
biochemical and cultural reactions and upon antigenic 
structures (Cowan and Steel, 1974). A useful primary 
differ entiation is made on the basis of lactose 
fermentation which is roughly correlated with 
pathogenicity. The coliform bacteria ferment this
sugar rapidly with the formation of acid and gas in
*
24h, while Shigellae and Salmonellae (essential 
pathogens) do not ferment it. Similarly, the dysentery 
bacilli or Shige 11 a%-, di v i de into two groups on the 
basis of fermentation of mannitol and are anaerogenic 
(Edwards and Ewing, 1972). While the Salmonella group 
in general produce gaseous fermentation with the 
exception of the typhoid bacilli.
A great variety of cultural reactions, not only
the conventional sugar fermentations, formation of 
indole from trytCphane eAic but various kinds of 
speciali zed tests such as utilization of tartaric 
acids and malonate and amino acid decarboxy1ase 
activity have been useful in the physiologica 1 
characterization of the Salmone11a and Shlgel1a 
group (Edwards and Ewing, 1972).
S c ro lo g .v ;
The technique of antigenic analysis have been
developed in the study of Shigella and Salmonel1»
groups and the antigenic composition o f theae groups
is perhaps better known than any other bacteria (Cowan
and Steel 1974). Three types of antigens are present
in the Salmonella group, two are associated with the
cell substance (somatic or 0 antigen and V v i ru 1 ence
I
antigen) while the third is the fiagella or H antigens.
Partial or complete Identification of serotype is 
carried out by the use of antisera containing 
appropriate antibodies. The serological 
differentiation of Salmonel1a strains is called 
Salmonella typing and requires a lot of skill and 
specialty.
In case of Shigel1a there are no fiagella antigens 
and the serotyping is relatively easy. However, in 
the past the nomenclature of the dysentery bacilli has 
been somewhat casual, and informal names such as Shiga 
bacillus, Flexners bacillus, irong bacillus and the
Hiss-Russel Y bacillus had wide currency (Edwards and 
Ewing, 1972). The name Shigella for the genus has
51
gained reasonably wide acceptance. International 
agreement has also been reached based on species name 
and serotypes as suggested by Ewing (1958) t l A  
introduce a welUcome order and stability.
J '
52
An t i b i o t i ca
Antibiotics are Chemical substances obtained from 
bacteria or fungi which in dilute concentrations are 
capable of inhibiting the growth of microorganisms or 
capable of destroying them (Pelczar et al.,1977).
The word antibiotic has come to refer to a metabolic 
product of one organism that is detrimental or inhibi-
tory to other microorganisms in ve.r..y.  s. .mall amounts 
(Garrod et al.,1981). Antibiotics were known by their 
activities long before they we re given the name by
which they are now call . Ml anuy years ago, the Chinese
use mo1dy soybean curd for the treatment of boils and 
controlled foot infections by wearing sandals furry 
with mold (Pelczar et al., 1977).
The first systematic search for, and study of ,.*ni tMotic 
(reported by Brock, 1974) resulted in the discovery
of actinomyceton in strains of actinomycetes. In 
1929 Alexander Fleming discovered the famous 
antibiotic named penicillin. Several thousand 
antibiotic substanceshave been isolated and identified 
since 1940 (Garrod et al., 1981). Many of them are of 
no practical importance yet, but a few have changed the 
entire concept of chemotherapy. These is no doubt 
that many more and possibly better antibiotics will 
be found (O'Brien et al., 1987).
53
M ade.pf action of antibiotics;
Antibiotics can inhibit or kill microorganisnis in 
one of several ways (Pelczar et al., 1977). A
(a) Inhibit cell-wall formation.
(b) Damage of the cell membrane (cytoplasmi c
membr ane).
S 5
(c) Interfere with protein synthesis
(d) Inhibit nucleic acid metFabolism.
A big setback to the use ntibiotics was the
discovery of antibiotics-resistance phenomenon.
Antimicrobial resistance in Man;
The use of antibiotics in human and Veterinary 
medicine for the treatment or prevention of infections 
has favoured the selection of antibiotic resistant 
organ i sms their wide dissemination in human
Population (Ogunnariwo, 1987). The first clinically 
serious consequence of antibiotic resistance to attract 
considerable attention was the widespread d i ss ein i na t i on 
in hospital of strains of S t aph.v 1 ococcus aureus that 
were resistant to penicillin by virtue of their ability 
to form an antibiotic-destroying enzymes (B-1actamase), 
and subsequently acquired resistance to several other 
ehern i ca 1 ly related antibiotics (Anderson, 1968).
54
By the early 1070s the gram negative bacilli have 
replace Ĵ . aureus as the most important cause of 
hospital acquired septic infections, and at least in a 
number of developed countries, widespread endemic A  
prevalence of multiple-antibiotics resistant strains 
of S.aures became uncommon (Parker et al.T 
Adegoke, 1983). A more serious Situation developed 
when antibiotics resistance became a common cause of 
treatment failure in diarrhoeal enteric disease 
(O'Brien et al., 1987).
Plasmid - determined transferable resistance was
detected with increasing frequency among £. coli. 
Shigellae and Salmonellae during the 1960s, and a 
few years later there were widespread epidemics of 
severe bacillary dysentery and typoid fever caused by 
strains that were resistant to several antibiotics, 
including the agent of choice for the treatment of 
these infections (Barrada and Guerrant, 1980).
In recent years, penci11inase-forming gonococci 
have shown considerable spread world-wide (Jaffe et 
al., 1981; Johnson et al., 1981) and several 
B-lactamase plasmids have been identified among them 
(Ansink-Schipper et al., 1982). Penci11in-resistant
55
strains of pneumococci became locally prevalent in 
South Africa (Jacobs et al., 1978) where they caused 
treatment failures and death of infants. The 
resistance was non-enzymatic and chromosomal ly 4  
determined, and was often accompanied by resistance 
to other antibiotics including tetracyclii
ch1oramphenico1
Until quite recently Vibrios with R factors coding 
for multiple antibiotic resistance were seen infre-
quently. Extensive epidemics of cholera have now
been reported from the United Republic of Tanzania 
(Mhalu et al., 1979), in which an initially sensitive 
strain became predominantly resistant within a few 
months, the R factors responsible for this indicated 
resistance to several antibiotics, including 
tetracycline the drug of choice for treatment.
Evidences have indicated that serious consequences 
of antit ics resistance were no longer confined to
urban hospitals but were being encountered increasingly
in the general population (WHO Scientific Working 
Group, 1983). There is also a greater prevalence in 
developing countries of resistance to easily available 
antibiotics such as ampicillin, tetracycline,
chloramphenicol and Sulfonamides, than is known to
56
occur in developed countries (Osoba, 1979). Survey of 
B-lactamases in Enterobacteria from developing 
countries revelead a corresponding greater variety 
of types, and of instances of multiple B-lactamases 
in the same isolates (Ogunnariwo, 1987)
Antimicrobial resistance in Animais;
It is well recognized that the administration of 
antibiotics to animals for any purpose (growth promo- 
tions, prophylaxis or subtherapy) lead to the accumula- 
tion of resistant bacteria flora (WHO Scientific 
Working Group, 1983). Antibiotics have been used for 
all these purposes for many years and it is difficult 
to separate the contribution made by each of the above 
(three purposesf to the pool'of resistant organisms 
in animals.
According to Edel et al. (1981) the danger of this 
pool is due to the fact that:
(i) Antibiotic-resistant pathogens common to 
animals and man may reach man by cross-
infection; and
57
(ii) Antibiotic-resistant non-pathogenic organism 
in an animal raay be passed to and colonize 
man, thereby carrying R plasmids whtcäi are 
subsequently - transfered to human A
pathogens or to indigenous flora in &
human body.
Many of the enteric gram negative org£anisms 
(e.g Salmonel1a species and some strains of 
Camp.vlobacter) infect man as well as animal 
(Topley and Wilson, 1983). The larger Proportion of 
Salmonella infections in m an &f e derived from eating 
contaminated meat, infected egg or milk and therefore, 
indirectly, from animal course. Whenever strains of 
antibiotic-resistant Salmonellae arise in animals they 
eventually reach man (Rowe et al., 1979; Lyons et al., 
1980).
The frequency of antibiotic resistance in various 
Salmonel1a species varies from one country to another. 
Linton (1981) revealed that while resistance to 
Sulfonamides and Streptomycin occured in up to 50% of 
isolates in the United Kingdom from 1958-1959, multiple 
resistance was rarely experienced in most species.
58
Where this did arise, it occured almost exclusively in 
S . typhimur i um. A survey of animal Salmonellae in 
North-East USA by New et al.,(1957) revealed thatt the
majority of _S_. typhimur i um, Ŝ. saint-paul and S , A
heide 1berg were resistant to three or more antibiotics 
including ampicillin, kanamycin and tetracycline in
addition to Sulfonamides and Streptomyrcciin.A S‘everal
y
&
< < ?
J '
59
authors (Lyons et al., 1980; Edel et al. 1981) 
attributed this resistance at least in part, to the 
continuing widespread use of antibiotics as feed A
additives and this may be a significant cause since 
antibiotics are often used at levels considerably 
higher than those recommended. 'S?
The antibiotics resistance s tatus\^fthe normal
gut flora is somewhat different. The oral
administration of antibiotics which iis the common
route of therapy, prophylaxis and growth promotion, 
invariably favours the selection of resistant strains
/ j
particularly from among the Enterobacteriaceae and 
other gram negative bacilli which then become prevalent 
(WHO Scientific Working group, 1983). Evidences now 
abound confirming that these resistant strains reach 
man via food chain. The most definite work has been 
done with £. coli. .The highest incidences of 
antibiotic resistant E. coli, were found in calves
(Howe and Linton, 1976; Adetosoye, 1980a) pigs and 
poultry (Linton et al., 1977) in situations where 
antibiotics have been widely used as feed additive.
60
Antibiotics are now not much used in preventing 
food spoilage because of high frequency o.f antibiotics
resistance developed by contaminat ing organistns (WHO 
Scientific Working Group, 1983). A
Properties of infectious drug resistance;
The original discoveries in the field of 
transferable drug resistance were made in Japan 
(Anderson, 1968) and followed the detection in that 
country from 1957 onward of a rise of drug resistaAc»e
in Shigellae. Most of the early work was done by the 
Japanese, one of the most a being Watanabe (1963)
whose review was the first comprehensive introduction
of the subject. Since then however, quite a large 
number of workers all over the world have contributed
to the knowledge •inX this field.
The properties of transferable drug resistance 
raay be summarised as follows (Anderson, 1968): The 
resistance may be to a single drug but is usually 
multiple. It is more commonly found among 
Enterobacteriaceae that is, Sh i ge 11a . Salmone11a 
F.scher i chia coli. Klebsiella, P ro t eu s  and related 
organisms inhabiting the animal or human intestines,
61
The genetic elements Controlling transferable
resistance are designated "R (resistant) factors"
and are borne on plasmids (Watanabe, 1963
The nature of Plasmids:
Plasmi. ds are extrachromosomal AgenSetic elements,
they are composed of DNA and are inherited yet 
dispensable and have the ability to replicate 
independent1y of the chromosome (Farar, 1979).
Plasmids are the physical entities responsible
for transmissible drugx rresistance (Falkow, 1975; 
Mitsuhashi, 1977; Jacoby and Swartz, 1980).
Structura11y , they are covalently closed circular 
molecule of double stranded DNA. Most plasmids 
ränge in size from a few million to approxiraate1y 
100 million daltons in molecular weight although 
a few are larger or smaller (Jacoby and Swartz, 1980). 
They are thus about one percent as large as the 
bacterial chromosome which they resemble in general 
Organization (Farrar, 1979; Jacoby and Swartz, 1980). 
Plasmids harbour genes which can be divided into four 
categories (Jacoby and Swartz, 1980): essential genes, 
genes involved in plasmid transfer (tra-genes), genes
62
concerned with interactions with other seif replicating 
genetic units or replicons, and genes that affect the 
host's cell interaction with the environment. The 
latter genes include those coding for enterotoxin pro-
duction, haemolysins, surface adherenicce antii gens
resistance to heavy metals, ability to ferment certain
carbohydrates and various other fumncct ions.
For some plasroid Systems, the resistance and 
transfer genes occur on sei. .reu mo1 ecu 1 es, each 
capable of independent replication. The transfer of 
such an aggregate mu1timo1ecular plasmid is achieved 
by mobilization of the Tra-R plasmids by its Tra 
Partner (Anderson^ 1968). In other plasmids, the 
resistance genes and transfer functions are combined 
in a single molecule which may or may not be able to 
dissociate into separate replicons.
Pt^amW. detect ion;
Plasmids can be detected by physical or genetic
techniques. A general method i. s centrifugat. l.on to
equilibrium in Ceasium Chloride gradients containing 
the dye ethidium bromide which binds differentially to 
plasmids and chromosomal DNA to allow their Separation 
(Helinski and Clewell, 1971). Recently, much simple
63
and more rapid techniques for plasmid visua1ization 
have been developed using agarose gel e1 ectrophoresis 
(Meyers et al., 1976).
Chromosomal or Plasmid mediated resistance:
Davies and Rownd (1972) suggested that
< P
micro-organisms acquired resistance to onlW y *on*e drug at 
a time and that organisms resistant to several drugs 
had arisen by accumulation of successive mutations. 
However, mutation and selection coul«\not explain the 
rapid increase in this incidence of drug resistance 
especially in Shigellae wh1i1 ch show resistance to as
many as four to six drugs. Transfer of multiple drug 
resistance in vitro by mixed cultivation of naturally 
occuring multipe drug resistant strain with drug 
sensitive ones have  ,proved conclusively that multipledrug resistancAe s due to the presence of
extrachromosomal resistant deterrainants or R factors 
(Watanabe and Fukasawa, 1961a).
Other plasmids act as fertility factors to promote
. .. Bacter
exchange of chromosomal genes orj bkoc c ioh ,>‘~iocins, 
metabolic enzymes, enterotoxins, virulence factors, 
resistance to heavy metals, to ul traviole'. * radiations 
and production of haemolysins (Jacoby and Swartz,1980).
64.
Methods of Plasmid transfcr:
Generally, there are three raethods of genetic 
transfer of Information in microorganisms, cell to 
cell contact or conjugation, transformation (naked DNA 
mediated) and transduct ion (bacteriophagemediated) 
Watanabe, 1966; Lacey, 1971). Conjugative plasmids can 
be detected genetically by testing for transfer of the 
property they deterraine to a suitable recipent by 
mating (Falkow, 1975). For example, to detect 
R-Dlasmid transfer to E. coli. an ant ibiotic-sensitive 
recipient made chromosona1 ly resistant by mutationtb 
nalidixic acid is mixed in liquid culture with the 
presumptive donor organism and then plated on media 
containing nalidixidic acid (which will prevent growth
of donor) together /wiAth an antibiotic that prevents 
growth of recipient except resistance transfer occurs. 
In this process pair formation takes place between 
donor and recipient cells by means of a specific 
structure synthesized by the donor, the sex pilli.
This attaches to the surface of the recipient cell 
and may serve as the conduit through which 
plasmid DNA passes from donor to recipient
65
(Anderson, 1968). THere are also "aggregates" consis- 
ting of a number of bacteria joined together by sex 
pilli. One strand of plasmid DNA passes into the
recipient cells and complimentary Strands are
' donor
synthesized in both^and recipient cells (Dunny et al., 
1978).
In transformation a drop of a solution of 
transforming DNA frora donor strain is mixed with the 
recipient cells. The treated cells are then spread 
on antibiotics-containing agar plates for selection 
(Ogunnariwo, 1987).
Transduction proceduresj Qemrploy bacteriophages 
e.g. phage epsilon in Sa1 mono 11a in carrying out 
genetic transfer from donor to recipient (Watanabe 
and Lyang, 1962) and phage PI in £. coli (Harada 
el at., 1963).
/ P
Conditions governing the freauency of Transfer 
in -C.Q.nLi.u ve plasroids;
The frequency of transfer of drug resistance is
the resultant of the interaction of a number of
influences (Anderson, 1968; Davies and Smith, 1978):
(a) The Recipient Strain: Different members of the
Enterobacteriaceae vary in their susceptibi1ity
i
66
to infection with a given R factor. In the case
of R factors in which the determinants are closely
linked with the transfer factor, this is probably
largely the result of the varying capacity of
different strains for accepting the transfer
factor encoded (Watanabe, 1963). £. coli K is
12
known to be an excellent recipient for R-factors 
of diverse origin. On the other hand &. 
t-VPhimurium type 36 is a relatively poor recipient 
for some R-factors originally found on £. co1i
and Shigellae but good rXectirpient for R-factors 
originally defined in .S., t.vphimur i um.
(b) The donor strain: Some strains seem to be 
more efficient donors that others. This 
character is presumably dependent at least,
in phar-t ron the state of the repression of
the transfer factor in the strain concerned.
e Ph.vsiological state of the donor and
recioi ent cel1s: Transfer of R-factors to
recipients cultures is usually low in
-5
frequency and raay be less than 10 per donor 
cell after mating for one hour. If the host 
cells are in suitable physio1ogica 1 state, 
e.g. log phase, adequate nutrient supply, etc
67
spread of resistance factor is enhanced in 
recipient culture.
This is caused by derepresion of the transfer 
factor concerned. The initial derepression
depends on the physio1ogica 1 state of thle doinor- 
recipient System. Epidemie spread of the transfer 
factor does not start until more than twelve hours
have elapsed after mixin? do recipient
trains (Anderson, 1968). then results in
rapid spread between recipient cells, so that 
fifty percent or more are infected within a few
hours.
( d ) The nature of 4the° linkaee between the 
determinants and the transfer factor?
A reversible association of Ampicillin
resistance gene and the transfer factor result in
a lower rate of transfer of the complete R factor 
than close linkage of the tetracycline resistance 
genes and transfer factor type, in which the rate 
of transfer of the determinant is that of the 
transfer factor itself.
V
68
(e) Ntm-SPecilic factors; For example, the density of 
cultures determines the frequency of contact and 
therefore the possibility of con jugat ion tietween 
donor and recipient cells and recipient celf" *hat
have already acquired R-factors. Sorae plasn 
transfer rauch more efficiently on solid than in
liquid medium so the mating
impringed on a filter for mo
conjugat ion (Jacoby and Swartz, 1980). For a few . 
plasmidsjtransfer is temperature sensitive and 
occurs more readily at 22 C than 37 C (Tarawaki 
et al., 1967). Thus both low and high incubation 
temperatures should be employed when attempting
However, at about
(Fa 1kow, 1975).
Incompat ibi 1 i t.v behaviour;.
Incompatibi 1ity behaviour is often used to 
classify plasmids genetically. Compatible plasmids can 
coexist in the same host, while incompatible plasmids 
cannot and so tend to displace each other (Chun et al., 
1984). •
Plasmids carrying resistance factors (R-factors)
found in Salmonellae and Shigellae have been grouped as 
follows (Jacoby and Swartz, 1980)
<?-
Plasmid Original Host Characters
Ri Salmonella paratyphi Dissociate into conjugalive 
resistance transfer factor 
RTF, r determinant, Am, Cm, 
Km, Sm, Watanabe and 
Fukasawa (1961a).
RlOo (also called Shigella flexneri TC or RTF; Cm, Hg, Sm, Su on 
NRI and R222) r-determinant. Watanabe and 
Fukasawa (1961a); Davies and 
r -
Rownd (1972).
R 10’Ri2,R13 Shigellae r determinants carry different 
R £ , R . marker: Am, Cm, Sm, Su 14 16 26
& Horada et̂ al., 1983).
L-T-2 Salmonella typhimurium r determinant: Sm, Tc, Cm, Su 
(Watanabe and Fukasawa, 1961).
> s
Am = Ampicillin; Hg = Mercury ions; Sm = Streptomcyin; Tc = Tetracycline; 
Cm = Chloramphenicol; Km = Kanamycin; Su = Sulphonamide.
70
Mechanism ol PIasmid-determined Resistance:
Plasmid determined resistance to antibiotics by 
four biochemical mechanisms (B e n v e n a n d  Davies, 
1973; Davies and Smith, 1978; Jacoby and Swartz, 1980). 
These are: •S~
(i) The drug is detoxified or inactivated;
(ii) Transport of the antibiotic into the cell is
blocked; 2?
(iii) The target site is alte red ;so that binding of
the drug is reduced or eliminated; or
(iv) The antibiotic inhibited step is by-passed.
The following are known mechanisms which are 
involved in the resistance against some of the 
populär drugs:
(a) S t r ep t omvc i n : Streptomycin is attacked
by\  two different enzymes viz: 3"-, 0-^* hobphottasef-rkse »
adeny1transferase (Jacoby and Swartz, 1980).
These enzymes when purified have been shown 
to be fairly small proteins, with submit 
Molecular weight:of 17 to 29 x 10D (Davies 
and Smith, 1978). They are found in the 
per i p 1asmi c scape between the inner and outer
cell membranes.
71
Since these enzymes permit growth in the 
presence of unmodified drug they are thought to 
provide resistance by modifying aminoglycoside at 
a faster rate than it can be transported into the 
cell (Dickie et al., 1978). Once modified the
aminoglycoside is no 1 onger
protein synthesis.
(b) Ampi ci11 in: A major Proportion of the resistance 
to B-lactam antibiotic such as ampicillin, 
carbenici11 in and Cephalosporins among strains of 
gram negative bacilli is produced by a variety of
extrachromosomal (plasmid or transposom mediated) 
B-lactamases, the effect of which is augmented in 
some strains by impaired diffusion of the 
antibiotic into the bacterial cell (O'Brien, 1987; 
Ogunnariwo, 1987). The enzymes are produced 
inducibly or cons t i t ixt i ve ly and are cell-bound
rather than excreted into the surrounding medium 
(Benveniste and Davies, 1973). More than twenty 
of these B-lactamases have been identified, but a 
few predominates, and one of these, the TEM-l-B- 
lactamase, is encountered more often than are all 
of others combined (Medeiros, 1984).
72
The B-lactamases are known to hyrolyse the
B-lactam ring present in amplicillin and other
related antibiotics (Ogunnariwo, ,0ß7' 'rK°
enzymes vary in molecular weight from 12 t
x 10 and, so far as they have been examin
iromuno1ogical1y distinct, except for
TEM-2 which cross react and appea
very few, perhaps, one araino acid (Ambier and 
Scott, 1978). The level of resistance 
encountercd is complicated by such factors as the 
Substrate affinity of the B-lactamase being 
produced, the extent of induction (if the enzyme 
is inducible), the location of the enzyme in the 
cell and the ease of entry of the antibiotic 
into the organism (Benvesnite and Davies, 1973). 
(c) Ch1orampheni co1: Since the introduction of 
ch1oramphenico1, the emergence of bacteria 
resistance to this antibiotic has been well 
documented. Okamoto and Suzuki (1965) were the 
first to show . the inactivatioo 
chloramphenicol in the presence of acetyl 
coenzyme A. The inactivation of
chloramphenico1 was due to acetylation of the
73
y i eld 3-ace t:
and Davies, ]
o n
nhc er; 
1[ > J L
~CL— ~CL'---■CLH^
( 1 I
OH M- (OH
^ 7 ^7 &
The structure of chlorampheni The arrows
point to the hydroxyl groups that are acetylated 
(Adapted from Benveniste and Davies, 1973).
The enzyme is synthesized constitutiv6ly and 
is located intrace11 ularly. 1t has a molecular weight
of 80,000 and consists of four identical catalytically 
inactive subunits each having a molecular weight of
20,000 (Benveniste and Davies, 1973; Jacoby and Swartz,
1980).
Only sporadic isolates of Salmone 11a t.vphi were 
resistant to ch1oramphenico1, which was agent of choice 
for treatment of typhoid since 1972, when there was a 
major outbreak of chloramphenicol resistant Salmone11a 
typhi in Mexico (Anderson and Smith, 1972). Sub-
sequent ly, strains of £. t.vphi sitnilarly carrying a
_ -0X-
74
ch1oramphenico1 resistance gene on a Inc HI plasmid and 
similarly resistant to Streptomycin, Sulfonamide and 
tetracycline have caused an outbreak in India and have 
become endemic in Vietnam and Thailand (O'Brien jlL al.. 
1987). The chloramphenicol-resistance gene was also
found on the InC F lme plasmids carried by the widely 
epidemic non-typhoid serotypes of S a1 mono 1la (Jacoby
and Swartz, 1980).
(d) Tetracvclines: Resistance to tetracycline is 
common in R enteric strains (Chopra and Howe,
1978). The fact that the gene mediating 
tetracycline resistance seems closely linked to 
that portion of the 11 factor which determines 
transmissibi 1ity may be the reason for its 
prevalence. &  tracycline is the only antiobitics 
for which the major mechanism of plasmid 
determined resistance is a block in drug uptake 
(Chopra and Ball, 1982). Sensitive bacteria 
accumulate tetracycline by two independent 
transport Systems one active and one passive 
(McMurry and Levy. 1978). Both Systems are 
partially inhibited in resistant cells. Unlike
75
most plasmid determined resistance mechanism in 
gram negative bacteria, tetracycline resistance is 
inducible ^sh^-?aand Howe, 1978). With induction, 
several new proteins are made and associate with 
the cell memberane, where they presumably i T  
interfere with antibiotics uptake. Infact, 
resistant cells possess an uptake, and perhaps 
and efflux System for tetracycline that is lacking 
in sensitive cells (McMurry and Levy, 1978). 
Tetracycline resistance genes are widely 
distributed among isolates of Ent erobacteriaceae in all 
parts of the world (Chopra and Howe, 1978).
The levels of drug_resistance con£er.e.d_-by R-factors-L 
The level of resistance attained by a strain 
carrying R factor depends on two constituents 
(Anderson, 1968); the host cell and the resistance 
determinant. Watanabe and Fukwassa (1960) pointed out 
that the same R factor may promote different levels of 
resistance in different hosts. A streotomycin R factor 
conferred resistance to more than 100 ug/ml of the 
drug on a Shigella strain, and to only 10 ug/ml on a 
strain of E ♦co1i.
76
Watson (1967) transfered R factors for multiple 
resistance, including that to Streptomycin, from 
Shigellae to £. coli K-12. The recipient cultures 
showed lower Streptomycin resistance than the donors. 
The differentft-in strep* ' ’ * " * re
however, of a smaller by
Watanabe and Fukasawa
(1967) demonstrated that mu1ti-resistance R factors 
endowed the £. co1i recipient with higher chlora- 
amphenicol resistant than that of shigellae donors.
The minial inhibitoryO'fchibitory concetration 
of ampicillin and pencillin for £. typhiraur ium of 
page type la resistance to A, S, Su and T described by
Anderson and varied between 64 and 128
ug/ml but was the ̂ same for both drugs in each wild 
strains K-12 infected with R factor from one of the 
strains of which the MIC was 128 ug/ml, developed an 
ampicillin MIC of 64 ug/ml, and a pencillin MIC of 
91 ug/ml, the difference being reproduceable.
These observations confirm that the level of
resistance confered by an R factor depends on both the 
R factor and the host cell. While extreme variations
77
from one hots to another exists, it is also cominon to 
find, in effect, the same level of resistance in thAe original host and in a new host. 
&
78
Mon-plasmid-mediated resistance in enteric bacteria
Although most mu1tiple-drug resistance in enteric 
bacteria is plasrnid linked, important types of 
resistance can be determined by genes located on the 
bacterial chromosome (Farrar, 1979).
Non-transferab,l e, apparently chromosome mediat0ed- 5
cephalospor ing beta-1actamases are found in many 
Strains of Escherichia coli. indole-positive 
Eroteus species; Enter Qba.c.t.e.L, Citrobacter» Serratia 
marcescens and Pseudomonas aerug inosa (Richmond and 
Sykes, 1973; Medeiros et al., 1984 Farrar and Newsome, 
1976). Virtually all strains of Serratia marcescens 
possess the chromosomal cepha1osporinase, and many also 
elaborate the p 1asmid-mediated TEM beta-lactamase 
(Farrar and O'Dell, 1976).
In £. aeruginosa. while much resistance is due to 
plasmid-contro11ed arainog1ycoside - modifying enzyraes, 
many gentamicin-resistance strains have been isolated 
from clinical infections which exhibit diminished 
accumulation of gentamycin but no detectable enzymatic 
modification of the antibiotic and no plasmids (Bryan 
et al., 1974; Bryan et al-, 1976). Strains 
resistant by the enzymatic mechanism are usually
79
highly res i tant^MIO 100 ug/ml) whereas those with
diminished pearmeabi1ity are less resistant to
gentamcyin (MIC 6-12 ug/ml) (Farrar, 1979)
PIasmid-mediated resistance and that due to 
chromosomal genes may not be entirely unrel
(Farrar, 1979). Transposable elements from plasmids 
can insert into the bacterial chromosome and can 
decrease or increase the level of functional activity 
of genes at or near the site of insertion (Saedler et
al., 1974).
For example, insertion _ nspo ,son into the 
chromosome might de-repress the synthesis of a drug- 
inactivating enzyme controlled by chromosomal genes, 
leading to an increase in resistance (Farrar, 1979). 
The prevalence of plasmids in medically important 
bacteria has undoubtedly increased since the beginning 
of the antibiotic era, and it appears that many of 
these possess transposable DNA sequences.
Lost of R Factor: The foregoing observations suggest 
four methods whereby an R factor may loose it 
transferabi1ity (Anderson, 1968):
(a) Segregation of the R factor during transfer so 
that only resistance determinants enter the
recipi ent.
80
(b) Segregation during division of the host strain so 
that some of the progeny receive deterrainants 
only.
(c) Transduction to a fresh host, in which the deter- 
minant only is carried over by the transducing
phage. ........................ f
(d) Destruction of the transfer factor by mutagens, 
without damage to the determinants also known as
curing.
Among the latter are many com£pounds that appear 
to function through their interaction with DNA and 
others that have different sites of action (Jacoby and 
Swartz, 1980). Known curing agents include ethidium 
bromide, acriflavine, acridine orange, chloroquine, 
guinone, daunomycin and methylene blue (Hahn and Clak, 
1976). * ‘ '
TransposQ.n.i t is known that many drug resistance
genes resides upon DNA sequence which can be 
translocated from one plasmid to another (Cohen, 1976; 
and Kopecho, 1967). Such transposons have been 
described which specify resistance to ampicillin 
(Hedges and Jacoby, 1974), tetracycline (Klechnar 
et al., 1975) and ch1oramphenico1 (Gottesman and
81
Rosner, 1975). Six of the wel1-established plasmid- 
determined beta-lactaraases in gram negative bacteria 
and four of the novel beta-1actamases are determined
by transposons (Jacoby and Swartz, 1980). These
■
include the transposons coding for three TEM-like 
beta-lactaraases in Pseudomonas spp. TEM-1 is 
determined by Tn 3 and TEM-2 by Tn 1 which shares 
considerable homology with Tn 3 (Ogunnariwo, 1987). 
Plasmids and Virulence in Salmonella and Shigella:
It has become increasinly evident that plasmids 
often play an important role in the pathogenic 
potential of variety of microorganisms. The pathogenic 
potential of Shigella f1exneri is directly correlated 
with the ability to invade and multiply within the 
colonic mucosa (LaBr ec et al., 1964). Genetic analysis 
has established that virulence in £. f1exner i is 
associated with several chromosomal loci (Formal et al 
1971; Gemski et al., 1972). Sansonetti (1982) 
established that all invasive £. f1exner i strains
irrespective of serotype, harbour a large plasmid of 
>140 megadaltons in size.
Also spontaneous variants that have lost this 
140 megadalton plasmid concomitantly became avirulent 
i.e. could neither invade HeLa cells or produce
82
kerato-con junct ivi t is in guinea pigs. Sitnilar genes 
are also known to be haboured by 120 megadalton 
plasmids (Maurelli et al . , 1985). At least seven
polypeptides (designated a through g) have been A
identified as unique products of the virulence S-
plasmids of Shigella spp and ent ero- invas i ve E_. 
coli (Oaks et al., 1986). Four of these polypeptides 
(a, b, c and d) are synethesized fron a 37-kilobase 
fragment of a cloned plasmid DNA that has the capacity 
to restore HeLa cell invasiveness in a Shigella 
recipient which has lost the 140 MDA plasmid (Maurelli
et al., 1988) .
In enterotoxigenic £. coli the following plasmids 
have been isolated and analysed; Enterotoxin plasmids 
(Both St and Lt); colonization antigen plasmids; Vir 
plasmids (which specificd a surface antigen as well as 
a heat labile, acid-sensitive, nondialyzable toxin
lethal f or rabbit, mice and chickens; and Col V plasmid 
(Eiwell and Shipley, 1980). Literature concerning the 
virulence plasmids of Shigella are few. Apart from 
those concerned with its invasive action (mentioned 
above) no plasmid has been implicated in the 
enterotoxin production of Shigella spp (Ellwell and 
Shipley, 1980).
83
ln the case of Salmonel1a a similar paucity of
Information is encountered. Possession of plasmid
has not been established with virulence. Butler et al.
(1979) transfered £. typhi plasmid to £. tvphimnr i um 
and the virulence of the R strain for mice wa:
assessed by intraperitoneal 50% lethal dose am
the number of organis'ms found in the spieen of infected
aniraals. The plasmid containing t.vphimur im strain
did not prove to be move virulent f ce. Elwell and •
Shipley (1980) inferred that the supposition that the 
epidemic R plasmids per se increased the virulence of 
their host bacteria lacks convincing supportive 
evidence. Even in a Situation where the presence of 
DNA regions specifying virulence enhancing properties 
has clear that in certain
circumstance, R (resistance) plasmid can be considered 
to be virulence plasmids. The concepts of virulence, 
that is, the extent or degree of pathogenicity, in 
terms of human disease is extremely difficult to 
quantify due to the raany contributing and interacting 
factors. There is no question, however that the
sudden and unexpected of multiple drug resistance in 
enteric pathogens seriously compromised appropriate
84
dical treatment with the resultant high morbidity 
(Anderson and Smith, 1972).
Immuno.lsmy _o£ Salmonella and h almonel 1 a vaccine: A  
Animal experiments have shown that it is possible 
to obtain a high degree of immunity in rabbits 
guinea pigs against intra-peritoneal inoculation 
(Burrows et al., 1968). Recovery from experimental 
enteric infection in the Chimpanzee is also associated 
with an immunity to subsequent challenge by the oral 
route, eighteen months after the primary infection 
(Gaines et al., 1960). The immunity is associated 
with the development of humoral antibodies such as 
agglutinins, precipitins and the like. Lysin is
also produced, and , like the Vibrio cholerae, the 
typhoid bacilli undergoes visible dissolution and 
disintegrat ion in the peritoneal cavity of the 
immune animal (Burrows et al., 1968> _
The Widal agglutination test has been an 
invaluable tool in serum diagnosis of typhoid fever 
(Levine et al., 1978).
The test is a macroscopic one and is carried out 
with both H and O antigens. The interpretation of a 
single such test must take into consideration
85
« c i 1iiary data such as a previous immunization or 
ittack of typhoid fever and the prevalence of endemic 
typhoid in the general population (Burrows et al. 
1968). It is therefore difficult to set arbitrary 
limits; in most instances an 0 titre of 1:100 and
an H titre of 1:200 raay be regarded as significant.
Typhoid vaccine has been used for many years 
to produce active prophylactic imraunity (Topley and 
Wilson, 1983). The vaccine consists of a saline 
Suspension of typhoid bacilli, usually 1000 million 
per milliltre, which are k i11ed by heat, pheno1, 
formaldehye etc. (Burrows et al., 1968). Triple 
vaccine consisting of para A and B bacilli in addition 
to typhoid bacilli (TAB) to the same total 
concentration bu t u  a ration of 2*1*1 has been 
coramonly used (Topley and Wilson, 1983).
Ques t < * 7 “ of the assay of the immunogenic
potency of typhoid vaccines, and their antigenic 
composition are raised perenially. There are 
discrepances between the results of field trials, 
studies of the experiementa1 diseases, and 
and immunogenic potency tests, and final answers 
are as yet possible. Typhoid fever remains a 
serious public health problem in many regions of
86
the »orld, and the typhoid vaccines which are 
««ailable are not wholly satisfactory.
Geraanier and Furer (1975) isolated a 
jilictose epimerase (galE) mutant of Salmone1Ia
:>chl (Ty 21a) and the result obtained with animal 
•odel indicated that this strain has the potential 
for use as live vaccine. Walulan et al. (1982) 
carried out a field trial to check the safety and 
stabiiity of the mutant strain and tested the
protection against typhoid fever af f onrded by three oral
doses. Earlier studies by Collins and Carter (1972)
have shown that live attenua.Otedv vaccine when 
administered orally, provided mice with better 
protection against subsequent challenge than to
inactivated vacci-n eys .\
The effectiveness of the Ty 21a vaccine was 
assessed by analysis of the incidence of typhoid fever 
in the subjects. The result of the follow-up (1 year
after) indicated that in the dosage schedule tested, 
the Ty 21a mutant strain found previously to be stable 
and safe, was protective against typhoid fever for at 
least one year, without any side effect like fever, 
malaise, headache and localized reactions at the site
87
of inoculation. These side effects have served to put 
the earlier parenteral vaccines out of reckoninj 
(Burrows et al., 1968).
The current commercially available vaccine formu-
lation incorporates 10 Q7 Ty 21a strain in gelatine 
capsule and is given with 0.8g of NaHCO to neutralize
gastric acidity (Scientific activities W3HOV-OMS, 1983).
Immunit.y to Shigella Infection and Shigella vaccine:
The development of an immunity effective to some 
degree is indicated by the relative resistance of the 
resident population of an endemic area of the acute 
disease which affects recent arrival, e.g.
"acclimatization diarrhoea" (Burrows et al., 1968; 
Steffen et al.,1988). This phenomenon is well known 
to residents of temperate clirnates visiting tropical 
and subtropical areas.
Antibodies, agglutinins, are found in response to 
infection with dysentery bacilli usually appearing 
after the sixth day (Topley and Wilson, 1983).
The diagnostic significance agglutinins is somewhat 
uncertain largely because "normal" agglutinins are
88
common. Normal serum commonly agg1utinates Shigella 
cLvsenteriae 1 in 1:20 dilution, but a titre of 1:40 
or higher is suggestive of infection (Burrows et *1., 
1968). The antibodies are apparently unrela l g -  
to effective immunity.
Unlike infections with typhoid and related 
bacilli, bacillary dysentery remains a localized 
infection, and the bacilli rarely penetrates 
beyond the regional lymphatics at the most (Topley 
and Wilson, 1983). AttemptsV to immunize humans orother primates with killed vaccines or even virulent 
organisms, administered by the parental route have 
been met with very little success. Several kinds of 
living vaccines administered by the oral route have
been used. One is made from bacilli which is 
Streptomycin dependent and therefore unable to multiply 
in the absence of the antibiotic (Formal et al., 1965). 
Such vaccines of £. flexner i have been field-tested in 
Yugoslavia and have been found to give significant, 
but type-specific , protection against the naturally 
occuring infection (Mel et al., 1965).
89
Polyvalent vaccines of hybrid strains of 
£» f 1gxngr i lb, 2a, and 3 and £. sonnei 1 given in 
two doses by oral route have been shown to produce 
a highly effective immunity against challenge with1A the 
virulent strains. Formal et al. (1981) transfered 
plasmid reponsible for form 1 antigen synthesis of 
£• sonnei conjugatively to established gal E £. t vohi 
strain. Serological studies revelead that the 
derivative strain produced the form 1 antigen in 
addition to the normal £. typhi somatic antigen.
Testing in mice demonstrated that the derivative 
form 1 gal Ŝ. typhi strain is protective against both 
S . sonnei and J3_. typhi _i'ha 11 enges . It was therefore
suggested that gal E _S_. typhi 21a oral vaccine strain, 
may also serve as a useful carrier for other antigenic 
determinantvVs Ot9  protect against Shigella infections.1
90
fcHAPTER t h r e e
ISOLATION .AND CHARACTERI2ATION QF SHIGELLAE AND 
SALMONELLAE INVOLVED IN DIARRHOEA
1NTRODUCTION 
Salmone11a and Shige11a isolation ai
chharacterization is a dynamic study and progress in
this area continues to be made. Both organisms are 
found in the intestinal tract of man and other warm- 
blooded animals as commensals of limited pathogenic 
Potentials associated with diarrhoeal disease (Topley
and Wilson, 1983).
Over the years a number of procedures have been 
developed to aidä ^quuiicck isolatiion and identification
from faeces. The importance of pre-enrichment of 
suspected Materials as a first step in the isolation 
of these organisms have always been emphasized. A 
number 'of enrichment broths have also been described 
(Harvey and Price, 1979). For Salmone11a enrichment 
the following have been used: Magnesium Chloride, 
malachite green, Strontium Chloride and Strontium 
selenite, Gram negative (GN) broth, Selenite F and 
Sodium tetrathionate broth (Harvey and Price, 1979). 
The choice of enrichment to be used to determined '
.91
by the specialist interest of the bacterio1ogist.
For example Selenite F is known to be essential for the 
enrichment of jLalmonel 1 a t.vphi primarily and other 
Salmonellae in faecel sarnples (Leifson, 1936; Harvey 
and Price, 1964). The same workers also demonstrated 
that Kauffman tetrathionate broth was more efficient 
as an enrichment medium for the isolation of Salmone11a 
from contaminated food and water. *  .
In the case of Shigella pre-enrichment of 
suspected material in Seleite F have been found to 
increase yield by 4.3 and 3.6 percent respectively 
(Sen, 1964; Bhat et al., 1971). Maximum number of 
isolations of both Shige 11a and Salmone1Ia organism 
was also obtained by Rollender et al. (1969) after 
initial pre-enrichment in GN broth.
Various media have been developed for the 
iso 1ation ^nad Identification of Salmonella and Shigella 
organisms from faecel sarnples. These include
xy 1 os e-1 ys ine-desoxycho 1 a t e agar (XLD), eos ine-me t hy 1 ene
blue (EMB) Hektoe'>C .s£n£tbv irrl ^'(1IEA) , desoxycho 1 ate-ci t rat e 
agar (DCA), Salmonellae-Shigellae agar (SSA) MacConkey
92
agar (MA) , Brilliant green agar (BGA),and Bismuth 
sulphate agar (BSA) (Goyal et al.t 1981). However roany 
of the selective and differential plating media are
infact so inhibitory as to prevent the growth of A
the very organism which they are were intende < «2«  -
promote (Rollender et al., 1969). SSA and BSA belong 
to this category (Bhat et al., 1971).
Since one medium is not sufficient for the 
isolation of enteric pathogens from faeces, Goyal et 
al.(1981) recommended a combination of MA and XLD or 
MA + HEA based on comparative studies using eight 
different media. Howev^^both HEA and XLD are known 
to be very expensive and are rare to comeby in Nigeria. 
Equally effective for the isolation of Shigella spp is 
the MacConkey-Tel1urite medium (1 ug/ml potassium 
tellurite in MacConkey agar) which has been used in 
the isolcatAio n of both Vibrio cholerae and Shigellaorganisms (Sen, 1964)„.
Generally, the differentiation and 
characterizat ion of the enteric bacilli is based upon 
antigenic studies. A useful primary differentiation 
is made on the basis of the lactose fermentation which
93
is roughly correlated with pathogenicity. The coli- 
form bacteria ferments this sugar rapidly with the 
formation of acid and gas in twenty four hours while 
Shigellae and Salmonellae (essentially pathogens) do
not ferment it. Similarly the dysentery baci5l2li. or
Shigellae divide' into two groups on the basis of
fermentations of mannitol and are anaerogenic 
(Ewing, 1958). The Salmonella group in general .
produced gaseous fermentations; typhoid bacillus is
typically anaerogenic.
A great variety of cultural and biochemical 
reactions including the conventional sugar
fermentations, motility, formation of Indole from 
tryptophane and various types of specialized tests such 
as the utilization of tartaric acids and malonate, 
araino acid decarboxy1ase activity etc. have been 
useful in the physiological characterization of the 
Sa lmoiit 1 1 a and Shigel 1 a groups (Edwards and Ewing,
1972).
Partial or complete Identification of Shigella 
and Sa 1mone11a is usually carried out by the use of 
antisera containing appropriate antibodies.
94
The vast number of different Salmonella serotypes 
(over 2,000 are known) and the numerous antisera 
required for the resognition of all these (a total of 
58 different "O" antigents and 72 "H"antigens) make 
Salmonella typing a "specia1ization" requiring a lot of 
technical skills and know-how.
A number of Salmonel1a species can further be 
subdivided into types on the basis of their 
susceptibi 1ity to distinct bacterophages (Garg and 
Singh, 1974). Since the types exhibit a high degree
of stability under natural cond itions, phage typing
is of immense value in epidemio1ogical investigations 
in tracing source of typhoid or paratyphoid fever.
In the case of Shigel1ae^phage typing is not very 
populär and serotyping is relatively easy. Based on 
somatic antigens four species of dysentery bacillus are 
r ecognized viz: £. d.vsenter i ae . £• f 1 exner i. S. bpydj \ 
and S .sonnei. These are further divided into various 
serotypes.
In this study the detailed biochemical characteri- 
zation of the Salmone11 ft and Shigella isolates was 
carried out in order to differentiate the isolates 
from other enteric bacteria.
95
Serological characterization was also carried out in 
order to "pigeon-hole" these isolates into respective
96
MATERIALS AND METHODS
Collection of samples and isolation of organisms:
A total of 10,000 faecal samples sent to the 
laboratory from patients with cases of provisiona11y 
diagnosed diarrhoea in three different hospitals in 
Ibadan, namely: The University College Hospital 
(6,000), State Hospitals at Adeoyo (3,000) and 
Ring Road (1,000) were screened for the presence of
Salmonella. and Singe. 1 la. 200 imp>llee:s were also
taken from diarrhoec piglets at the Teaching and 
Research Farm University of Ibadan. The later were 
collected by using sterile cotton wool swabs. 
Cultivation techai_gjAe_&
A loopful of each stool sample (preferably from 
blood and muAcoiyd area if any) was inoculated on platesof MacConkey and Deoxycholate citrate agar. Also bijou 
bottles containing 4ml Selenite F broth were similarly 
inoculated to enrich the isolation of any Shige11a and 
Salmonella if present. All cultures were incubated at 
37 C for 24 hours.
The Selenite F was later subcultured into fresh 
DCA and one MacConkey agar plates containing 1 ug/ml
97
potassium tellurite (MTA) and incubated at 37 C for 
another 24 hours. The plates were later read and 
non-lactose fermenting colonies were sei ect ed.
Purification of Isolates;
Cultures sharing predorainantly non-l, ac^tose  
fermentation were subcultured on fre sh DCA to ensure
purity. Pure cultures were streaked onto nutrient 
agar slopes and kept on the shelve and later studied
in detai1.
Biochemical characterizatio Isolates;
The biochemical characterization of the isolates 
was according to Edward and Ewing (1972) and Cowan and 
Steel (1979) as follows:
(i) Fermentation of Carbohydrate: The isolates were 
tested in batches to ensure better
reproducibi 1ity. They were innoculated into fresh 
agar plates. A descrete pure colony of each 
' S * o late was inoculated into carbohydrate broth 
containing a Durham tube. The medium consists 
of nutrient broth plus 0.5^ of the particular 
carbohydrate e.g. glucose, plus Andraiwindicator. 
Since the end products of carbohydrate
98
fermentation are acid or acid and gas the 
indicator revealed production of acid and the 
inverted vial trapped the gases where produced. 
The carbohydrates employed for this test were!
glucose, lactose, mannitol, xylose . s
dulcitol, salicin, rhamnose, sorbi arabinose
raffinose, adonitol, maitose and inositol.
Test for H.vdrogen sulphide: With the straight 
wire loop one of the selected colonies was 
carefully picked and inoculated into a tube each 
of Kliger Iron Agar ^(KIA) by stabbling the but and 
streaking the slope. A test tube of peptone water 
as well as a plnte of MacConkey agar plate were 
innoculatedi for purify. All the cultures were 
incubated at 37 C for 24 hours.
Hydrogen sulphide was produced by organisms 
capable of reducing Su1phur-bearing compounds such 
as Sodium thiosulphate present in the medium. The 
hydrogen sulphide reacted with iron salts in the 
medium to form a black precipitate of ferric 
sulphide. The presence of black colour along 
the inoculation channcl only was taken as a 
pos i t i ve resu 11.
99
(i i i) Test_Lax_Indole Production: A twenty four hour
broth culture of each of the isolate was used. 
About 1 ml of Kovacs reagent was delivered into 
the broth culture. The production of Indole from 
the metabolism of tryptophan by the bacterial
enzyme typtophanase was detected by th S O J  
development of a pink to red colour. Ä > N  
(iv) C.vtochrome oxidase test; A piece of Whatman No. 2 
filter paper in a Petridish was impregnated with a 
few drops of freshly prepared oxidase reagent 
(1.0% tetramethyl parapheny1ene hydroch1oride).
A loopful of the growth of the isolate on a agar 
plate was smeared onto a small area of the 
reagent-impregnated paper. A positive reaction 
was denoted by a dark purple colour within 30
seconds. Positive and negative Controls were 
set up usiinng Pseudomonas aeruginosa and
Escheri chi a coli which gave positive and negative
eactions respectively for this test.
< 5
(v) Urea test: A slope of Christensen’s. urea agar was 
innoculated heavily with the isolate. It was 
incubated overnight and examined. The presence of 
enzyme urease, which hydrolysed urea to ammonia,
100
was detected by a change of colour of phenol 
red indicator in the medium from straw or yellow 
(acid) to pink or purple (alkaline). Pale pink 
colour was disregarded according to the 
manufactuer's instruct ion.
(■ vi)v  Citrate utilization test: Koser's citroat~e< broth
in a bijou bottle was innoculated with the isolate 
using a straight wire and later incubated and 30
C. The test.detected those organisms which were 
capable of utilising citratein the form of its 
sodium salt as the sole C source. Organism 
capable of utilising citrate produced alkaline 
metabolites which changed the pH of the medium, 
resulting in a change in colour of the bromothymol 
blue indicator from green (neutral) to deep-blue 
(a 1 ka1i ne).
(vii) Mot i1i tv: Motility was performed by the
conventional hanging-drop technique. Each isolate
< 5was grown in peptone water and incubated at 37 C 
overnight. One drop of the broth culture was 
placed in the centre of a cover-slip. A ring was 
raade in the centre of a microscopic slide with
plasticine.
101
The slide was inverted over the coverslip so that
the ring encircled the culture. The slide was 
quickly turned so that the coverslip was then on 
top. The organism was examined for mobility 
using the X10 and X40 objectives.
Decarboxylase reaction; Other biochemical tests 
included lysine decarboxylase, lysine deaminase, 
ornithine decarbox1ase; Arginine dihydrolase.
For the decarboxylase reaction, tubes of the four 
media (arginine, lysine, ornithine and control) 
were inoculated through the paraffin layer with 
the aid of a straight wire. They were incubated 
and examined daily for four days. Formation of a 
violet colouration gave a positive decarboxy1ation 
reacti on.
red (MR) reaction: Glucose phosphate (MR) 
medium was inoculated with each isolate and 
incubated at 37 C for 2 days. 2 drops of Methyl 
red solution was added, shaken and examined. 
Formation of a red/orange colouration proved 
a positive reaction.
102
Voges Proskauer reaction: After reading the MR 
reaction the same culture was employed for the 
VP test. 0.6 ml 5% <x-naplthol solution and A  
0.2ml 40& KOH was added. The tube was shakcn and
examined and 1 hr later, a strong red coloi was
observed in positive reaction.
KCN test: Bijou bottle containing KCN broth was
inoculated with a loopful of an overnight broth 
culture of the isolate. The bottle cap was tightly 
screwed down and incubated for up to 48 h. at 37 C 
Turbidity (indicating growth) was observed in 
positive cu 1 tur eso
Serological Identification of Isolates;
Slide agglutination test was performed on 
each of the isolates as follows: A loopful of 
saline solution was placed on a clean glass slide. 
To this was added a small amount of the pure 
culture of the isolate to be identified and the 
later was thoroughly emulsified on the saline 
solution until it was faintly turbid. A loopful 
of the commercially prepared agglutinating sera 
(supplied by Wellcome Research Laboratories, 
England) was placed beside the bacterial emulsion
103
on the same slide. The two were later mixed using 
the inoculating loop. The slide was rocked 
gently back and forth to afford proper mixing of
an t i serum and bacteria. The mixture was obser ved
against a white background in the presence of a 
good light source. Agglutination or clumping of 
cells indicated a positive reaction.
ln case of Salmonella Identification, 
Salmonella polyvalent "O" (A-G) serum and 
polyvalent A (Composit e "phase 1 and phase 2") 
was employed. , s y
The Shigellae were classified into Shigella 
dvsenteriae (Group A), Shigella flgxneri (Group B) 
and Shigella bo.vdi i (Group C) by using 
commercially prepared agg1utinating sera obtained 
from Wellcome Research Laboratories, England.
icSS5'
104
RESULT
Of 10,200 stool specimens cultured, 31 isolates of 
Shigella and 22 isolates Salmonella were made. A  
gftlaiLLal Morpholog.v and Staining; Shigella on DCA were 
round, pale, 2-3 millimeter diameter, slightly convex
colonies with entire edge. When viewed against a dark
background, they were whitish an«d slightly opaque. 
Salmonel1a appear like Shigella on D.C.A. .
The difference lies in the producttion of hydrogen
sulphide in the medium. Both bacilli were gram
negative rods closely resembling and indistinguishable 
frora the coliform bacteria. No particular arrangement 
of the cells was apparent on microscopic examination. 
No capsule was apparent and spores were not formed. 
Table 3.1 (Appendix 4) shows the biochemical features 
of the organisms encountered in the study.
Carbohvdrati? fermen tat ion ; Organ isms later identified 
as Shigella were found to produce acid but no gas 
in glucose, mannitol, arabinose and trehalose. Other 
carbohydrates including lactose were not attacked.
The other non-lactose fermenters showing acid and 
gas production in glucose, mannitol, dulcitol,
105
sorbitol, arabinose, xylose, Rhamnose and Maltose 
were later identified as Salmonel 1 a t.vphi . In 
general the Salmonel1a were found to utilise more 
carbohydrate than Shigella.
Hydrogen sulphide Production: All the Salmonella 
species were found to give a positive reaction in the 
Kliger's medium as indicated by the regulär formation 
of black precipitate of ferric sulphide along the 
inoculation channel. The Shigel lLa i s<olates did 
not produce hydrogen sulphide,ÜST
Indole Production: The Salmonella isolates were found 
to be indole negative as well as the majority of the 
Shigel1a which were positive. In other words the 
ShiKej ja organisms gave variable results to the 
indo1e t es t.
Oxidase Production: All the isolates reacted 
negatively to the oxidase test. This shows that 
neither SalmonelIa nor Shigella produce cytochrome 
oxidase enzyme and hence failure to give a purple 
colouration when the oxidase reagent was applied to 
a smear of the culture isolates on filter paper. 
Urease Production: None of the isolate was found to 
be urea positive as there was no familiär cherry-red 
colour developing in any of the urea slants employed
in the test.
106
Citrate ut i1i zat ion: Most of the Salmonella isolates 
(except those later identified as Salmone 1 1 a t.vph i ) 
were able to utilize citrate as a sole source of carbon
whereas all the Shigella spp were citrate negatil vg.
Mot i 1 i t.v: All the Salmonella tested in this study were 
found to be motile whereas all the Shigel 1a isolates 
were non-motile.
Decarbox.vl ase Production; Organisms later identified to . 
be Shigella failed to produce decarboxy1ase enzymes, 
hence argini. ne, lys, ine and or ni thine were unaffected.All Salmonella isolates weres pPos itive .for Arginine, 
lysine while some gave variable result when tested on 
ornithine.
ECM-U.t_i.Li7-atIon: AllXs trains failed to grow in the 
potassium cyanide broth after incubation for 48 hours.
MR and VP tests: The Salmonel1a isolates were positive 
for the methyl red and negative for Voges Proskauer 
reactions so also were the Shigella organisms.
Of the thirty-one Shigella isolates, twenty-four 
were serotyped as Shiee 1 1 a f 1 exneri : four Shigel 1. ft 
d vs ent er i ae and three Shigel 1 a bo.vd 1 1 . The Singel la 
f1exner i were mainly type 6, 2, 3, 1 and 4 while 
£. d.vsenter i ae were of types 2 and 3.
107
Nine isolates of Salmonella were identified 
as Salmonella typhi. The remaining isolates were
108
DISCUSS10N
In this work MacConkey agar (MA), Desoxycholate- 
citrate agar (DCA) and MacConkey-Te11urite agar (MTA) 
were used for the isolation of Shige11a and Salmone11a
present in faecal samples taken from
The choice of these selective media was made on common 
sense ground. Xy1 ose-1ysine desoxycho1ate agar (XLD) 
and Hektoen enteric agar (HEA), both superior to the
above three are known to be very expensive and very 
rare to come by in this countr
DCA has been shown to . -ior to. MA (Bhat et
al., 1971) and is readily available. According to 
Rollender et al. (1969) the sodium-deoxycholate 
content allows for the inhibition of coliforms 
without decreasingj, the ability of the medium to 
support the growth of Sa 1monei1a and Shigella 
organ i sms MacConkey agar contains bile salt
whicl ' also inhibitory to gram positive organisms
and in combination with potassium-te11urite its 
selectivity is enhanced for the isolation of Shigella.
Typical non-lactose fermenters appears as 
colourness on both DCA and MacConkey. Shigella 
organisms appear as grey-centered colourless
109
colonies on Te 1 1 ur i t e-MacConkoy agar. ilowever, 
Identification of Salmone11a and Sh i g o11a based on 
tnorpho 1 og i ca 1 appearance, is subjective. While 
both pathogens ordinally produce colonies of typical 
aspects on selective medium, it may be altered by 
growth in close association with other organisms. Also, 
sometimes these pathogens produce colonies of a typical 
appearance for reasons that are not entirely clear.
Thus the differentiation of enteric pathogens based on 
their inability to ferment lactose may not be 
infallable because once a while lactose fermenting 
colonies of Salmonella do como up. Although not 
encountered in this study, the Shigella sonne! have 
always been recognized as late lactose fermenters.
This investigation established that Shigellae are 
the more frequent causative agents of diarrhoea in 
people whose stools were sampled. Sotne of the 
Shigellea were isolated on freshly prepared 
Desoxycho1ate citrate agar (DCA) after preliminary 
enrichment in selenite F broth. Previous work by 
Adetosoye and Rolitu (1986) found the isolation rate 
of Shigella in stool samples to be higher (42.1 
perscent) when compared with that of £. coli (33.7 
percent) and Salmonellae (24.6 percent) respective1y .
110
ln tlii s study KUIH percent of the isolates were 
Shigellae while Salmonellae acounted for 40 percent.
Of the two hundred swab samples of diarrhoeic piglets 
processed only one grew Shigella while there was no 
Salmonella encountered. Shigella was previously 
thought to be a pathogen of man and primates only, but 
has since been isolated from non-human source (Formal 
et al., 1958). Nyaga et al. (1985) reported the 
isolation of £. d.vsenter i ae from domestic fowl in 
Kenya. Ibu et al. ( 1987) also isolated £. f1exner i
from diarrhoeic piglets in Jos. It can be assumed 
that these organism were acquired from contarainated
f eeds.
Among the Shige l1 l1 a isolates identified £. f1exner i 
was the most p. reval ent as twenty-four of tlhe thirty-one
isolates were so identified. This is in ia greement
with the work of Ogunbi (1971), Rahaman (1!984), and 
Adetosoye and Rotilu (1986). In the experience of 
these workers £. f1exner i ranked higest in the 
freguency of isolation among other Shigellae from 
clinical specimens. Mutanda et al. (1979) also 
reported that in East African countries, namely:
111
Kenya, Tanzania and Uganda, £. flexneri was the 
most frequently isolated species, followed by 
£. sonnei- £. bo.Vi 1 came third 5.» (f^enteriae 
was least. Usually one particular species, dominates 
in a Community or country . For example in the 
developed countries such as Britain, U.S.A and J apan,
£. sonnei accounted for most of the cases of 
shigellosis (Roseberg et al., 1973). In this study 
late lactose fermenting £. sonnei colonies were not 
encountered throughout the investigation. Earlier 
report by Adetoyose and Rotilu (1986) also did not 
indicate the presence of S.. sonnei in any of the stool 
samples screened. The failure to isolate &. sonnei may 
be due to the fact that the organism is prevalent only 
in the temperate countries.
Overall there seems to be low Shigella and
Salmonel1JaL ifni susspected diarrhoea stools screened in 
this worJc, Odugbemi et. al . , (1982) reported 3.3 
percent Shigella and 0.9 percent Salmonel1a when six 
hundred and seventy four stool samples were examined in 
Lagos. Earlier, Ogunbiyi (1971) obtained 8.5 percent 
prevalence in the number of Shigella isolates. This
112
consistently low percentage of isolation may be 
attributed to the fact that there are numerous 
other etiological agents that cause diarrhoea apart 
from these two organisms. For example it is known 
that rotavirus is an important etiology of diarrhoea in 
children (Kuraar et al.t 1984) and well over 80 percent 
diarrhoea in infants have been attributed to Rotaviral 
infection. Escherichia coli has also been implicated 
in cases of serious diarrhoea afflicting man and 
animals (Adetosoye, 1980a). Another reason is that 
only limited number of piglets were sampled. In the
cou rse of this investigation many lactose-fermenting £. 
coli showed up repeatedly on the isolating plates but 
since they were not the primary aim of this research 
they were usually ignored and such plates discarded. 
Undoubted some of these E. coli might have been the 
agent of diarrhoea.
Twenty two Salmonellea were found in all the stool 
samples screened. This is not surprising because 
infection with Salraonella organisms is known to be 
widespread, and apart from clinical Salmonellosis,
113
both man and animals are known to be carriers to 
Salmonellae. Many publishcd work on Salraonel1ae 
in Nigeria between 1956 and 1962 emanated from 
some medical expatriates working in the University 
College Ibadan, notably P. Collard; R, Sen; S. Lapage;
D. Montefiore; R. Johnson and W. Plowright. Some of 
the Salmonel1a strains isolated were from patients 
suffering from various ailments other than typhoid.
Of the two hundred and eighty two pathogens isolated 
from Veterinary Clinic of University of Ibadan 
between 1971 and 1975, Salmonel1a accounted for 17.3 
percent (49 isolates) of the total number (Falade, Ojo 
and Ogunnariwo, 1977). In this work forty percent of 
the total number of isolates were found to comprise of 
S. t.vphi arid othe;r SSalmonel 1 a species.
Accoirr„ddiingpredominantr . s. ..to Collard and Sen (1957) the most pe cies of Salmonella in Ibadan were S .
X a
t.vvpphhi and £. &a£gLaMm!a. In this present work £. typhi 
accounnited for forty one percent of the total number of
Salmone1a isolates. It thus confirms that cases of 
typhoid fever is still very prevalent in Ibadan. For 
example in India, Saxena et al. (1983) found £. 
tvphimurium and £. t.vphi to be the most prevalent of 
all the Salmonella serotypes encountered.
114
The number of human lives claimed by Salmone11a 
infection is staggering:, Veterinary Sa lmone 1 1 os i s is 
also a big threat to the animal industry. For example 
in Britain 14,351 human and 11,682 Veterinary 
infections were recorded between 1968 to 1980 (Edel et 
al., 1981), The age group at the highes^risk were 
infants less than one year old and adults in the senior 
age bracket. Most of the human infections were known 
to occurs in the hospital on person-to-person spread.
In animals, the usual souf-ce of infection is through 
contaminated feeds. The habit of promoting growth by
feeding animals with an1t1 i biotics has 1ed to the
selection for resistant strains.
Persistant,  ™report s of isolation of. S.alm.one.llae and
Shigellae in our environment calls for urgent attention
by health authorities in this country. Although it may
never be possible to eliminate Salmonella and Shigella
oragnisms completely from our environment and farming
set up, concerted efforts should be made to rainimise
the spread of these two deadly organisms by way of
Provision of good water supply. The current campaign
on hygenic living should be encourages since
"cleaniness is next to holyness. In the Veterinary
practices a lot of Controls need be introduced to curb 
the dissemination of these pathogens.
115
The low level of Shige 1la isolates encountered in 
piglets may be due to the fact that<j)Shigel 1 a is not 
normally a pathogen of animals.
(ii) The small sample size
(iii) The hygeine Status of the environment in- £Xwh,i i  ch.
the piglets were raised. , j
s
\  <f
j y
116
CHARTER 1-OUR
i-M££CI.lQUS DRUG RESISTANCE IN SH1GELLAE AND SALMONELLAE 
-LSQLATED FRQM DIARRHOE IC CASES
INTRODUCTION
Shigellosis and Sa 1mone11osis are endermaii c in- i
various parts of the world includil ing Nigeria, ̂ SThe
causative agents of these diseases are and
Salome 1la spp. respectively.
In the past, the Sulphonamide drugs were • the drug f
of choice for bacillary dysentery (Shige11osis) 
(Cheever, 1952). Other drugs which have been found 
useful include Tetracyclines, Ampicillin, Trimethoprim­
sul phamethoxazo 1 e and Nalidixic acid (Panhotra £.1 
&!•. 1985). O  '
In the case of SalmoneI1a infections, chloramphe-
nicol has been the most successful drug. The 
recommended dose in adult is 500mg every four hours 
(Mandat, 1979). Other drugs found useful in the 
treatment of Salmone11osis include Ampicillin 
(Roberston &]_. , 1986); Co-1 r imoxazo 1 e (Akinkugbe 
et al . . 1986) Amoxycillin (Pillay, £_L ad.., 1975) and 
Furazolidone (Hezog, 1976).
117
resistant Salmonel1a typhi in Nigeria by Njoku-Obi 
and Njoku-Obi (1965), other workers have reported a 
wide-spread incidence of antibiotic resistance in 
enteric pathogens in this environment (Ojo, 1973; 
Adetosoye, 1980; Ibu et al., 1987; Olukoya et a 
1988b). Such resistant strains carry genes encoding 
Products that inactivate those antibacterial agents; 
keep them from reaching their target sites or provide 
alternaives for those sites that have been blocked. 
Some of these antibacteria1 resistance genes are on 
the bacterial chromosome, but the majority are on 
extrachromosomal genetic elements called plasmids that 
may transfer themselves 0  other strains or species of 
bacteria (Jacoby and Swartz, 1980). •
The original[ dU i1scovery of transmissible drug
resistance was made by Japanese workers in 1959 
(Ochiai, et/ad., 1959). The t r ansrai ss i b 1 e agents 
responsible for drug resistance are known as 
resistance factors or R factors (Mitsuhashi, 1960). 
Although the commonest transmissib1e resistance pattem 
was against Sin, TC, CM, and Su, combinations of 
resistance to four, three or two of these drugs and 
single drug resistance were known to be transferable
(Datta, 1965).
118
Evidence that R factors are widespread comes 
from raany sources and they have been found wherever 
they were looked for (Rangnekar et al., 1983; 
Adetosoye and Rotilu, 1986; Olukoya et al., 1988b)A. 
In this work, the drug susceptibi1ity patterns
of Shigellae and Salmonellae isolated from diarrhoeic 
cases in Ibadan were investigated. The isolate . were 
also screened for the presence of transraissible 
resistance plasmids. The later were identified and 
characterized in Chapter 5.
ö&
I #
/
119
MATERIALS AND METHQDS 
Bacterial isolates:
Fifty two bacterial isolates from diarrhoeal 
cases were employed in this study. They comprised of 
23 £. Il.e.xneri, 4 £. dysenter iae. 3 £. boydi i . Otheerr:s 
are 9 £.. tvohi and 13 Salmonella spp. The isola*t -e-s
were maintained on nutrient agar slants prOiort t to use.
Ant imi crobi al susceot i bi 1 i t.v testing: \  V '
1. Disc diffusion method: The modified method of 
Bauer et al. (1966) was employed.
Five colonies of each pure bacterial culture 
were inoculated into 5ral sterile nutrient broth 
(Oxoid) in a bijou bottle. The broth cultures 
were then incubated overnight at 37 C and diluted 
five times to give an approximate bacterial 
concentrat ion of lO^cellsper ml . (de t ermi ned by 
Miles and Mistra method).
SThe diluted broth culture was asceptically 
poured and spread on the whole surface of freshly 
prepared sensitivity test agar (Difeco). Surplus
Suspension was decanted from the surface of the 
agar plates which were allowed to dry at room 
temperature for 3 to 5 minutes. Later multo
1 2 0
discs Oxoid Code No. 3857E containing appropriate 
concentrations of the antibiotic substances 
(Appendix 1) were placed on the agar with sterile 
forceps. The disc were spaced far apart to prevent 
overlapping rings of inhibition and gently pressed 
down to ensure contact with the surfac e to the 
agar. For a series of tests, £. SLQ.U NCTC 10418 
was used as sensitive control strain. Plates were 
incubated at 37 C for 24 hours» . ,
After overnight incubation, the zones of
diameters (including thl«e  6nun <discs) were measured
with a ruler. A reading of 6mm indicated no zone 
of inhibition. T̂ ie end-point was taken as 
complete inhibition of growth as determined by the 
naked eye. The diameters of zone of inhibition 
were compared with those of control strains.
(See Appendix 5).
Minimal inhibitor.v concentratiQn_(Mi-Ci 
Determination;
The MIC of each of the antibiotic was deter­
mined for each of the isolates in order to obviate 
the shortcomings of the traditional disc diffusion 
test which is prone to Problems (Bailey £1 aj_. *
1981
121
Stock Solutions of Chioramphenico1, ampicillin, 
Streptomycin and tetracycline were respectively made in 
sterile distilled water. The concentration of the
stock solutios was 2000ug/ral. Serial dilutions of eia ch
antibiotic wa*s  carried out in 10 tubes, the first o<f/ ­
which contained 0.128 ml of the Stock solution of the
antibiotic and 9.872 ml of nutrient broth to give a
concentration of 128 ug/ml of the antibiotic. After
thorough mixing 4.936 ml content of the first tube was
asceptically transfered into a second tube and mixed
with an equal volume of nutrien 9 ?>r oth to give a
concentration of 64 ug/ml. This procedure was repeated 
for the next eight tubes. o  oncentrations of the 
antibiotics in the tubes were 128, 64, 32, 16, 8, 4, 2, 
1, 0.5 and 0.25 respectively. One millitre of 8 hours 
old nutrient broth cultue of each isolate was diluted 
with 4ml of nutrient broth. 1 ml of this diluted 
broth culture was innoculated into the tubes containing 
the serially diluted antibiotics above and incubated at 
37 C for 24 hrs. E. co1i 10418 was used as a control. 
The Minimal Inhibitory Concentration of the antibiotic 
under test is the lowest concentration of the
antibiotic which inhibited the organism.
122
ConjggatiQii--(-interrupted mating) experiments:
Plasmid transfer experiment was performed using 
the Standard procedure. £. oo1i K 12 5 25 resistant
‘X
to 200ug/ml nalidixic acid (received frora Dr. A 
Adetosoye) was used as recipient. The donors were the 
Shigel1a and Salmone11a isolates which have been found 
by the disc diffusion method (above) to be resitant to 
ampicillin, tetracyc1ine, Streptomycin and 
chloramphenicol either signly or in combinations.
The donors and the recipients were grown 
separately in Brain Heart Infusion broth for four 
hours. Aliquot of 0.5ml of eah donor cell was 
mixed with 1ml of recipient cells in sterile Bijou 
bottles. To this was added 3.5mls of freshly 
prepared Brain Heart Infusion broth and incubated 
overnight for 18 hour at 37 C without shaking
(Adetosoye and Rotilu, 1988).
Selectisn methp.dJ-
The selection was carried out using freshly 
prepared MacConkey agar plates to which has been 
incorporated various antibiotics formulations 
(see Table 4.1 below).
123
SELECTIVE MEDIA QF FIVE FQRMULATIONS
Anti- AMP TE C S Na < ? -
biot i c
Med ium 
1 25 'V1'00 
2 25 00 
3 100  
4 25 100
5 & 100
Key: Amp: Ampicillin, ö
Te: Tetracycline,
C: Chloramphenicol
S: Streptomycin,
Na: Nalidixic acid
X 1
Selection nlates: On this medium only £. cg.il 
transconjugants that has acquired resistance to the 
drug(s) incorporated into the medium grew.
124
Test for TransconJugants;
After inoculating the selection plates with the 
mixed cultivation broth, the plates were incubated for 
24 hours at 37 C. Colonies were picked from the 
selection medium and tested for antimicrobial 
susceptibi 1ity by disc diffusion method to establish 
the transfer of donor resistance determinant.
125
RESULTS
Zones of Inhibition produced in a 1awn of 
organism piated from diluted overnight broth culture 
were measured (Table 4.2a) (Appendix 5). The
resistance patterns of the isolates to the
used is shown in Table 4.2b
The resistance of the Shigella isolates to each 
of the four major antibiotics werle of the #ollowing
order: Twenty eight of the isolate%«were resistant to
Streptomycin, twen twenty one
to Ampicillin and
The Salmonelli on in
number of resistani d u  a m ,  vn a. «g^• IS strains
were resistant to Streptomycin, 8 to tetra'cycl ine, 8 
to ampicillin and 8 to chloramphenico1. None of the 52 
isolates was susceptible to all the antibiotics tested.
Overall, twenty one drug resistance patterns were seen 
in the 52 isolates. However, 10 patterns accounted for 
78$ of the total (Table 4.2b). The isolates were 
resistant to minimum of one and maximum of seven 
antibiotics. The most common pattern was 
T-CT-F-A-S-C-Te (20$), followed by T-CT-F-A-S-Te (10$) 
while four other resistant patterns including CT-S
scored 8$ each
126
The seven drug- resistanfcLpattern was more common among 
&lLLgS-l-l.a iso 1 ates (approx. 33%). Also pattern 
T-CT-S-Te-C and T-CT-A-S-Te-C both accounting for 16% 
were not encountered in the Salmonella isolates s -  
On the other hand patterns CT-F-S
and CT-S which occured prominently among Salmone1Ia 
isolates were not encountered within the Shitrel 1 a 
i so 1a t es.
Table 4.3a, b and c represent the comparative 
results of in vitro susceptibi 1ity tests on four 
commonly used antibiotics against the fifty-two 
isolates of Shige 11a and Salmonella. Ampicillin was 
found to be most active against Salmonella spp. The
antibiotics inhibH ;ed all the isolates but one
at a concentration nn   tof 64ug/ml. At 8ug/ml more than 
40% of the isolates were inhibited (Table 4.3c). 
Streptmycin and tetracycline showed identical
activites. At 32ug/ml almost all the Salmonella 
isolates were inhibited. The activity of 
ch1oramphenico1 was slighly inferior to the three 
others. At concentration of 64ug/ml 3 of the isolates 
were found to be resistant. At concentrations greater 
than 128ug/ml one of the Salmonella isolates was not 
susceptible (Table 4.3a).
127
Overall, none of the antibiotics was found to be 
inhibitory at concentration lower than lug/ml.
The picture encountered in the Shigella isolates 
was significanly different. There was a consistently 
high resistancivalue throughout (Fig. 4.3a and b). T he
level of ampicillin resistance for Shigella was very 
high. Twenty-six of the isolates (87 percent) had MIC 
of >32ug/ral. At 128ug/ml twenty-seven percent of the 
isolates were uninhibited. The resistance of the 
Shigel1a isolates to the other antibiotics was found 
to be high. Twenty-One of the 31 Shigella isolates
were not susceptible to 32„  uNg/yml of tetracycline. 
in vitro activity of Streptomycin and chloramphenicol 
were similar as there were resistant Shigella organisras 
encountered at all the concentrationsemployed. For 
example, none of the isolate was inhibited at 4ug/ml of 
ei ther ant i biot ic.
Table 4.4a shows the resistance patterns of some 
of the isolates to four commonly used antibiotics and 
the deterininants transfered on mating each donor with 
JJ. co1i K12 525, a lactose fermeter resistant to 200ug
t
of Nalidixic acid used as the recipient.
128
All the forty-two isolates screened transfered 
ampicillin resistance (100 percent). Twenty-one 
isolates (50.0 percent) transfered two determinants 
either A-Te or A-S or A-C. Seven (16.6 percent) 
transfered three determinants either A-Te-S oi 
None of the isolates transfered four determinants. 
Among the Shige 11a species the chloramphenicol 
determinant was transfered at a low frequency as only 
three (10 percent) of the isolates were able to 
transfer the determinant as against six (42.8 percent) 
recorded in the Salmonel1a isolates. In all the cases 
where the resistance determinants were successfully 
transfered, the resultant transconjugants showed 
resistance to all the antibiotics whose determinants 
were transfered,„  S
129
DISCUSSION
Antimicrobial susceptibi Iity testing provides 
Information by which therapy can be selected or
modified by adjustment of the dosage of the \
antimicrobial agent already being administerd.
By the broth dilution method it was detsecytTed in
this investigation that tLh e minimal inhisjbiQtoyry 
concentration (MIC) of Ampicillin to the Salmone11a 
species were generally fairly high For example most 
of thera were inhibitqd at concentrations between 4 to 
128ug/ml (Table 4.3a and 4.3c). In an earlier work, 
Rotilu (1985) (Personal communication) found that all 
the Salmonella isolate ks investigated were inhibited 
by ampicillin at a cconcentration of less than 30ug/ml.
Generally, the level of resistance attained by a strain 
carrying R factor depends on the host cell and the 
resistance determinants (Anderson, 1968; Chopra et al., 
1978). Thus there is Variation from one bacterium to 
another. The higher MIC figures observed in this 
investigation when compared to Rotilu work may be due 
to the following reasons:
(i) increase in seiective ' antibiotic pressure due to
indiscriminate use
130
(ii) incorapl ete dosage k
V
(iii) fake drugs that abound and
(iv) increasing seif medication among the populace.
The Shigellae investigated in this work were 
generally more resistant than the Salmtoone l1 l1 ae (Table
4.3a and 4.3b) as significant number of theerma were not 
inhibited at antimicrobial concentration of 128ug^ml. 
Previous work in Ibadan by Rotilu (1985) (unpublished) 
showed that the Shigellae were highly resistant to many 
antibiotics, including ampicillin, Streptomycin and
/ v f
chloramphenico1. Chun et al. (1984) described a 
Streptomycin R factor confering resistance of more than 
l,000ug/ml of the drug on a Shigella strain and to only 
lOug/ml on a strain of E_. coli. These workers pointed 
out that the same R factor usually produce different 
levels of resistance in different isolates.
It may be pointed out in passing however that the 
results of in vitro antimierobia1 tests is usually at 
variance with the response of humans to antimicrobial 
therapy. Ilost factors remain the major determinants of 
the outcome of antimierobia1 therapy (Weinstein and 
Dal ton, 1968). .
131
Such factors like adherence of bacteria to epithelial 
surfaces, susceptibi 1ity of bacteria to phagocytosis, 
Chemotaxis of neutrophills and bactericidal activity of 
human serum play a significant role on the outcome of 
antibiotic therapy (Washington, 1979).
The fifty-three isolates (31 Shigella and 22 
Salmonella) studied share twenty-one resistance 
patterns to eight antibiotics employed. Previous 
report by Adetosoye and Rotilu (1986) established 
nine patterns of resistance to six drugs by Salmonel1a.
Shigella and £. co1i isolates Ethiopia,
1
Gebre-Yohannes and Habte-Gabr (1984) found nineteen
drug resistance patterns in three hundred and sixty
Shigella isolates. The multiple drug resistance
Problem occurs worldwide and is asq^result of 
indiscriminate use of antibiotics.
The subtherapeutic use of the antibiotics 
created indiscriminate selective pressure so that 
pathogens become resistant to a particular antibiotic 
or a related one and could transfer such resistance 
to other bacteria. In the opinion of many workers 
(Simmons and Stolley, 1974; O'Brien et al., 1987) 
many of such drugs need no longer be used.
132
Among the Shigella spp., &. f1exner i has been 
associated with unusually high drug resistance. Gebre- 
Yohannes and Habte-Gabr (1984) had twelve resistant 
types on investigating one hundred and eighty-two £.
flexneri isolates. ..................... ^
The practical use of multiple drug resistance 
survey is to guide treatment in areas or farms where 
laboratory facilities are unavailable (Noworyta, 1972),
In such areas, information on drug resistance patterns, 
preferably linked to specific geographical areas, could 
be helpful in selecting appropriate drug therapy.
Antibiotic resistance usual ly is unpredi because
patterns change abruptly. However, in Po 1 and^Noworyta 
(1972) was able to delineate the "Western resistant" 
part o f ? oland from the "Eastern sensitive" part.
It is doubtful if such a corollary can be made in this 
country because of constant intermingling of the 
populace and lack of large-scale systematic studies.
However continuous survei1lance of drug resistance in 
enteric pathogens (e.g. Shigella. Salmonella and £. 
coli) should be encourage in order to guide doctors in
I
the choice of drugs. It should be emphasized, however, 
that antimicrobial therapy has not been a solution to
the control of Salmonellosis and Shigellosis. Thls is aainly 
due to the problea'of infections drug resistance.
133
—  *. . r ' ince
It has been shown that members of the fam i l1 y 
Enterobacteriaceae including Salmone11a and 
harboured R-factors which are transferable to sensitive 
recipients (Anderson, 1968; Adetosoye and Rotilu, 1986; 
Olukoya et al., 1988b). The autotransferabi1ity of R 
plastnids in this study was very high as all the 
isolates transferred single or multiple resistance 
deterrainants to £. co1i K12 recipient. Many reports 
have indicated such high percentage of resistance 
transfer. Chun et al. (1984) recorded 73 percent 
transfer in Shigellae isolated in Korea. Rotilu (1985) 
(unpublished) reported 89.5 percent transfer in fifty- 
seven isolates of Salmone 1 la. Shigella and £. 0 .0 1 i from 
this environment (Ibadan area).
However, in vivo transfer of plasmid does not go
c A
on at this prolific rate due to the pH of the gut, 
oxidation-reduction .potentia1, volatile acids, 
antagonistic metabolites in the gut and presence of 
other bacteria among other Parameters (Watanabe, 1963). 
But one can safely assuine that transfer is relatively 
efficient between genetically related bacteria.
134
The accepted model for the transfer of R-factor by 
conjugation suggestcd that the donor bacterium made a 
temporary contact with recipient bacterium by means of 
specialized hair-like appendages or pilli through which 
one of the replicated R-factor is transfered to the
recipient cell while the other is retained by ±thxe donor
(Anderson, 1965). v X
The molecular weights of These R-fva Sct/ors 
t
encountered in the work were later determined
135
T AB L E 4 . 2B
PREVALENCE OF ANTIBIOTIC RESISTANCE IN SHIGELLA AND 
SALMONELLA IN FAECAL SAMPLES OF DIARRHOEIC PATIENTS
A
Pattern of Resistance Salmonella Shigella Total %o  ooff  TIostoalla te
T-CT-F-A-S-C-Te (7) 10 20
T-CT-F-A-S-Te (6) 3 10
T-CT-S-T-C (5) 8
T-CT-A-S-T-C ( 6 ) 4 8
CT 4CT--FS-S 4 44 88T-CF-F-S-C-Te US( 6 ) 1 2 4
CT (1) 2 4
CT-F-A-S 4 2 4
T-CT-F-S-Te 5 2<< 1 2
CT-F-A-S-Te-C 6 & 1 2
T-F-S-Te-C 5 1 2
CT-F-S-Te 4 1 2
CT-F-S-Te-C 1 2
CT-S-C-Te ^ 1 2
CT-F-A-S-C 5 1 2
T-CT-S-C-A-Te 6 1 2
CT-A-S 3 • 1 2
CT-S-C 3 1 2
CT-Te-S 3 2 4
Te 1 1
KEY: T = Trimetroprim Sulfamethoxazole;
CT = Colistin sulphate; F = Nitrofurantion;
S = Streptomycin; C = Chloramphenicol;
Te = Tetracycline; A = Ampicillin.
136
TABLE 4.3A
IN VITRO ACTIYITY OF ANTIBIOTICS AGAINST 30 SHIGELLA AND 22 SALMONELL/ 
Number of Isolates inhibited at different concentrationa of the antibiotics ug/ml
V « •
AMPICILLIN
137
TA ULI? 4.3b
IN VITRO ACTIVITY OF ANTIBIOTICSAGAINST 30 Shigella Isolates
Organisms
(No. of 
Isolates Antibiotics Percentages of isolates with MIC (ug/ml)
1 2 4 8 16 32/» 128 >128
Shigella spp , Ampicillin 0 0 0 6.7 0 13.3 36.7 73.3 100
n = 30
Chloramph- 0 0 0 3.3 10.0 26.7 56.7 66.7 100
enicol
Strepto­ 0 0 0 33.3 53.3 63.3 88.3 100mycin I
Tetracyc­ 0 0 ?&.0 16.7 30.0 56.7 63.3 100
line
H
o
J '
138
Table 4 .3c
INVITRO ACTIVITY OF ANTIBIOTICS AGAINST 22 Salmonella
ISOLATES
Organisms 
(No of 
Isolates) Antibiotics „Pe rcentage of,  isolates with MICS S(u*jg/ml)
1 2 4 8 16 64 128 >128
Salmonella Ampicillin 0 18.2 36.4 40.9 ff.3 95.5 100 0
spp.
Chloramph-
enicol 0 0 22.7 50.0 68.2 72.7 86.4 90.9 100
n = 22 Strepto­
mycin 0 0 13.6 r27.3 63.6 95.5 100 0 0
Tetracy­
cline 0 13.6 36.4 77.3 95.5 100 0 0
Ä
4 ?
/
139
TABLE 4.4a
CONJUGATION: DETERMINANTS TRANSFERED ON MATING
EACH DONOUR WITH E. COLI K 12 Nar
140
i n
Resisttraanncsec opnhjeungoatnytpse of 
Isolate Identification
No. AMP TE S C25 25 25 25 A
59 Salmonella spp. +
60 S. typhi +
64 T? VI . +
65 Salmonella spp. + +
66 S. typhi + r
67 Salmonella | + : +
42 isolates were screened.
+ = growth of trnnsconjugants on selection plate 
- = no growth of transconjugants oh selection plate
< £ •
142
£ilAi?.TER fl VE
IS.QLAT1QN AND CHARACTERI2ATI0N OF PLASMIDS IN SHIGELLA 
AND SALMONELLA 1S.QLATED FRQM DIARRHOEAL CASES
INTRODUCION
Plasmids are extrachromosomal genetic material
found virtually in all bacterial species (Anderson and
Threlfal. 1974). Plasmids har:bbou ried by members of th
Enterobacteriaceae mediate the transfer of a variety 
of genetic determinants, including those of drug 
resistance, haemolysin and enterotoxin synthesis,
colicinogeny, heavjfy  metal tolerance, resistance to
ultraviolet irradiation, carbohydrate ferraentation, 
hydrogen sulphide synthesis and other metabolic 
characters (Anderson and Threlfal, 1974). They have
also been divided into thiry or more groups by method 
of compatibi 1ity characters (Jacoby and Swartz, 1980).
cPsla.smids generally vary in size ranging from a
^  f e(w million to approximately 100 million daltons
(Jacoby and Swartz, 1980). Those coding for
resistance and virulence are known to be fairly large 
in size. The invasive ability of Shige1 1 a f 1 exner i.
143
has been shown to be due to possess ion of 140 
megadalton plasmid (Sansonetti et al., 1982).
Plasmid profile analysis has been useful as an 
epidemio1ogica 1 tool in investigating outbreaks of 
enteric disease (Riley and Cohen, 1982). When used as
a finger-print for a strain, the plasmid profile may
A A
aid in differentiation of strains or identifying the 
source of infection (Taylor et al., 1982). Such 
differ entation may serve as a means of identifying 
related and unrelated Salmonella and Shigella. Thus 
in outbreaks in which there was a strong epidemiolo- 
gical association with a common exposure (e.g. a 
particular food or water) one would expect that 
Salmonel1a or Shige 11a which are resistant to many 
antibiotics would usually be the same genetically. 
However,p 1 asmid profile analysis often proved this 
assumption to be false (Holmberg et al., 1984). It has 
also been demonstrated that plasmid profile analysis is 
as specific as phage typing in identifying related 
samples and either method is clearly superior to
144
antimicrobial susceptibi 1ity testing (Holmberg et 
al., 1984). The acquisition of new plasmid 
may also 1ead to complete change of phage type.
A case in point was a change from type 204 to type
193 by £. t.vphimur im implicated in bovine salmone 1 1 os i s
in Britain (Willshaw et al., 1980). When first
isolated in 1974 the organism was resistänt to
su1phonamide and tetracycline but >.«e r acquired
two more plasmids which coded for resistance to five
more antibiotics with concomitant change in phage type.
Over the years a number of methods have been 
evolved in the isolation and characterization of 
plasmids DNA. These methods involve three basic 
s t eps:
(i) Growt. h  of the bacteria in a suitable medium;
(i i) Amp1ification of the plasmid;
(iii) Harvesting and lysis of the bacteria and 
purification of the plasmid DNA.
The general approach is to remove enzymatica 11y or 
physically any rigid cell wall and then to lyse the 
cell with a detergent usually SDS (Sodium deodysyl 
sulphate). Nucleases are inactivated an’d the DNA 
is then recovered by ethanol precipitation.
145
Purification methods involve preferential removal of 
long Strands of- DNA from the covalently closed circular 
strands of DNA. Determination of the plasmid DNA
molecular weight is usually done by use of 
polyacri1amide gel e1 ectrophoresis followed by staining 
with ethidium bromide (Birnboin and Dolly, 1979).
In this country there is little information on 
plasmid profile analysis of Shigellae and Salmonellae, 
associated with diarrhoea. Thus the aim of this 
present study is to:
(i) isolate andfcharacterize the plasmid content 
of tho.se organisms with a view to comparing 
them with those encountered elsewhere.
(ii) identify and characterize the antibiotic
resistance (R) plasraids present in transcon- 
ugants obtained in Chapter 4.
146
MATERIALS AND METHODS
Bfl.g-t.exia - L - is .p ia te s ;
All the 53 bacterial isolates obtained from 
diarrhoeal cases were employed in this study. They 
comprised of 24 Shigella flexneri. 4. S .dysenteriae.
3. £.. hoydi i» 9 S..lyt>hi and JJL Salmonella spp. (Table 
3.1). One of the isolates (&. f 1 exner i ) was obtained 
from diarrhoeic piglet. For the characterization of 
R plasmids, eleven of the transcönjugants obtained in 
Chapter 4 were employed.. These were carefully
selected so that each of resistant determinant
o O
successful ly transfered was represented (Table 5.2).I
An £. coli strain (V517) containing multilple plasmid 
obtained from Dr. Olukoya of Dept. of Genetics and 
Oncology, Nigeria Institute for Medical Research, 
Yaba, was used as size reference.
Plasmid LNA isolation procedure;. This was carried out 
by the method of Birnboin and Dolly (1979)., but with 
certain modifications. Since the foremost objectives 
was to screen for the presence of plasmids, certain 
procedures seemed unnecessary. Treatment with RNase 
was omitted, since the concentration of NaOH used was
147
sufficient to remove RNA. As the phenol required 
re-disti11ation, Chloroform alone was used instead of 
the pheno1-chloroform mixture.
Bacteria were grown overnight in Trioticase Soy 
Broth (TSB). One and a half millilitres of the 
!S $ ,h 
culture was poured into an Eppendorf tubi
centrifuged at 8,000 x g for 2 mins. ^ T'hSe merdi.um was
removed by aspiration and the bacterivalV pellet was
resuspended in 100 ul of an ice-cold solution of 50mm 
glucose, 10mm Tris (ph 8.0). The mixture was held for 
5 min. at room temperature hen 200 ul of freshly
prepared 0.25N NaOH, 1% sodium dodecyl sulphate (SDS) 
were added respective1y . The tube was closed and the
contents were mixed by inverting the tube rapidly two 
or three t imes. The tube was held on ice for 5 min.
after which 150 ul of an ice-cold solution of potassium
acetate S(p2H v4.8) was added. The tube was held on
ice Iop min. and then centrifuged for an additional
5 .. 4 C. The supernatant was transfered to a
clean tube and an equal volume of Chloroform was added. 
The contents were mixed on vortex mixer and then 
centrifuged for 2 min. The supernatant was transfered 
to a new tube and two volumes of 95% ethanol were added
to precipitate the DNA
148
After mixing on a vortex rnixer the tube was held 
at room temperature for 2 min. The tube was then 
centrifuged for 5 min. at room temperature and then 
the supernatant was removed. The liquid in the tube 
was allowed to drain away by placing it in an inverted 
Position on a paper towel. One millilitre of 7035 
ethanol was added after vortex. Mixing again briefly, 
the tube was centrifuged for 5 min. -«The supernatant 
was removed and the tube. was inverted to allow the 
pellet to dry. The pellet was resuspended in 50 ul 
of TE buffer (pH 8.0). #
Agarose gel electrophc s i s  o f BM L
The procedure d< ibed by Meyers et.al (1976) was
modified as follows:
0.7% agarose (supplied by Pharmacia, Sweden) was 
dissolve in Trisborate buffer (8.9mM) Tris base, 2.5MM 
disodium EDTA, and 8.9mM boric acid. A dye solution 
consisting of bromocresol blue (0.0735), SDS (735) and 
glycerol (3335) in water was added at 5 ul per sample to 
the DNA samples prior to e1 ectrophoresis. Electro- 
phoresis was carried out in vertical lucite slab gel 
apparatus. The dimension of the gel were 9.6 by 
14.2 by 0.6cm.
149
Sample wells were made by use of lucite comb 
with 14 teeth, each 0.508cm wide and spaced by 0.478cm. 
The power source was a Heathkit regulated high voltage
power supply, model IP-17, and e1ectrophorejssis;  was 
carried out at 60mA,' 120V, for 2h or until the dy e
neared the bottom of the gel. The gel was then 
strained in a solution of ethidium bromide in water 
(0.4ug/ml) for 15 min. Direct visualization of the 
DNA bands without removal of gels from glass tubes 
was made possible by illuminating the gels with a 
long wavelenght u. v. light (Black-Ray B-100A, 
ultraviolet products, Inc.). The gels were later 
removed from the glass tubes, i1luminated with a 
short-wave mineral light (uvs-54, ultraviolet Products 
Inc., Calif.) and photographed using type 57 film 
(ASA 3000) with a Polaroid mp-3 hand camera equiped
with Wratten K2 and 25 filters.
150
RESULT
Table 5.1 shows for each isolate, the number and 
size of different plasmids. It is seen from the table 
that most of the isolates contained more than one 
plasmid. Some of the Shigella isolates contained 
between eight and ten plasmids. One of the Salmonel1a 
isolates (No. 9) harboured nine plasmids while others 
contained between two and six plasmids per isolate.
Altogether, a total of one hundred and thirty , 
plasmids were isolated from twenty-nine organisms and
they belong to forty distinct types. By using plasmids 
of known molecular weights, the molecular weights of 
the plasmids isolated from the bacteria were determined 
to be within the ränge of 2.0 x 10& D 55.5 x 10*° D 
most of them however are less than 10 Daltons.
Figure 5.1 (a & b) show the agarose gel electro- 
phoresis of R plasmids from the transconjugants. It 
revealed the presence of R plasmid as well as one to 
five other plasmids gained during conjugation (Table 
5.2). The R plasmids ränge in size between 2.2 - 38 
Mda1. The ampicillin resistance determinant (which all 
the isolate transfered) was carried on plasmid with
151
molecular weight of 13.3 Mdal. Others are 
Streptomycin 36, tetracycline 38 and ch1oramphenico1 
4.0Mdal. The isolates showing triple resistance
patterns carry plasmids raging in size between A  
2.2-13.3 Mdal. < ? -
152
DISCUSSION
The agarose gel eIecctrophoresis method for 
detecting covalently closed circular plasmid DNA and 
the estimation of plasmid mass is sensitive and does 
not require radio isotopes or ultracentrifugation.
The method is simple and it employs readiily available
reagents. In practice, ordinary bench centrifuge can
be used for spinning to separate theScell wall 
!
materials for DNA, A "black and white" ordinary 
camera and roillimetre rule was used to record results.
The method has been applied in the analysis of plasmid 
components of clinical isolates of £. coli. ]£. 
pnenmon i a . £. f reundi and a host of other bacteria 
(Meyers et. al, 1976 and Olukoya et. al. 1988 a, b).
In the present study it was found to be effective 
in carrying out the plasmid profile survey of all the
Salmonel1a and ShigelIa isolates screened.
The migration of covalently closed circular (CCC) 
plasmid DNA were related to the molecular weights and
single brands of DNA were obserVed for each plasmid
»
specie (Fig. 5.1 a & b). The molecular weights were 
found to ränge between 2.0 and 55.5 x 10^ daltons. 
Most of the plasmidshave their molecular weights 
below 20 megadaltons (Mdal).
153
The data presented in Tahle 5.1 also indicated that 
nearly all the Shigella isolates possess extremely 
small plasmids ranging from 2.0 - 3.0. Mdal in size.
Tacket et al. (1984) investigated the plasmid profiles 
of 136 Shigel1a isolates twelve of which contain only 
small cryptic plasmids of low molecular w eight.
Many of the isolates investigated in this wofk contain 
a number of plasmids which were common indicating that 
few Shigella clones account for Shigellosis in this
country. This is in contrast to ie work of Tacket 
et. al (1984) who demonstrated that Shigellosis in
Bangladesh patients was by large number of
Shigel1a clones possessing a variety of distinct 
plasmids. It was also found in this work that the 
plasmid profile of isolate number 2 (£. f1exneri)
recovered from piglet was identical with those isolated
from human ces. This finding is in agreement with
the work of Wilshaw et al. (1980) who demonstrated
sirailar plasmid profile in epideraic strains £. 
t.vph imur i um isolated from man and animal sources. The 
phenomeon suggests that the Shigella isolated from 
diarrhoeic piglet must have been contacted from
handlers.
154
Consistent absence of very large molecular weight 
plasmids (>55.5kb) in all the Shiare 1 1 a and SaImone 1 1 a 
isolates was noted in this work. Similar studies by 
Olukoya et.al (1988a) failed to demonstrate appreciable 
nuraber of large molecular weight plasmids in most of 
the strains Of enteric pathogens isolati n Lagos.
However, the large plasmids have been associated with 
the enteroinvasive Shigella (Kopecho et al., 1980; 
Sansonetti et al., 1982 ; Tackette^al., 1984). In 
an earlier investigation using Sereny test (Sereny, 
1957) four out of the fourteen Shigella isolates
screened were found to be i invasive. They caused 
purulent keractoconjunctivities in guinea pig models, 
however subsequent plasraid profile studies fails to 
reveal any large plasmid in these invasive isolates#
»’ .. . Laii c i- - ... ". Two reasons
can be adduced for this: either the isolates do not 
harbouT these large plasmids or they have been lost 
during storage. It is known that loss of Shigella 
invasineness occur in storage in association with 
loss of large molecular weight plasmids (Kopecho et 
al., 1980; Sansonetti et al., 1982). The isolates
155
employed in this work were stored for lenght of time 
varying between 6 raonths to 2 yeaij. It could be 
assumed that they might have lost their large 
molecular weight plasmids.
EighKt  of,  the nine Salmonella isolates ^screened
contained between two and six plasmids. on 1 y
exception was isolate number 9 with as many as nine 
plasmids. The isolate might have acquired the 
extra plasmids promiscously from other bacteria in 
in the environment. Acqui stiio n of new plasmids have
been demonstrated in .£. typkimiuri um (Willshaw et al.,
1980) leading to resistta.n„ce to more antibiotics.
Two of the Salmonella isolates (65 and 67) carry 
three identical plasmids and hence are genetically 
related. This is suggestive of a common source of 
infection/in both patients from whom these isolates 
were obtained.
When compared witli plasmids ‘ isolated from 
America by Holmberg et al. (1984) and Tacket et al. 
(1984) there is a ma'rked difference in the molecular 
weights of the plasmid DNA contents of the Salmonel1a 
and Shigella isolates. Those encountered in this 
work are of smaller size (ranging from 2.0 to 55.5
153
Mdal) whereas those in America are larger (ranging 
from 2.0 to 200Mdal. This difference may be due to 
the fact that different strains are responsible for 
enteric diseases in different communities. This also 
emphasizes the value of plasmid profile studies as an
epidemiologica 1 tool in investigatind outbreaks of
diarrhoea.
The sizes of resistance (R) plasmids detected 
in this work were also fouhd to be small and Va ry 
between 2.2 and 13.3Mdal. The exception are 
tetracycline and Streptomycin R plasmids (Molecular 
weights 36 and 38Mdal respective1y ). Previous work by 
Olukoya et al. (1988b) ind icated a ränge of between 3.3 
to 6.5Mdal for most determinats and 24Mdal for 
tetracycline resistance. Ana-Vincete and Da Alraeida 
(1984) also reported the presence of 36 Mdal R plasmid 
in Salmonel1a agona isolated in Rio-de-Janeiro.
It thus appear that most R plasmids vary in size.
Many are of small molecular weights while others are 
fai rly large in size.
157
Generally, plasmids confer resistance to raany 
antibiotics by a variety of inechanisms including 
coding for enzymes which modify or destroy such 
antibiotics.
&
/
158
TABLE 5.1
Number and size of different plasmids among Shigellae and Salmonella
Isolates
159
61 2.5
5 S_. dysenteriac l 14 25.0
22 14.0 
25 «j^.O
31 Q m  8-0
35 O  5.7
5.0
# 3.0 2.5 
v 64 2.4
boydii 14 25.0
21 15.0
24 12.0 • 
27 10.0
30 8.5
34 6.8
41 5.0
&
55 3.0
64 2.4<£ • 67 2.3
' 7 44.0
7 # flexneri 3 8 12 29.024 12.0
32 7.5
55 3.0
< 5 64 3.4
71 2.2
76 2.0
19 S. flexneri 2 2 75 2.0
80 2.0
-------------------------
% .
160
20 S_. flexneri 6 4 23 13.0 
61 2.5 
71 2 . 2  
78 2 .
21 _S. bovdii 3 25 
35 J r 5 
60 < %  6-:2.5
22 S. flexneri 6 3 22 14.0 
73 2.0 
80 2.0 
26 S. dysenteriae 3 s 7 4 4012 29.0
62 2.5 
72 2.1 
76 2. 0
27 dvsenteriac 47 3.5
52 3.0
28 S. flexneri 2 53 3.0
30 S_. bovdii 30 8.5
32 7.5
40 5.0
51 3.1
32 jS. flexneri 2 6 10 34.0
25 12.0
30 8.5
41 5.2
61 2.5
70 2.2
161
1G2
163
Fi g . 5. Agaros e -̂ el e 1 ec t rophorese i s of R. plasmids 
(and otherj) transfered to co 1 i K,x Nar during 
conjugation. From the left, column 1-2, Amp; 
columns 3-4. Amp and Strep, columns 5-6, Amp and 
Tet, columns 7-8, Amp and Chi (See also Table 5.2) 
*O* Iolumn 9  C d l L-' pla•» sml ’ids ö f kA 'wn, s' 1.2«&2>.w(st&nda2?d)
164
-5 5 .5  cdal 
_36 .Q indal
-'3.5 î dai4 A " *  
7.4  adä^*'*’ 
- 5.6
- 5 . i . w i l
mdal 
^ *0.6 adal
Fig. 5.1b Agarose gel e1 ectrophoresis of R plasmids
(and othersJ) otra nsfered to JR̂  co) * K12 Na r duing conjugat ioi rom left column 1 E_. coli containing 
plasmids of known size (Standard) column 2-3, Amp- 
Strep-Tet, and column 4 Amp-Step-chl (See also 
Tab 1 e 5.2)
165
TABLE 5.2
✓
PLASMID PROFILE’ OP TRANSCONJUGANTS
Column on Plasmids (Mdal) Resistance R plasmid
agarose
JSä______ A
1 13.3, 36, 40 AMP 13.3
2 3.1, 7.4, 13.3, 36 13.3
3 2.8, 5.2, 7.4, 13.3, 36 Amp + Strep 13.3, 36
4 13.3, 36 Amp + Strep 13.3, 36
5 13.3, 38 Amp + tet 13.3, 38
6 13.3, 38 Amp + tet 13.3, 38 
7 0.8, 3.1, 4.0 Amp + Chi 3.1, 40
8 3.1, 4.0, 7.4, 13.3,
36, 55.5 Amp + Chi 13.3, 3.1, 40
9 0. 8, 1.0, 3.3, 5.6, 13.1 
36, 55.5 Standard
10 2.2, 3.1, 4.5, 13.3 Amp + Strept 
tet 13.3, 2.2, 3.1
11 2.2, 3.y ß . 6, 13.3 Amp + Strep 
+ Tet 13.3, 2.2, 3.1 
12 1. /T 2.2, 3.1, 5.6 Amp+Strep 
+ Chi 2.2, 3.1
&
166
CHAPTER SIX
V1RULENCE QF SHIGELLA SPEC1ES ISOLATED I *
DlARRHOIEC CASES
INTRODUCTION
Shigella is a gram negative bacteri
faraily EntSfobacteriacea. It causes a dysentery-1ike
disease and dairrhoea referred to as Shigellosis. 
(Topley and Wilson, 1983). Shigellosis appears 
to be a two-organ disease; a jejunal fluid secretion 
caused by entcrotoxins (Formal et al., 1983) and
acute bacterial dysentery
(Keusch, 1978).
The enteroti _ first described
in 1903 and purified in culture filterate of Sh. 
d.vsent er i ae (Keusch et. al 1972a)^ £. flexneri and 
£. sonnei (Keusch and Jacewicz. , 1977). The toxin 
was found to cause histological alterations in the 
colonic mucosa as well as transudation of fluid in the
i
rabbit illeum (Keusch et al., 1972b). Its mechanism of 
action was found to be different from that of Vibrio 
cholerae and Escherichia coli in that it does not 
produce any marked increase in adenylate cyclase or
167
cyclic AMP (Flores et al.t 1974 Steinberg et al.,1975). 
It does not cause depletion of goblet cell mucus 
(Steinberg et al., 1975). Howev.er, like £. coli Aand V. 
cholerae enterotoxins, intestinal receptors have also 
been demonstrated for Shigella enterotoxins in rabbits 
(Bresson et. al, 1984; Fuch et al., 1986; Mobassaleh et 
al., 1988).
The pathogesesis of Shigella infection has been . 
studied in other animal models. Starvation, opiates 
and Streptomycin pre'treatments have been used singly 
and in combination to compensate for the natural 
resistance of animals such as guinea-pigs, rats and 
mice to challenges with Shigellae (Grady and Keusch, 
1971). However in the highly manipulated animals, the 
infection o differs from the usual clinical pattern 
of human disease and knowledge gained from such studies 
have been subject of controversy (Keusch et al., 1976).
Rhesus monkeys (Branham et al., 1949) and human 
volunteers (DuPont et al., 1970; Levine et al., 1973) 
have also been used in the investiagation of the 
virulence of particular strains of Shigella. It is 
known that Shigella possess two potential pathogenic
168
modes of action. These are invasiveness and entero- 
toxigenticity. Levine et al. (1973) established that 
the primary virulence determinant of Shigella is the 
invasive capacity of the organism as strains which 
cannot penetrate and multiple within the colonic 
epithelial cells do not cause disease in humans.
Among the procedures available <?vtesting the
ability of Shigellae to penetrate epithelial cells and 
cause ulcerative lessions, is the Sereny test. This 
measures the capacity of the organism to cause kerato-
Conjunctivitis (Sereny, Invasion of Heia cell
monolayers has also been used as a practical assay 
method (LaBrec, et. al., 1964).
The rabbit illeal loop model discovered by De and 
Chatterjee (1953) has been used extensively to assay 
for heat-labile and heat-stable enterotoxins produced
by bacteria associated with diarrhoea (Arms et al., 
1965; Moon et al. 1970; Fuch et al., 1986). Apart 
from being an in vivo assay method, the ileal loop 
model affords the opportunity to measure directly 
the amount of fluid secreted by the rabbit small 
intestine upon expoture to enterotoxins.
1G9
These investigations were carried out to study 
the virulence of Sh i cre 1 I a species isolated from cases 
of diarrhoea. Attempts were also made at inducing 
diarrhoea syndrome using pretreated laboratory animals
170
MATERIALS AND METHODS
Test for invasiness - Seren.v test:
ßacterial strains: Fourteen isolates of Shigella
recovered fron) cases of diarrhoea were used.
The isolates were maintained on nutrient agar slants
prior to use. Broth•  cultures containjinQg yapproximate 
6 x 103 Shigel1a/ml (determined by the Miles and 
Mistra oiethod) was prepared by growing the organism in 
nutrient broth and incubating overnight.
With the aid of sterile Pasteur pippete one drop 
of the Suspension was introduced to the right eye of 
individual guinea pijj. The left eye served as conrol. 
Each guinea pig was kept in separate cage and examined 
daily for four days for evidence of eye infection 
characterized by swelling, eye-ball depression, 
accummulation of fluid, mucus or blood and corneal 
opacity. The isolates which produced corneal opacity 
were later used to induce Shigellosis by oral 
administration on pretreated guinea pigs and mice.
Experimental Shigellosis_in Guinea pigs and mi.c.e.1.
Bacterial strains: The organisms used in the study 
were those which produced keratoconjunctivitis in 
guinea pigs (Sereny test positive). They included
171
isolates No. 1 (&. flexneri 3), 3(£. flexneri 6).
5 (£. d.vsent er i ae) . and 7 (£. bo.vdi i ) . The isolates 
were kept on nutrient agar slants prior to use. 
Pre-treatment of animals for experiment: TheB<A
animals employed were grouped into three categories 
according to their pre-experimental tresaattmmeents.
Group 1 consisted of 5 adult guinea pigs which
were not treated; V
Group 2 consisted of starved mice 
(deprived of food but given water ad. lib.).
Group 3 consisted 5 starved and calcium carbonate 
treated mice (fed with 25 mg CaCo^ suspended in lml. 
distilled water to neutralize stomach acidity prior to
the adrainistration of bacterial culture) (Formal et 
al., 1958).
The three groups of animals were then fed with
lml. of 24 hr old nutrient broth culture containing 
30 x IO-*1' ml of the isolates as determined by Miles 
and Mistra method. One animal in each group was 
not fed and thus served as the control.
172
Anal temperature: of cach of thc treated animal 
was taken daily for seven days. Faecal samples of each 
animal was also examined. Failure of the animal to 
develop diarrhoeic Symptoms (rise in body temperature 
as well as watery or bloody mucoid stool) withi n 72 
hours was taken as a negative pathogenicity
As s SL.Y- f .o.f—ente r p t <?x i g en i c i t y ;
Bacterial strains: Twenty nine strains of Shigella 
were employed in this assay. The strains were 
those isolated from individual diarrhoeic case.
They comprised of six serotypes of S . d.vsent er i ae . 
twenty of £• flexneri and three of £. IlGZdÜ*
The strains were maintained on nutrient agar slants, 
kept at room temperature prior to use.
Preparation of crude bacterial en.terflli?xiiil_ A loopful 
of each bacterial strain was subcultured on nutrient 
agar and five colonies were inoculated into 2Oral 
of Tryptotpft/Soy Broth and incubated at 37 C for 24 hrs. 
The broth culture was centrifuged at 18,000 rpm for
30 mins. at 10 C. The filterate was divided into two
portions - one portion was heated at 65 C for 30 mins. 
while the other was left unheated. Both portions were 
kept at - 20 C until needed.
173
The control organisms were Y. cholera! 
(i) Ogawa 19339;
(i i) Ogawa 21140;
(iii) Classical 154; and 
(iv) Escherichia coli 36004.
Animais: Five Newzealand rabbits
and 2.1 kg were used. They were d .
were allowed to drink water ,&d 1 i b . twenty four hours 
prior to surgery.
Surgical procedurei The rabbit was anaesthesized for 
surgery by injecting pentabarbito1 through the ear vein 
to effect. The technique of preparing intestinal
segments was similar to that described by Moon et al.
(1974). All little incission of about 8cm long was
made on the linea alba. The small intestine was
carefully brought out and the duodenum was identified
About 10ml of warm sodium Chloride solution was 
injected into the lumen of the i11eum and squeezed 
through into the caecum. Ligatures were made with 
catgut carefully so as to avoid desruption of 
mesenteric capillaries. The intestine was ligated
to form series of approximaItely 10cm segments
174:
interrupted by 5cm sutures. An average of ten 
ligated illeal loops were made in each rabbit 
(Figs. 6.2 and 6.3).
Assay for heat Labile.(LT) enterotoxin; With 
of a syringe, 1ml of the unheated (heat 
toxin was injected into each of the alterna 
loops. The interrupted 5ml loops were 
uninoculated. The first and last ligated intestinal 
loops contained the control, (LT) toxins i.e. V.cholera 
Ogawa 19339. The intestine was later replaced into
the abdominal cavity and the ncis sion closed into two
layers b„y  continuous suture.»
The rabbit was keä(p, H a warm place for 18 hours.
Later it was killed by injecting pentabarbitoi. The 
incission was opened and the intestine brought out.
The results was recorded as follows:
Fluid accummulation evaluation; The volume of fluid in
each ligted loop was measured in millilitres using a
graduated syringe. The length of the loop was measure
in cm with a ruler. A ratio of Vo1ume was determined
Length
and values of 1 and above was regarded as positive LT.
175
Assa.v for heat stable (ST) enterotoxin; Fresh rabbits 
were used and the surgical procedure described above 
was followed. One millilitre of preheated enterotoxin 
(held at 65 C for 30 mins.) was ’injected into each of
the larger (10cm) loops while the interrupted 5cm loops 
were kept uninocu1ated.
Escherlchia coli 36004 enterotoxin was employed as
positive control. This was injected into the first 
loop. One millilitre of Tripticase Soy Broth was 
inoculated into the last loop.^JThe operated rabbits 
were returned to their cages and killed 18 hours later 
by injecting pentabarbitoi.
Fluid accummulation evaluatlonl The volurae of fluid in 
each ligated loop was similarly measured in millilitres 
by using a syringe. The length of each loop was
measured i A ration Volume was determined and 
Length
va 1 ue of 0 .4 or above was regarded as positive for ST. 
Histopathological evaluationl Appropriate segments of 
small intestine were carefully excised from rabbits 
during postmortem. Tissuej were fixed in 10% neutral 
buffered formalin, embedded in paraffin and sectioned 
at 5u. Sections were stained with Mayer's hematoxylin 
stain with 1% alcohol eosion as a counter stain and by 
the Brown and Brenn modification of the Gram stain for 
bacteria in tissue. The gross appearance of the
176
RfiSAiLI
Table 6.1 shows the result of invasive
capabil1i t i es of fourteen Shigella isolates empl i
the Sereny test. Four of the isolates (Nos. 1,3,5,7)
were found to be invasive in that they induced
mucopurulent discharge frora the cornea of inf ected
animals (Fig. 6.1). In three of the foü^cases the 
effect became visible after 48 hours, first as red- 
dening of the eyes. This later developed subsequently 
into inflamed purulent Keratoconjuctivitis. It is of 
interest to note that the four isolates coraprised of 
two £. f 1 exner i ; one £• dga.c.njtfjJ*ft and one £. boxdil- 
The remaining ten isolates were found to be negative
ornea
Exp e r i .niea, higellosis in Guinea oigs and Mice;
our invasive Shigella isolates failed to
iarrhoea when fed orally into pretreated guinea 
mice. A case of mucoid stool was encountered
in one of the pretreated mice but on culturing the 
stool on MacConkey, it failed to grow Shigella. There 
was also no appreciable change in the anal temperature
of the animals.
177
Assg.v f 9f Ent crQUtjLigcni ci t.Y__ (ileal Io op ligation
tes t);
Introduction of enterotoxins of Shigella into the 
lumen of the ligated segments of small intestine 
living rabbits frequently resulted in an accumulat1ion 
of exudates to a varying degree causing dri 
dilation of the Segments (Tables 6.2 and 6.3; Figs. 6.2 
and 6.3).
In the assay for heat labile enterotoxin, six of 
the twenty-nine isolates tested caused fluid 
transudation and elicited positive response in segments 
of the rabbit small intestine. These were four jj>. 
f lexner i . one &. d.vsenter i ae and one £. bo.vi i . The 
remaining twenty-four isolates only caused slight 
increased in th»e intestinal fluid volume in varying
proportions (Table 6.2; and Figs. 6.2 and 6.3).
In the >toslt s involving the he'at stable enterotoxins 
four out of the eight isoI*t?8 randomly selected 
r ec o»,rad,ed positive responses (Table 6.3). The
four isolates comprises of three &. f 1 exner i and
one S . bo.vdi i .
One isolate, Shigel1a boydi i (No. 30) was found 
to produce heat labile and heat stable enterotoxins 
respectively (Table 6.2 and 6.3).
Prominent histological changes were noticed in all 
the Segments studied. This generally revealed haemorr­
hage, hyperaemia, generalized necrosis of the mucosa, 
cellular infi1teration and oedema.
LÜ-S-tog-ft t hg Lag-yi.
V. cholerae +ve coniro 1_113.39_;
Section of small intestined revealed degeneration 
of the mucosa and moderate amount of neutrophilic 
infilterati ons.
S. flexneri 6 (+ve for Seren.v and tv.e. Ent e.rotpxin).
The sections of the small intestine revealed mild 
glandular degeneration with moderate lymphocytic 
infilter .‘- T
S . eria +ve for fluid accumulatioa (■EiiLerotox i -
T’hi s section showed submucosal oedema and 
haemorrhage. There is also mild neutrophilic 
infilterate in the submucosa.
S. bovdii +ve for fluid accumulation (En t ero tox ijcen i c ) 
This section of the intestine shows degeneration 
of the mucosa, submucosal, oedema and neutrophilic
infiltrati ons.
17 C
DISCUSSION
In previous investigations conducted by Silva et 
al. 1982 involving fifty eight strains of Shige1j1_a>i 
twenty seven of thera were able to induce
keratoconjunctivits while the remaining thirt[ y one were
negative in the test. The relatively large number 
of avirulent isolates (Sereny negative) encountered 
in this study may be due to the fact^he isolates 
have been maintained on slants f f a i r 1 y long period
(over six months) before being tested. Thus, they may
have lost the plasmids enc for invasineness.
f
Recent evidences have demonstrated a high frequency of 
of spontaneous loss of high mole.cular weight plasmids 
coding for virulence in strains of Shige11a when ' 
they were maintained on stock medium for long periods 
(Sansonet t i J O l ., 1982; Silva et al., 1982; Takabashi 
et al.,
[n tÄis study six of the twenty-one Shige 11a 
isolates employed in the rabbit illeal loop ligation 
tes^^to assay for heat labile enterotoxin were found to 
produce significant fluid accumulation in the ligated 
intestinal loops of the rabbits. Four of these were
1 8 0
2- lljE&ne.ri , One £. d.vsenter iae and £. bovdi i (Table 
6.2 and 6.3; Fig. 6.2 and 6.3). Previous workers have 
been able to demonstrate fluid accumulation in the 
ligated iluero of experimental rabbits (Keusch et at., 
1972 and Moons et al., 1974). In the case of 
Shigellae, positive response has been limited to £. 
dvsenteriae and S.flexneri (Keusch et al. 2) and 5..
sonnei isolates (Keusch and Jecewicz, 1977). ShigeI1a 
bovdi i has not been reported to cause fluid 
accumulation in ligated illeal 1
Four of the eight isolates screened for heat 
stable toxin gave positive response in rabbit illeal 
loop test. There has been no previous report of heat 
stable toxin production by any of the Shigella species 
tested. Keusch e ‘X1 . (1972a) obtained a negative
response when S-hiXgel1a toxin was heated for 30 min. at 
90 C. This^^sult agreed with that obtainecf by
Adetosoye x(1s98/1 ) (Unpublished data)% when £. coli 
enter Pto «  n filterate heated at 99 C for 30 min. gave 
negative result in ST assay whereas when these 
filterates were heated at 65 C for 30 min. fifty-four 
out of 100 filterates produced positive result 
(Adetosoye et al., 1984). Thus it could be said that
65 C is the Optimum temperature for ST assay.
1 8 1
The li i s t opa t ho 1 og i ca 1 changes obscrved from ileal 
sections exposed to LT enterotoxin show that generally 
there was necrosis, haemorrhage and cellular infilter- 
ations (mostly neutrophils) of the lamina. Oedema of 
the submucosa was observed. This is in agreement with 
the work of previous investigators including Arms et 
al. 1965, Levine et. al. (1973) who found similar 
histological alterations. Keusch et al.(1972a)
demonstrated that £. d.vsent er i ae 1 enterotoxin produce 
alterations and extrusion of epi theli al cel1s
into the intestinal 1umen whrevn ‘inoculated into the 
ligated illeal loops of rabbits. These workers also 
found large numbers of transmigrating lymphocytes and 
considerable nuclei debris scattered throughout the 
lamin©. propria. Goblet cell depletion which is a 
special f eAture  of V. cho1era enterotoxin was not
encouni with the Sliige 11 a isolates employed in this 
study. Earlier investigators (Steinberg et al., 1975) 
also failed to demonstrate the depletion of goblet
The mechanism by which diarrhoea is caused by 
Shigellae has always been a subject of controversy.
It is hard to believe that the prominent necrosis and 
ulceration does not totally reflect an important 
feature in the overall diarrhoea syndrome (Grady and
1 8 2
Keusch, 1971). The question is whether these changes 
precede the mucosal demand for more fluid to excrete 
or are independent and caused by another mechanism. 
Studies of intestinal, perfusion done in monkeys 
infected with Shigel 1 a d.vsenteriae demonstrated that 
there were consistent transport abnormalies of salt 
and water occuring in the colon (Rout et al., 1975).
The magnitude of this colonic absorption defects 
correlates with the degree of bacterial invasion of 
the small intestine. Fluid is secreted in the jejunum 
and diarrhoea is manifested when the colon is unable to 
absorb fluid entering it frora the small intestine 
(Formal et al., 1983).
The absence of bacterial invasion of the colon 
suggests that enterotoxins may be responsible for the 
jejunal fluid secretion (Formal et al., 1983). Infact 
the argument in favour of bacterial invasion as a 
necessary Step in diarrhoea pathogenesis seems to have 
been mitigated. 1t has recently been learnt that non­
invasive but colonizing enteric pathogens such as 
enteropathogenic serotypes of E. coli (which do not 
produce eit her heat labile or heat stähle E. coli 
toxin) and strains of Vibrio cholerae deleted of genes
183
for both A and B subunits of cholera toxin, do cause 
diarrhoea and produce a toxin closely related to 
Shigella toxin (Levin et al . , 1983; O'Brien and 
La-Veck, 1983).
Toxin binding studies have implicat ed t\ le presence
of,  d ̂eve1opmenta1 ly regul. ated toxin rec<e5pt3ors to fluid 
secretory response (Bresson, 1984; Fuchs et al., 1986). 
In studies involving neonates and adult rabbit 
Mobassaleh et al. (1988) provided the first direct 
evidence of a role for the "Gb " glycolipid-binding 
site for a functional receptor for Shigella toxin 
raediating enterotoxin (fluid secrtory) response 
of the small intestine. Future research will 
undoubtedly unravel the actual boichemical pathways 
leading to the production of fluid in the intestine.
It is noteworthy to mention the inability of 
provoking diarrhoea in laboratory animals fed with 
Shigella isolates. None of the isolates could be 
recovered from the faeces of orally infected animals 
< 5
which had been pretreated by starvation and application 
of calcium carbonate; neither was any febrile condition 
noticed. A number of reasons have been advanced for
the seemingly ineffectiveness of oral inoculation of 
pathogenic organism is mice and guniea pigs.
184
Thfes^include the antagonism of normal intestinal 
enteric flora to Shigella (Freter, 1962).
There have also been publications on colicin and other
antibiotic substances produced by certain £. co1i which 
inhibit the growth of other bacterial species in the 
gut (Freter, 1956). The action of immune antibodies 
have also been implicated as a protective mechanism 
(Adamus et al., 1980).
Starvation, opiates and Streptomycin pretreatment 
have been used singly and in combination to compensate 
for the natural resistanc%'of animals such as guinea 
pigs, rats and mice to challenges with Shigellae (Grady 
and Keusch, 1971) o  However in this highly manipulated 
animals, infections often differ from the usual 
clinical pattern of human disease and knowledge gained 
from such studies have been subjet of controversy 
(Keush et al., 1976). .
This investigation has conclusively revealed that 
some Shigella organism are capable of causing diarrhoea 
through their invasive actions. Some of them also have 
the capability of elaborating both heat stable and heat
labile t ox i ns.
185
TABLE 6.1
Invasiveness (by Sereny Test) of Shigella isolates from clinical
diarrhoea cases
A
Isolates RES ULTS A5 (t e r
Number ISOLATE EYE 24hr 48hr 72hr 96hr
1 2. flexneri 3 RIGHT +++ +++
2 S. flexneri 1 f * . - -
3 S. flexneri 6 ft ++ +++ +++
4 S. flexneri 2 If - - - - .
5 S. dvsenteriae 1 LEFT - + ++ +++
6 S. bovdii 
7 g. bovdii P + •+ + +++
8 S. dvsenteriae o< RIGHT - -
- -i
29 S. bovdii f - - - -
22 S. dvsenteriae 6 ft - - \ -
33 S. flexneri 2 f - - -
36 S. flexneri 2 f - - - -
37 g.. flexneri 4 ft - - - -
44 S. flexneri . 6 ft •
N
KE, Y: + = Positive Sereny Test i.e. production of
m u c o p u ru le n t  d i s c h a r g e / r e d d e n ln g  o f the eye.
Negative (See fig. 6.1)
186
TABLE 6.2
Enterotoxigenicity of Shigella isolates using
the ileal loop 1 ligation test, (Heat labile enterotoxin)
Isolate Loop Isolate Lenght of Volume Volume Remark
No. No. oop (cm) of fluid Lengthratio
Control 1 V. choiera 6.5 7.0 1.07 +ve
Og.19339
ff 2 E. coli 5.5 0.3 -ve
36004
2 3 &. flexneri i - ! 3.5 0.5 -ve3 4 S. flexneri 6 SK 5.6 1.01 +ve16 5 S. flexneri 1 4.1 0.7 -ve
29 6 S.dvsenteriae 2 5.0 4.0 0.8 +ve
20 7 S. flexneri 6 6.5 1.5 0.2 -ve
27 8 S. dvsenteriae 6.0 3.0 0.5 -ve
19 9 S. flexneri 5.0 4.0 0.8 +ve
23 10 / S. flexneri 5.0 2.0 0.4 -veControl 11 E.. coli 36004 7.0 1.5 0.2 -ve
ff V. choiera 
19339 5.0 5.5 1.01 +ve
26 S. dvsenteriae 4.5 l.Ö . 0.22 -ve
*•14 §.. flexneri 5 5.0 2.0 0.4 -ve
187
Table 6.2 Contd.)
Isolate Loop Isolate Lenght of Volume Volume Remark
No. No. loop (cm) of fluid Length(ml)
17 15 S.- flexneri 2 6.0 2.5 -ve
30 16 S. bovdii 6.0 6.5 1.08 +ve
6 17 EL- bovdii 5.0 2.5 ( ‘0.5 -ve
32 18 S. flexneri 2 5.6 N.44 -ve
44 19 S. flexneri 1 6.0 22.50S 0.33 -ve
46 20 S. flexneri 2 6.0 2.0 0.33 -ve
N.T 21 S. flexneri 2 N T  ' N.T N.T N.T
4 22 JEL- flexneri 2 2.5 0.4 -ve'V <
26 23 S. flexneri 2 1.5 0.24 -ve
Control 24 Classical V. 1.5 0.25 -ve
cholera 15~4
If 25 V. cholera 
Ogawa 21140 6.5 2.0 0.31 -ve
33 26 S. flexneri 2 5.5 4.0 0.8 +ve
48 27 S_. flexneri 2 3.0 1.0 0.33 -ve
18 28 S. flexneri 4 5.0 2.0 0.4 -ve
45 29 S. flexneri 2 4.5 3.0 0.66 -ve
49 30 S. flexneri 6 4.0 4.0 1 +ve
188
5 31 S. dvsenteriae 5.0 2.0 0.4 -ve
21 32 S.. bovdii 5.5 2.5 0.4 -ve
34 33 S- flQxneri 2 6.0 2.5 0.4- -ve
29 34 S. dvsenteriae 5.5 1.5 0.24 -ve
37 35 §. flexneri 4 5.0 1.5 0.24 -ve
38 36 S. dvsenteriae 5.5 1.0 <^"0.18 -ve
KEY
N.T = Not tested 
+ve = Values greater than 1 
-ve = Values less than 1
189
TABLE 6.3
Enterotoxigenicity of Shigella isolates using
the ileal loop ligation test (Heat stable enterotoxin 
i.e filterate heated to 65 C for 30min)
Isolate Loop Length Vol. of Volume Remark
No. No of loop fluid /JLength (cm) (me) _______
20 37 g. flexneri 4.0 1,2 0.3 -ve
19 38 g. flexneri 3.5 0.4 +ve
26 39 g_. dvsenteriae 5.0 ,5 0.24 -ve
24 40 _g. flexneri 5.2 4.0 0.79 +ve
17 41 g. flexneri 5 3.5 0.6 +ve
30 42 £. bovdii 3.1 0.75 +ve
44 43 g.. flexneri 1.3 0.3 -ve
27 44 g. dvsenteriae 5.0 1.3 0.26 -ve
C E_. coli 36004 3.5 1.5 0.4 +ve&
< r
190
v
V Fig. 6.1 Keratoconjunctivitis in Guinea pig
caused by £. flexneri (isolate 3)
191
Fig. 6.2 Ileal loop 1 igationtest. Nos 1-10 
show S10hcimg ella spp inoculated into each segment.
192
sF>igN.. 6.3: Ileal loop ligation test. C = Control organism (AL* cho 1 era Og. 19339).
Nos 26-32 shows SU»gglJL& spp 
inoculated into each 10cm segment.
C = Y- oho 1 era 26 and 30 are positive 
g . f 1 cxnerJ. 1 27 , 29 are negative ; 
ft . f 1 exner i ; 31 negative £. dvsent CT l &£ and
32 negative £. lumlll-
193
CHARTER SEVEN
SUMMARY AND CQNCLUS1QN
In this study, stool satnples from diarrhoeic 
patients attending outpatient clinics in three 
different hospitals in Ibadan were screened for 
aetiological agents of diarrhoea» such as Salmone1Ia and
Shigella. The hospitals were: The University College 
Hospital and States Hospitals at Adeoyo and at Ring 
Road, Ibadan. Samples were also collected from 
diarrhoeic piglets at the Teaching and Research Farm, 
University of Ibadan. The latter were collected using
sterile cotton wool sw„bs.
Selenite F was used as an enrichment medium to 
enhance the isol ... on of Salmonella species while
potassium tellur•itVe was added to MacConkey agar for
the isolat>1oOn -irn  Shigel la. Deoxychol,ate Citrate 
Agar (DCA) and MacConkey agar were employed as 
sei ec t i ve medi a.
he biochemical characterization of the isolates 
was performed according to the method described by 
Edward and Ewing (1962) and Cowan and Steel (1974). 
The Shigellae were further subdivided into various
i‘J4
serotypes with commercially prepared agg 1 u t i na t i ng 
sera. The Salmonellae were also classified into 
j>. typhi and Salmonella spp.
The isolation frequencies of both Shige 11a and 
Salmone11a were generally low. However, this work
established that Shigella organisms are more prevalent
than SalmonelIa in human diarrhoe in this ronment.
In this investigation 31 Shigella isolates and 22 
Salmonel1a organisms were isolated. The distribution 
pattern of the serotype showed that S .f1exner i was the 
most frequent (24) followed by £. d.vs ent er i ae. (4) and 
£. bovdi i (3), £. sonnei was not isolated. One of the 
Shigellae was isolated from diarrhoeic piglet and was 
identified as £. fIexner i. The later did not show 
any unusual characteristic frora other £. f1exner i
isolates. The piglet may have become infected through
ingestion of feed or water contaminated b.v* Shigel la 
organismsV By comparing the number of different 
o
speciel isolated in this work one can conclude that 
£. f1exner i is more prevalent than other serotypes in
this environment
195
Ainong the Sa J mone 11a species encountercd in this 
i nves t i ga t i on S . t.vphi predominates on the average as 
one out of every three Salmonellae isolate^. Other 
Salmonella soecies apart from tvphoid hacilli . \
accounted for roughly two-thirds of the total number. 
They are all potential agents of diarrhoea in man and
involves the animal reservoir. Their modae ofS trans­
mission may be through food (or water) in which multi­
plication usually takes place. In 557s country and 
other developing countries many lives are lost annually 
due to typhoid and other Salmone11a infections. This 
can be checked by Provision of good drinking water, 
better hygenic, living conditions and enforcement 
of public health control measures. The latter include 
prevention of suspected carriers from handling food 
stuffs meant färSsale to the public. Also effective 
immunization campaigns will serve to reduce the 
incidenc.e iqn typhoid fever.
The antibiotic susceptibi 1ity of Shigellae
and Salmonellae isolated from diarrhoeic human beings
in this work was investiagated. The disc diffusion
« '
as well as the Minimal Inhibitory Concentration (broth 
dilution) procedures were employed. With the disc
diffusion method a total of twenty-one resistance 
patterns to the eight antimicrobial agents used for 
the tests were observed. Some of the iso l a t es  were 
found to be resistant to four, five, s ix: and s<even
drugs. The mos t common resistant p a t t e ^ w a s  T-CT-F-A- 
S-C-Te and it accounted for twenty percent of the total 
resistant patterns. This resistant pattern was more 
common among the Shigella isolates. On the other hand 
pattern CT-F-S and CT-S which occured prominently among 
the Salmone11a isolates were not encountered with the
ampicillin for SaImone11a iso,loautneds  twhe“re tgheenMer,aclolfy high 
as most were inhibited at concentrations between 16 to 
128ug/ml. C g  e Sh i ge 11a organisms were even more 
resistant v»s a significant number of them were not
inhIilbuliwlted  at concentrations higher than 128ug/,ml.
A larger percentage of the Shigellae were inott. • 
sursceptible to Streptomycin at 128ug/ral than chlora-
mphenicol and tetracycline at the same concentration. 
It was observed that at concentrations lower than 
8ug/»al virtually all the four drugs were ineffective.
197
The practical use of drug resistance survey is to 
guide clinicians in the choice of drugs to administer 
in treatment. However, this investigation has shown 
that most of the antimicrobial agents investigated are 
not useful as a result of drug misuse.*  TTfhtee  eeir aergence
of multiple drug resistance phenomenomn has been blamed 
on indiscriminate use of antibioties either as 
prophylaxis, for treatment of diseases and as well as 
growth promoter (Anderson, 1968).
Such resistance can Zither be chroraosonally or 
plasmid mediated (Jacoby and Swartz, 1980). ln this 
investigation, some of the resistances were found to be 
transmissib1e and therefore were extrachromosomal.
The ampicillin resistance determinant was 
transfered in every isolate to £. co1i k,.» . Chlora-
/ V r  ^
mphenicol, Streptomycin and tetracycline resistance 
de lants were also transfered along with Ampicillin
eSistance determinants in some of the isolates. From
< t5h«ese results it was shown that some of the resistance 
determinants were chromosomal since not all deter­
minants were transfered to £. coli K12.
198
The plasmids coding for these resistance were 
characterized using gel electrophoresis. They were 
found to ränge between 2.2 and 38Mdal. The chlora- 
mphejücol R factor was found to be located on the 
4.0Mdal. plasmid, ampicillin 13.3, tetracycline 36 
and Streptomycin 38. The ampicillin resistance 
plasmid was present in all except one of the trans- 
conjugants screened. The triple resistance (A-S-T 
and A-S-C) determinants were general ly found on low 
molecular weight plasmids (between 2.2 and 5.6Mdal). 
In addition to their R plasmids most of the isolates 
carried other plasmids which were transfered to the
reci pi ent (Fig. 4.1^.
In this wor 4k,, tweeinty-one out of thirty-one 
Sh i ge 1 1 a and eighf* out of twenty-two Sa lmone 1 1 a 
isolates were discovered to habour plasmids whose
number vsariy yfrom one to ten per isolate. The 
Shisgecrypt?ll a isolates exhibited a 1-arge number of smallic plasmids and a few large ones. The sizes of 
e plasmids ränge between 2.0 and 44.0Mdal. The
Sa 1mone 11a species possess fewer plasmids whose sizes 
ränge between 2.0 and 55.5Mdal. This result is in
199
agreement with the work of Olukoya et al. (1988a) who 
isolated a significant number of of small cryptic 
plasmid whose size ränge between l-45Mdal from strains 
of pathogen £. coli. SalmQngll.ft spp, I.g.r,S.i P. i a, spp, 
Shieel 1 a s p p, CamP.Y 1 obacter spp and ü^isser
gonorrhoeae. 0 3
The virulence of the Shigella iso/lates from 
diarrhoeic cases was investigated in this work. Three 
different experimental approaches were employed in the 
investigation. First, oral inoculation of mice and
guinea pigs was carried out in order to produce 
Shigellosis in the anomal models. Second, the invassive 
capabilities of some of the isolates were investigated 
by inoculating known numbers of the isolates into the 
cornea of experimental guinea pig models (Sereny test). 
Lastly, assay for enterotoxin production was carried
out usin/g lIirgated ileal loops of experimental rabbits. 
Oral inoculation was unsuccessful as none of
hen Satnim als came down with Shigel,l, osis. Previous
estigation by Freter (1962) failed to produce
Shigellosis in guinea pig. The reason advanced for 
that was the antagonism of normal enteric flora to 
the inoculated Shigella. Kowever, four out of the
200
fourteen isolates used in this work produced kerato­
conjunctivitis in the guinea pigs used in the 
experiment. These findings agreed with those 
others (Sereny, 1957 and LaBrec, 1964) who produced
keratoconjunctivitis in guinea pigs inoc ted
Ö S with
S.hig.e 1 La cultures.
Six of the twenty-nine Shigella isolates screened
produced heat-labile toxin in thl e i 1 eial loop of rabbit.
The toxin was accompanied by histological changes such
as haemorrhage, glandular dlegeraltion with moderate 
lymphocytic infilterati Furthermore, four isolates
produced heat stabl
& terotoxin as culture filterates
of these i so 1 a t es* heat ed to 65 C for 30 mins. dilated 
ileal loops of rabbliit with the accumulation of fluid. 
One of the isolates, identified as &. bovd i i also
produced _ heWat labile enterotoxin.
Fl'om these studies it could be concluded that the 
pathogenicity of Shigella is indeed a two-way process 
asion of epithelial cells and toxin production. The 
latter Step has been confirmed by recent discoveries of 
the presence of toxin receptors which mediated fluid 
secretory response in neonate and adult rabbits
(Mobassaleh et. al., 1988). Future research work
201
would unravel the actual biochemical pathways leading 
to the production of fluid in the intestine during 
diarrhoea episodes.
This investigation has opened new areas- Of
research naraely:
(1) Epidemio1ogica 1 use of plasmid prorile in 
differentiating and identifying strains of 
bacteria involved in enteric infections.
(2) Mode of action of Shigella toxins.
(3) Biochemical pathways which occur in the 
enterotoxin induced by Shigella in cases of 
diarrhoea.
(4) Developmment t*  o fC varccine against f 1 exner i
which i.S the most common in this environment.
$
202
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250
APPENDIX I
ANTIBIOTICS CONCENTRATION
Ampicillin (Amp) 25 mcg
Colistin Sulphate (CT) 10 "
Naiit̂ idic acid (Na) 30"<-
Nitrofurantion Compound (F) 200^^
Chloramphenicol (C)
Streptomycin (S) ^ . 2 5  ■
Tetracycline (TE) 5 5 0  ■
Co-Trimoxazole (SXT) O /  25 "
&
&
A
251
A p p e n d i x 2.b
Scheme for bulk dilution of antibiotics in the macrobroth or agar dilution 
uscd in suscoptibility tosting (modifiod from Ericsson and Shcrris 1971).
Conc. of ‘ vVo l,(ubm)e Sterile Total Intermediate Final conc.
Tube antibiotics taken distilled Vol.water . conc. (ug/ml at 1/10 inNo. (ug/ml or (ml) (ml)added (ml) i.u/ml) agar plateu.i/ml)
1 2000 12.8 7.2 20 128
2 1280 3.0 3.0 6 1268400 64
3 1280 2.0 6.0 8 32
4 1280 2.0 14.0 16 16
5 160 3.0 3.0 6 8.0
6 160 2.0 6.0 40 4.0
7 160 2.0 14.0 J ? 20 2.0 , 
8 20 3.0 3.0 ^  y 10 1.0 ,'V
9 20 2.0 0.1, 5 0.5
10 20 2.0 14.0 16 2.5 0.25
< /
For agar plates take 5ml of intermediate conc. add 45ml molten agar cooled 
to 45-50 C; mix and pour two plates each to obtaim sensitivity agar with 
indicated final conc.
a = initial stock antibiotic conc. prepared from power or ampoule. 
b = Volume of the stock conc. taken
c = Volume of distilled water added to 'b' to obtain total volume.
252
TABLE 3.1 (APPENDIX 4.) 
BIOCHEMICAL CH AR ACTE RI STIC OF ISOLATES
Sh. boydii
253
TABLE 3.1 (con td . )
n
CO c
0d oa «3
• f i :G
U \•Us ©c© G T3 ©
t-, ©u © ©u •©ß
■>o>1
X Xg
o
© 0) Ja c
0o) >4 ax Xc XG XG G •Q
S F •GI’O .C j
oa © © © © s
£ F sr= SF CO CO £ F CÄO|G wJC|G äCO K CJ=O3|
10 16 17 18 19 20 21 22
Arabinose A
Dulcitol
Inositol
l.actcse %
Mallose c
Mannitol - t[A/ J
Ithamnose S>
Sulicin 
Sorbitol 
Sucrose ' V
l rehalose A
Xylose 
I udole
Voges Proskaur
flexneri
254
TABLE 3.1 (Contd . )
%
o co
% r <u >» u<D U0> ua> u •H ■oo c C c < f-4£ X X X Cu 0c) >£o»l<u X X
wl w| co c£o IXJ 
,0) <U J3|<Gu J=|oc c -CI<Cu £
c£o £ F co CO co 5 F co C£O CO
. 1 io 16 17 18 19 20 21 22
Urease
h 2s 0
Methyl red - f
Arginine %
dehydrolase S>
Lysine decarbonxy- 
lase
Ornithine
decarboxylase
flexneri
TABLE 3.1 (Contd.)
Gram -ve, Straight 
rod
Motility
Growth in KCN
medium
Citrate as a C
source
Carbohydrate utili- 
zation
Gas from glucose
Acid from:
Glucose
Adonitol
Arabinose
Dulcitol 
Inositol ‘
256
Lactose
Maltose
Mannitol
Rhamnose
Salicine
Sorbitol
Sucrose
Trehalose
Xylose
Indole
Voges Praskaur 
Urase
Methyl red
i i i i CtOo Sh.flexneri 6
to Sh.
i i i i flexneri 3
to Sh
i i i i CTi dysenteriae
t-<ol Sh > X ,  
00
1 I I I dysenteriae
O
to O3
i i i i ' 100 &fle xner^i 2 
CtOo s h , J r C
t7oI
i i i i dysenteriae -O
i Sh. boydiii i i
00 Sh.
i i i i H» flexneri 6
0t0o Sh
i i i i flexneri 2
oCoO Sh.
J o - flexneri 2
00 Sh.
V  ' ' ' flexneri 2
oo Sh.
, , , , CT> flexneri 2
00 Sh.
1 I I I flexneri 4
1 I I I  _ c00o Shdysenteriae
»2S •
Arginine dehydrolase 
l.vsine decarboxylase
"mithine decarbo­
xylase
258 < ? -
TABLE 3.1 (contd .)
i T
fr* CO <o ccuuco CcOu CaO, cCoU Q& JC # CO CO
5 *Qce c> 0c) C0) C>>u OJ C
OJ
• X M X X >> I EoÄ 0) a>
co C C q0) qa> Ul aS-SCL JL _LO -LCl -ca
45 46 48 49 11 12 13 15 52 _EL2_ -5-i
Gram -ve, straight 
rod 5 ?
Motility
Catalase X:
Growth in KCN
medium
Citrate as a C +
souree $
Carbohydrate utili- A
zation:
Gas f«rom glucose $
Acid from: 9 6
Glucose AG A3 AG AG A AG AG
Adonitol &
Arabinose A A G AG AG AG AG AG
Dulcitol * AG AG AG AG
Inositol AG AG
259
TABLE 3.1 (Contd.)
Cf
ft f
Ot CcOx
t ft Q
CfOt fCat
S CDO tu
< k u G> fatj fOtJ fDtJ cQJ #  'S 
Ul
c<u o >-> >> >> o o
c  
CfOt
•
ÖCJ cfot  
I X
Caj
  E E Ecf
X
ot  G«J Gm3 l C"cOö CO CO CO "CrOa CcO3 
45 46 48 49 11 12 13 15 50 51 52 53 54
Lactose
Maltose A-G AG AG AG AG AG AG AG AG
Mannitol AG AG C b AG AG AG AG AG AG
Uhaimose AG AG AG AG
Sulicin
Sorbitol AG AG AG AG AG AG
Sucrose
Trehalose A ) A G AG AG AG A G AG A G . AG„ AG
Xylose AG AG AG AG A J C A AG AG
Indole
Urase
Methyl red 
* II2 S %  :
<?-
260
TABLE 3.1 (Contd.)
cCou
a
CO 3 Ca
a «®  aa Qi a
u t-4 u u O * a0) 0) oi 9 s \ s CO C
aO
c oX g G G
CQ)
Xd> DX X < t
G <vG G _____4 joJ _toJ
45 46 48 49 11 12 5 T 50 51 52 53 54
Voges Proskaur
&
Ar ginine dehydro- 
lase O
Lysine decarboxy- 
lase A
Ornithine decarboxy- 
lase /
2 C
v
261
TABLE 3.1 (Contd.)
a
3  « a
a JS aa a a
co CO aXi QG> ■'S
CO aCO jC Xi
c al >a>l
CO
DJ 
>> -OE Eo
W| w’S
—ai w cn
55 56 57 58 59 60 61 62 63 64 65 66- 67
Gram -ve, straight 
rod
Motility
Growth in KCN
medium
+
Citrate as a C +
source
Carbohydrate utili- 
zation:
+ +
Gas from glucose AG A > AG' A - A- AG A AG
Acid from:
Glucose AG AG AG AG
Adonitol
Arabim AG AG AG AG
Dulcit AG AG AG AG
In«
ii
ccn
Salmonella
n spp
S. typhi
0cn5
Salmonella
cn spp >HW
V» ► **>  J >  > Salmonella M
wo   + i l l °  O i O>  
cn
 1 °  °  o 1 oo spp W
ts3
•  0to5 
Salmonella
cCn
O
O spp 3o
> S. typhi
&
1 >   1 ' > '  > 05
+ 1 , 1 - 0 ,  , o  1 o
S. typhi
+ 1 1 1  >  O>   ' I ' 1 >  o>  1
f Salmonella 
t r i spp
S. typhi
C0O5
n A
05 s . H £ m  K
Salmonella
c0n5 spp
8■4?
S. typhi
*
Salmonella
05 spp
o v
+ + +
V V o v
OV OV D\
OV
o v
V V
ov o v
Lactose
Maltose A-G AG AG AG AG AG AG
Mannitol AG A AG AG A AC AG
Rhamnose AG AG AG
Salicin
Sorbitol AG AG AG AG AG
Sucrose
Trehalose AG AG AG AG AG AG
X yloae AG AG AG A AG AG
Voges Pioskaur
Indole /
>
Urease -
Methyl red + + + 1
S + + + ' + h 2s AG AG AG AG AG
i + + uuii Salmonella
spp
S. typhi
+ + + Ucnl • v \ /
cn Salmonella+ + + -«3 spp >H
JSaÄlm.
CO
oYnella mf  0t5o
i + + COs  1 spp CO
Salmonella
i + * + to ■> spp Oo3
+ * S. typhio
o; S. typhi
• - W
c Salmonellatno spp
' &  * S. typhi
ccon
O S. typhii
Salmonella
1 + + cUln spp
cn S. typhi 
+ + -t \cn
•
Salmonella
1 + + cn
•* spp
Arginine dehydro- 
lase
Lysine decarboxy- 
läse
Ornithine decarboxy- 
lase
TABLE 4.2A APPENDIX 5
RESULTS OF ANTIBIOTIC SENSITIVITY TESTING OF O - 264
SALMONELLA AND SHIGELUA ISOLATES
Co-Trimo Colistin Nalidixic. Nitrofu- Strepto­ Chloram- Tet Ampici­
xazole sulphate acid rantion mycin phenicol lin llin
ISOLATE SXT 25 CT 10 Na 30 F 200 S 300 Chi.
E. coli 10418 16mm 16mm 16mm 15 mm 16mm 16mm
Sh. flexneri 6 R 6 R 16 S 8 R 6 R 6 R 6 ,R 
" 6 R 8 R 16 S 6 R 6 R 6 R 6 R
11 R 6 R 13 R 6 R R 6 R 10 R 
Sh. dysenteriae 9 R j F6 R 14 R 6 R 16 S 6 R 14 R
16 S 6 R 13 R 6 R $ 6 R 6 R 11 R 
s . flexneri 6 R 16 S 11 R 6 R : : 6 R 6 R 14 R
s . flexneri 6 R 10 R 13 R 6 R 18 R 6 R 6 R 
6 R 12 R 11 R 6 R 6 R 6 R 6 R 6 R
6 R 14 R 16 S 21 R 6 R 6 R 6 R 21 R 
6 R 11 R 18 S 6 R 18 R 6 R 16 R
6 R 11 R 14 F < >17 S 6 R 6 R 6 R 17 S 
S. boydii 16 S 16 R 13 :F 9 R 6 R 17 S 8 R 14 S
S. flexneri 13 R 6 R 14 R 18 R 6 R 6 R 6 R 6 R 
6 R 9 R 14 R 10 R 6 R 6 R 9 R 6 R
S. dysenteriae 14 R 13 R 14 R 16 R 6 R 6 R 6 R 6 R 
6 R 6 R 16 S 11 R 6 R 14 R 9 R 6 R
S. flexneri 6 R 6 R 13 R 11 R 6 R 6 S 6 R 6 R 
S. dysenteriae 6 r 12 R 18 S 6 R 6 R 9 R 6 R
S. boydii R 13 R 10 R 6 R 11 16 S
J * (i R R

2 G 6
i Co-Trimo Colistin Nalidixic Nitroiu- Strepto­ Chloram- Tetracyc­ Ampici-
xazole sulphate acid . rantion mycin phenicol line u m
ISOLATE S X T  25 CT 10 N a  30 F  200 S 300 Chi. TE 50 AMP 25
16mm 16mm 16mm 15mm 16 mm 16mm 13mm i/ ^14 m m
57. Salmonella spp 16 S 10 R 13 R 13 R 6 R 18 S 16 s 13 R
58. tt !! 6 R 6 R 11 R 11 R 6 R 20 S i 9 R 6 R
59. ?! tt 21 S 14 R 21 S 21 S 6 R 20 S R 18 S
GÜ. S. typhi 16 S 14 R 11 R 16 S 6 R  * 18 S V * \ 6 s 14 S
61 tt « tt 16 S 14 R 16 S 18 S 6 R 16 R 16 S 16 S
62. Salmonella spp 16 S 11 R 13 R 16 S 18 S 18 s 19 S 16 S
63. S. typhi 6 R 11 R 11 R 16 S 6 R 20 s 16 S 16 S
64. tt tt 16 S 11 R 14 R 16 S 6 119 s 17 S 16 S
65. Salmonella s p p 6 R 9 R 12 R 13 R 6 R 16 R 14 S 11 R
66. S. typhi 6 R 9 R 12 R 12 R 6 R 6 R 6 R e; R
67. Salmonella spp 16 R 9 R 12 R 11 R 6 R 6 R 6 R 6 R
0