F University of Nigeria Research Publications

Aut

hor

OGUIKE, Joseph Ukachukwu PG/Ph.D/91/12644

Title

Studies on Aeromonas species isolated from Diarrhoeal and Non Diarrhoeal Stools and Environmental sources in parts of Enugu State, Nigeria

Facu

lty

Biological Sciences

Dep

artm

ent

Microbiology

Dat

e

March, 1999

Sign

atur

e

Lw." .- h! .. STUDIES ON AEROMONAS SPECIES ISOLATED FROM '# r 4 DIARRHOEAL AND NON DIARRHOEAL STOOLS AND i : ~ V I R O N M E N T A L SOURCES IN PARTS OF ENUGU STATE, NIGERIA.

JOSEPH UKACHUKWU OGUIKE PG/Ph . D/9 1 / 1 2644

A THESIS S U B M I T T E ~ TO THE DEPARTMENT OF MICROBIOI.OGY, IJNIVERSITY OF NIGERIA NSUKKA.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS ]:OR TI IF, AWARD OF THE DEGREE OF D~C'IUR OF PHILOSOPHY (Ph.D) IN MlCRODIOLOGY

MARCI-I, 1999

SUPERVISOR:- PROF. I I.C. GUGNANI DEPARTMENT OF MICROBIOLOGY, UNIVI3<SITY 01: NIGLI<IA, NSUKKA.

CERTIFICATION

Mr. Joseph Ukachukwu Oguike, a post-graduate student in the Department of ti-- * t . Microbiology, has satisfactorily completed the requirements for the degree of Doctor of

Philosophy (Ph. D) in Microbiology. The work embodied in this thesis is original and has

5 1- not been submitted in part or full for any other diploma or degree of this or any-other

University.

DY J.A.N, Obeta Head of Department Department of Microbiology University of Nigeria, Nsukka.

Prof. H .C . ~ u ~ n a n i (Supervisor) '

Professor of Microbiology Dept. of Microbilogy University of Nigeria, Nsukka.

DEDICATION

This work is dcdicatcd to millions of rural Aliican childrcn who have lost thcir livcs as a

consequence of DIARRIIOEA.

ACKNOWLEDGEMENT

I am deeply grateful to my supervisor for his good guidance, patience and critical review

of all aspects of this work, inspite of his crowded academic commitments.

I would like to express my thanks and deep appreciation to Dr. Ade Adelugba of the

Veterinary Research Centre Vom-Jos and Dr. C . Odibo of the department of Microbiology,

Nnamdi Azikiwe University Awka, who not only placed the use of their laboratory facilities at

my disposal, also spared their time from their busy schedules to put me through the tissue culture

work and chromatographic analysis of my samples respectively. I think words are grossly in

adequaete with which to express my gratitude to both of you. I am also grateful to Prof. A.

Okoye of the dept. of Vet. Anatomy for helping out with the photographs of my tissue culture;

Prof. I.U. Obi of the department of Crop Science University of Nigeria and Prof S. Olaitan of

the department of Vocational Technical Education UNN for their wonderful co-operation in the

statistical analysis of this work. Dr. Cha of the department of Vet. Microbiology for his immense

help in the Rabbit ileal loop and suckling mouse techniques.

I remain very grateful to Prof. A.N.U. Njoku-Obi for setting me on the right path in my

academic pursuit. I am also gratehl to His Lordship Prof. Bishop E. Iheagwam, of the Diocese

of Egbu for all his encouragements and moral support, Professors Ray Anyadike, Steve

Oboegbulem, Cletus Aguwa and S. Onyiriuka for their keen interests in the progress of this

study. I also wish to thank qll my colleagues in the department of Microbiology, particularly Dr.

Chris Irqegbu for his immense contribution in the completion of this work. I shall ever rcmain

grateful to Prof. C. Okafor and his wife Dr. (Mrs.) Joe Okafor for their invaluable help and

support in the completion of this work. My thanks also go to Mr. Joe Mbawuike for his friendly

and sincere assistance. I also express my gratitude to Mr. S. U. Ndumadi for the pains in typing

the manuscript into computer and the the entire members of MEST Computers, Onuiyi for their

nice hospitality.

I wish to express my appreciation to my late mother Angelina who saw me start this

study, but never lived to see the end of it; my appreciation also goes to my late brothers Job and

Solomon and may the Good Lord receive their Souls.

My deep and profound gratitude go to my loving wife Francisca and my children Nkechi,

Amaka, Ikechukwu and Chizomam for their patience and having to bear with my frequent

absence from home, during the course of this work. What else shall I say, than to .pray The

Almighty God to protect, guide and help you all to achieve higher goals than I have done. I am

also immensely grateful to Rev. Fr. Patrick Walsh a Catholic Priest of the Holy Ghost

Congregation who showed me the light of education. May the Good Lord Bless and keep you.

Above all I thank the Almighty God for all his mercies and guidance throughout the

course of this work. To Him go all the praises and all the thanks and to Him also be the glory.

Joe. U. Oguike Department of Microbiology Univerity of Nigeria Nsukka.

- . TITLE .. . . .. CERTIFICATION . . DEDICATION . . ACKNOWLEDGEMENT TABLE OF CONTENT LIST OF TABLES .. LIST OF FIGURES .. LIST OF PLATES .. ABSTRACT .. . . INTRODUCTION .. LITERATURE REVIEW. .

TABLE OF CONTENTS

Taxonomy .. . . . . . . Isolation .. . . .. . . Culture media . . . . . . Cultural characteristics .. . . Identification .. . . . . . . Typing of Aeromonas .. . . Serotyping of Aeromonas .. . . Typing by Polyacrylamide-Gel . . Molecular typinglother special methods BiochemicaVhybridization typing .. Epidemiology of Aeromonas infections Host risk factors . . .. . . Water as main source of infection .. qood as a source of infection . . Clinical features of Aeromonas infection Systemic spread .. . . ..

,'

PAGES

1 . . ll ... ill

v X

xii xii xiv 1 9

2.6.0 Mechanism of pathogenicity . . . . . . . . .. 29 2.6.1 Adherence/invasiveness . . .. . . . . .. .. 29 2.6.2 Production of enterotoxin .. . . . . . . . . . . 33

2.6.2.1 Aerolysin .. . . . . .. . . . . .. . . 33 2.6.2.2 Cytotonic enterotoxin . . . . . . . . .. . . 34 2.6.2.3 Cytotoxic enterotoxin . . . . . . . . . . . . 35 2.6.3 Haemolysins .. . . . . . . .. . . .. . . 36 2.6.4 Endoproteases . . . . . . . . . . .. . . 38 2.6.5 Sodium channel inhibitor . . . . . . . . . . . . 38 2.6.6 Genetic control of virulence .. . . . . . . . . . . 38 2.7.0 Antibiotic susceptibility .. .. . . . . . . . . 39 2.7.1 Antibiotic resistance .. . . . . . . . . . . . . 39

MATERIAS AND METHODS 3.1.1 Source of clinical specimen (faeces)

3.1.2 Source of environmental samples .. 3.1.3 Method of collection (faeces) . . 3.1.4 Collection of environmental samples 3.2.0 Isolation methods (faeces) .. . . 3.2.1 Culture of environmental samples . . 3.2.3 Colonial morphology . . . . 3.3.0 Identification methods . . . . 3.4.0 Biochemical tests .. . . .. 3 S.0 Haemagglutination (HA) test . . . . . . . . . . 50 3 S.4 Haemagglutination Inhibition (HA 1) test .... . . . . . . 51 3.6.0 Haemolytic activity .. . . . . . . . . . . . . 52 3.6.1' Preparation of culture fiee supemate . . . . . . . . 52 3.6.2 Preparation of red blood cell syspension for haemolytic activity .. 52

'3.6.3 Haemolytxassay .. . . . . . . . . . . . . 53

vii

Detection of Cytotoxin .. .. . . . . . . . . Preparation of monolayer cell lines (Vero cells) .. . . Methods of toxin detection .. . . . . . i . . Cytotoxin assays .. . . . . . . .. . . . . Rabbit intestinal loop (ileal loop test) .. . . . . . . The suckling mouse test for enterotoxin .. .. . . . . Gel filtration analysis . . . . . . . . . . .. Determination of protein concentration .. . . . . Estimation of the molecular weight of the partially purified toxin Antibiotic susceptibility tests . . . . . . . . . . Detection of betalactarnase activity .. . . .. . . . .

Prevalence of Aeromonas in faecal samples . . . . . . Occurrence of Aeromanas species in the environment .. . . Age-wise distribution of Aeromonas from human stools .. . . Sex distribution of Aeromonas in diarrhoeiclnon diarrhoeic stools Seasonal variation of Aeromonas species (1992 . 1995) .. . . Age and sex distribution of Aeromonas species from diarrhoiec stool Other enteric pathogens isolated from same specimens during the period .. . . . . . . . . . . .. . . Prevalence of Aeromonas as sole enteropathogens . . . . Distribution of Aeromonas species in diarrhoeic and non diarrhoeic subjects .. . . . . . . . . . . . . Haemagglutination activity of Aeromonas species from diarrhoeicl non diarrhoeic stools .. . . . . . . . . . . . . 79 Haemagglutination of environmentalisolates of b

Aeromonas species . . . . .. .. . . . . . . 84 Haemogglutination patterns of Aeromonas with various animal erythrocytes .. ' .. . . . . . . . . . . 84

Haemagglutination inhibition activity of Aeromonas species .. Haemolytic activity of Aeromonas isolates fiom diarrhoeicl non diarrhoeic stools on human cells . . .. .. . .

Haemolytic activity of Environmental isolates of Aeromonas species on Human RBCs.. .. . . . . . . Haemolytic titres of Aeromonas species with variuos animal erythrocytes . . . . . . . . . . . . . . C y t o b assay of culture supernates on Vero cells . . .. Distribution of cytotoxin producing Aeromonas species in diarrhoeiclnon diarrhoeic stools .. . . .. . . . . Distribution of cytotoxin producing strains from the environment Enterotoxin production by culture supernates fiom diarrhoeic stools in the ileal loop .. . . , . . . . . . . Enterotoxin production by culture supernates of non diarrhoeicisolates Enterotoxin production by culture filtrate of Environmental isolates Fluid accumulation in the ileal loop by culture supernates of Aeromonas species . . . . . . . . . . .. Enterotoxin production by the sole enteric pathogens using the suckling mouse technique . . . . . . . . . . Enterotoxin production by the sole enteric pathogens using the Rabbit ileal loop method . . . . . . . . . . . . Relationship between the virulence determinants of Aeromonas spp. from diarrhoeic and non diarrhoeic faecal isolates .. . . .. The virulence determinants of Aeromonas species isolated as sole enteropathogens .. .. . . . . . . , . . . gesult of the gel-filtration analysis .. . . . . . . . . Standard marker proteins .. .. . . . . . . . . Molecular weights of peak protein fractions in test samples . .

4.33 The elution of protein fi-actiondactivity from supemates of A hvdrophila and A sobria .. .. . . . . . .

4.34 Susceptibility patterns of Aeromonas species isolated as sole enteropathogens . . . . .. . . . . . .

4.35 Betalactarnase production by Aeromonas isolated as enteropathogens .. . . . . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . . CONCLUSION .. . . .. . . .. .. . . . . APPENDIX .. . . . . . . . . . . . . . . .. REFERENCES . . . . . . . . . . . . . . . .

LIST OF TABLES TABLE:

The biochemical identificatioddifferentiation of Aeromonas species ..

Isolation of Aeromonas fiom faecal specimens and environmental samples . . . . .. . . .. . . .. .. .. The percentage occurence of Aeromonas species in faecal specimens and environmental samples . . . . . . . . . . . . . . Prevalence of Aeromonas spps in diarrhoeic stools of various age groups .. . . .. . . . . . . . . . . . . Prevalence of Aeromanas in non diarrhoeic stools of various age groups Sex distribution of Aeromonas isolates in dianhoeic/Non dianhoeic stools .. . . . . . . . . . . . . . . . . . . Enteric pathogens isolated fiom 1 150 faecal specimens during the period (1 992 - 1995) .. . . .. . . .. . . .. . . .. Distribution of Aeromonas isolated as sole pathogens fiom the stools of patients .. . . . . . . . . . . . . . . Aeromonas species isolated fiom diatrhoeic and normal healthy subjects (Non dianhoeic) percentage distribution of different Aeromonas species Haemagglutination of Human Group 0 red blood cells by Aeromonas isolates from diarrhoeal stools, . . . . . . . . . . . . Haemagglutination (HA) of Human Group 0 red blood cells by Aeromonas isolates fiom Non dianhoeal . . .. . . . . . . . . Haemagglutination activity of environmantal isolates of Aeromonas Haemolytx activity of Aeromonas isolates from diarrhoeic stools . . Haemolytx activity of Aeromonas isolates from Non diarrhoeic stools .. .. .. Haemolytx activity of Environmental isolates of Aeromonas species

Cytotoxic activity of culture fiee filtrates of Aeromonas spp fib,,: dmrhoeic stools .. . . . . . . .. . . . . . .

Cytotoxic activity of culture supernates of Aeromonas spp. from stools of non diarrhoeic subjects .. .. .. . . .. .. . . 101 Cytotoxic effects of environmental culture free supernates of Aeromonas on Vero cells. .. . . . . . . . . . . . . . . 102 Enterotoxic activity of culture supernates of Aeromonas spp fiom diarrhoeic stools on rabbit ileal loop .. . . . . . . . . . . . . 103 Enterotoxic activities of culture supernates of Aeromonas spp from non dianhoeic stools on rabbit ileal loop .. . . . . . . . . 105 Enterotoxic activities of culture supernates of environmental Aeromonas isolates on rabbit ileal loop .. ., .. . . . . . . . a 107 Enterotoxic activity of Aeromonas species recovered as SOLE Enteropathogens . . . . . . . . . . . . . . . . . . 109 Fluid accumulation in rabbit ileal loop as a result of the culture fiee filtrates of Aeromonas isolates recovered as sole enteropathogens fiom stools .. . . . . . . . . . . . . . . . . 111 Enterotoxic activity of the culture fiee supernates of Aeromonas isolated as sole enteropathogens in faeces using the suckling mouse technique .. 114

Frquency of the virulence determinants of Aeromonas species isolated . . . . . . from Diarrhoeic and Non- dianhoeic stools ... . . . . 115

The virulence determinants of Aeromonas spp. isolated as SOLE pathogens from diarrhoeal stools . . .. . . .. .. . . . . 117 Absorbance readings at OD 595 rim, the diastatic index, and the elution volumes of active fractions of _A hydrophila a . . . . . . . 120 Absorbance readings, the diastatic Index and the elution volumes of the active fractions of A sobria .. .. . . . . , . . . . . 12 1 Standard proteins with logarithims of their molecular weights and their elution volumes on Gel-filtration using Bio-Gel P-4 .. . . . . 123 Antimicrobial susceptibility profile of 68 Aeromonas strains isolated as SOLE enteropathogens .... . . . . . . . . . . . , 129

xii L.

3 1. Betalactamase production by the Ampicillin and Amoxycillin resistant strains of Aeromonas . . .. . . . . . . . . . . 130

LIST OF FIGURES FIGURE:

A simple scheme for the preliminary identification and differentiation of Aeromonas from Plesiomonas .. . . . . . . . . . .

Distribution of Faecal isolates of Aeromonas for 4 years period (1992 - 1995) .. . . . . .. . . . . . . . . . . Seasonal distribution of Aeromonas spp. isolated from human faeces during the period (1 992 - 1995) .. . . .. . . . . .. Distribution of Aeromonas isolates f r o m di$oeic stools according to age and sex.. . . . . . . . . . . . . . . Haemagglutination patterns of Aeromonas species with hfferent types of erythrocytes .. . . . . . . . . . . . . . . Haemolytx titres of Aeromonas supernates with erythrocytes of different animal species . . . . . . . . . . . . . . . . Standard (marker) proteins with logarithms of their molecular weights and their elution volumes on Gel-filtration using Bio - Gel P.4. .. . . Elution profile and enterotoxic activties of protein fractions of _A hydrophila . . . . . . . . . . . . . . . . Elution profile and enterotoxic activities of protein fraction of A sobria

LIST OF PLATES PLATE

1. Beta haemdysis of A hydro~hila on blood agar plate . . . . . . 9 1 2. Uninoculated (normal) appearance of "Vero" cell line .. . . . . 98 3. Cytopathic effect of culture free supernate of cytotoxin producing

... Xlll

Aeromonas sp, on Vero cell line .. . . . . . . . . 99 4. Fluid accumulation and loop distention by enterotoxin producing

Aeromonas sp. on rabbit ileal loop (positive ileal loop test) . . . . 104

5. Enterotoxic activities of some active fractions of A hydrophila on rabbit ileal loop .. . . . . . . . . . . . . . . 127

XIY

ABSTRACT

TOPIC: S'lUDlES ON AEROMONAS STRAINS ISOI,A'TEI> FROM DIARRHOEAL AND NON-DIAKKI IOEAL STOOLS AND ENVIRONMENTAL SOURCES IN PARTS OF ENUGIJ STATE, NIGERIA.

Aeromonas species are being increasingly recognised as potential pathogens in diarrhocal

diseases world-wide. This work investigates the prcvalcncc of Aerolnonas specics in

diarrhoeal and non diarrhoeal subjccts and also in the environment. Faecal specimens li-om a

total of one thousand one hundred and fifty diarrhoeal and healthy subjects were examined

for the presence of ~nesophilic Aerolnonas species. Five hundred and iifty environmental

samples, mainly water from over head tanks, watcr stored in pots, tap water, streams and

few assorted vegetables and "Aghara" (Solanuni sp.) from the local markets were also

similarly examined. The isolales were studied in detail lor their biochemical and physiological

~liaract~ristics. Out ofo~lc thousand one hiuitlred ant1 fifty I.accal specimens csamincd, 157 (1 3.6%)

yielded Aeromonas specics. The prevalence in diarrhoeal stools (1 7.9%) was significantly higher

than that in non diarrhoeal stools (5.2%) (PC 0.05). l'hc prevalence of Aeromonas in the

environmental sa~iiplcs was 9.0% hydrophila coinpriscd 56% of thc f'xcal isolates and

50% of the environmental isolates. A sobria comprised 28% and 40% and A caviae

cornpris& 16% and 10% respectively. Majority (54%), of the faecal isolates cram the

diamhoeic stools, 45% of no11 diarrhoeic isolates and 60% of

environmental isolates exhibited haemagglutination properties. IIaemolysin was observed in

80%, 55% and 90% of diarrhoeal, non diarrhoeal and environmental isolates respectively.

Culture liltratcs of 66.2% of faecal isolates and 62% of environmental isolates were

cytotoxigenic on "Vero" cell lines. Culture filtrates of' the faecal isolates (70%) exhibited

enterotoxic activities using the rabbit ileal loop method, while 66% of the environmental

isolates showed enterotoxic activities by the same method. The culture filtrates of all the 68

isolates recovcrcd as sole pathogens in diarrhocic stools produced enterotoxin ill the rabbit

ileal loop, whilc with the suckling mouse technique 66 (97%) out of 68 produced

enterotoxin. Column chromatographic anaysis of partially purilied culture supernates of A

hydrophila and A sobria revealed 34 protein fractions each with enterotoxic peaks at

fractions 1 1 and 12 respectively. All the Aeromonas isolates rccovercd as sole pathogens

ucre susceptible to the Aminoglycosides, Cefi~roxime and Floxacin and resistant to the

penicillins. I3etalacta1nase production was demonstrated in majority of thc resistant isolates.

The current study shows that Aeromonas specics with potential virulent hctors can be

isolated from the stools of all age groups whether healthy or diarrhoeal, and also from the

environment in Enugu State. It .is therefore possible that Aeronlonas isolates from

environinental sources, may play a significant rolc in the epidemiology of Aero~nonas b

associated diarrhoeal diseases in humans, and particularly in children, in the study area.

CHAPTER 1

INTRODUCTION

The genus Aeromonas comprises Gram negative, catalase positive, oxidase positive,rod

shaped bacteria that are capable of both respiratory and fermentative metabolism of

glucose. They are ubiquitous and facultatively anaerobic and reduce nitrates to nitrites.

The genus has been classified in the family Vihr- , , (Baumann and Schubert, 1984);

but Colwcll 4.(,1986,) noting the low level of DNA relatedness of V i b r i ~ and

Aeromonas proposed a new family called Aeromonadaceae.

Aeromonas species vary considerably in the shape and size of the cells; with some

strains appearing as short

cells, may also be present,

rods, while others appearing as thin and filamentous curved

but these are readily distinguished from those of Vibrio. Their

size rang6from 0.3-1 pm in diameter and 1-3Spm in length Motility when present is by a

single unsheathed flagellum, although many strains produce lateral flagella in young

culture.-exist singly, in pairs or short chains. b

The genus Aeromonas contains two well separated groups (Popoff, 1984). The

first group consists of a single psychrophilic, non motile species, A Salmonicida which is 1

a strict parasite of salmon and trout under natural conditions and is the causative

organism of the economically important furunculosis disease of salmon and trout, but not

a pathogen of man. The second group consists of mesophilic motile strains of which

initially three species were recognised. A h~drophila, A sobria and A caviae. (Popoff gt

al, 1981). Subsequent DNA hybridization studies have shown that there are at least 11 -

species of mesophilic aeromonads, seven of which are of taxonomic standing. Most

human pathogenic strains fall into the following hybridization groups. Groupl. (A

hvdrophila), group 4 (A caviae) and group8 (A sobria). Other species have also been

associated with diarrhoea] disease, including A veronii (hybridization group X) and A

schubertii (Hickman- Brcnner gt id, 1988).

Although motile aeromonads may be clearly differentiated into groups by DNA

hybridization techniques, there is no simple means of differentiating these groups by

biochemical reactions. Furthermore, species other than A hydrophila may only be

differentiated by means of a large number of tests and for this reason, many laboratories

continue to group all motile aeromonads in the general category of A hydrophila group or b

complex (Hickman - Brenner 1988). Other motile Aeromonads are recognised

pathogens of reptiles (Marcus, 1'971) amphibians (Shotts al, 1972), fish (Trust and

sparrow 1974, Heuschrnann-Brunner, 1% and Nzeako 1990) and cattle (Wohlgemuth

al 1972). In humans, the organisms are recognised as the cause of diseminating infections -

in the immunocompromised hosts (Davis gt 1978: Wolf 1980; Ellison and

Mo~tow, 1984) and are also wound pathogens (Davis a al 1978).

In recent years, Aeromonas has received increasing attention as an agent of food

borne diarrhoea1 disease in other wise healthy people (Champsaur gt 1982; Gracey a al

1982; Janda a 1983; Agger 1985; Palumbo a 4 1985a). However, the role of

Aeromonas as an enteric pathogen is not fully clarified and the genus is best described as

a "putative enteropathogen" (Varnam and Evans 1991) The basic difficulty in relating

Aeromonas to diarrhoeal disease, lies in the fact that while it is fairly easy to establish

statistical and epidemiological links, feeding studies with human volunteers have failed to

confirm a pathogenic role (Holmberg et a1 1986; Morgan and Wood 1988). In six

.episodes, a statistically significant relationship was established. Furthermore, there are

only a few cases where a causal link between a food contaminated with Aeromonas and

cases of enteric disease exists (Agbonlahor d, 1982: Ibe and Onyemelukwe, 1994).

The results or seven epidemiological investigations, each carried out in a different

country, into the relationship between Aeromonas and diarrhoeal disease have been

reviewed by Morgan and Wood (1988). In six episodes a statistically significant

relationship of these studies was established in six, between Aeromonas and diarrhoeal

disease, the exception being the Italian study (Figura al, 1986). However, in work not

covered . . . by the review, Millership a (1983) also failed to establish a relationship . .

between Aeromonas and diarrhoea in England, while in Thailand a statistical relationship

existed among visitors but not among native Thais (Pitarangsi a d, 1982). Of the studies

indicating a positive relationship between Aeromonas and diarrhoea, the most persuasive

was a very large, age matched control study (Burke a d,1983), which found that

Aeromonas was the most coinmonly isolated bacterial pathogen from children suffering

from diarrhoea.

Although the results of feeding experiments appear to contradict the

epidemiological evidence, Morgan and Wood (1988) believe that the administration of the

challenge culture in bicarbonate solution, a standard procedure with other

enteropathogens is in-appropriate for Aeromonas. It should also be appropriate that the

recovery of microorganislns from diarrhoeal stools does not prove a causative role and

that Aeromonas, like other organisms, may have a purely commensal role. It is likely that

confusion arises from the fact that some Aeromonas strains have a primary causative role

in diarrhoea, while others are, indeed commensals (Stelma, 1989). However, while

agreeing with the contention of several authors that the recognition of more virulence

determinants will permit the entero pathogenicity of the organism to be fully understood,

is most certainly correct, the status of Aeromonas as merely a putative pathogen cannot

currently be challenged (Varnam and Evans 199 1).

Mesophilic Aeromonas species are common organisms in the environment, .

especially in water and sewage (Schubert 1991,

untreated and treated drinking water, raw beef,

fresh vegetables (Palumbo a al, 1989) .Fricker . .

Ljungh 1991). It has been suggested that foods

Havelaar al, 1992), and also occur in

pork, lainb, fish ,sea food as well as in

and Tompsett 1989 and Waldstrom and

are contaminated by the water used, for

example, to wash carcasses in processing plants or to wash fresh vegetables during

preparation. Faecal contamination of meat during slaughtering process is also possible

and studies by Gray and Sticker (1989) indicate that the faecal carriage rate of

Aeromonas in pigs and cows is about 6-8% Most motile Aeromonas species are

psychrophilic and thus they will grow at refrigeration temperatures (Beuchat 1991).

THE BACKGROUNDIOBJEXTIVE OF THE STUDY.

In Nigeria, most slaughter houses are littered with cow dungs, with pigs roaming

around these houses. In addition the intestines of the slaughtered cows are processed and

sold to people for the preparation of delicacies in hotels and restaurants e.g "Ngwongwo"

or pepper soup. w i t h the escalating costs of meat in Nigerian markets nowadays, people

now go for the processed intestines of cow meat.

It is possible for an improperly cooked Ngwongwo or pepper soup made with

these processed intestines to be a likely source of infection to nian.Water supply is a

perennial problem in Nigeria particularly in the dry season. During the rainy season,

some villages collect ground water in small shallow wells both for drinking and'for other'

domestic chores. It is quite possible that faecal contamination of these ground flowing

rain water, washes the cow dungs along into these shallow wells, thus constituting a

major source of contamination by mesophilic aeronionads. Futhermore, Okafor and

Nzeako (1985) and Nzeako (1990) confirmed the presence and pathogenic potentials of

Aeromonas species in Nigeria fresh water and smoked fish. Subsequently the consumption of

fresh water and smoked fishes may also add to the spread of these aeromonads to man. In

studies on the aetiology of human diarrhoea in developed countries, Aero~nonas species

have been isolated from 1-3% of faecal samples collected

from patients. Drinking water and food are the suspected vehicles (Kirov 1993).

Although there have been many reports of acute diarrhoea associated with

Aeromonas species in faecal samples (Trust and Chipman, 1979; George et al; 1985,

Agger gt d, 1985 and Gluskin a 4; 1992) Aeromonas species have not become generally

accepted as enteric pathogens. This may be because, incorrect laboratory identification

has led to failure to isolate Aeromonas species in patients with diarrhoea, while strains

isolated from patients with diarrhoea have not been tested for enterotoxigenicity.

There is paucity of information on Aeromonas in relation to diarrhoea disease in

Nigeria. ~ ~ a r t l from the reports of Eko and Utsalo (1989) in the south Eastern part of

Nigeria and a case report each from South Western and Enugu States of Nigeria

(Agbonlahor gt a; 1982; Ibe and Onyemelukwe 1994) no detailed epidemiological

investigations on the aetiological role of of Aeromonas species have been attempted in

Nigeria Also the virulent factors of Nigerian strains of Aeromonas spccies havc not been

investigated.

The present work, therefore aims to.

(1) investigate the prevalence of Aeromonas species in stools of diarrhoeic and non-

dib-hoeic children and adults.

(2) Study and establish their role in the aetiology of diarrhoea and possible

relationship with environmental strains.

(3) investigate the virulent factors associated with Nigerian strains of Aeromonas

species implicated in diarrhoea1 disease.

(4) Determine the in vitro susceptibility patterns of Aeromonas strains to different '

antimicrobial drugs. *

CIIAIYTER 2

LITERATURE REVIEW

The genus Aeromonas comprises Gram-negative, catalase positive, oxidase

positive rod shaped bacteria, thal are capable ol' both respiratory and fermentative

metabolism of glucose. The genus has been wrongly classified within the family

"Vibrionaceae" (Baumann and Schuberl 1984), but lately Colwell a (1986), noting the

low level of DNA relatedness of Vibrio and Acromonas have proposed a ilcw family the

"AEROMONADACEAE" Aeromonas are autochthonous to aquatic environments. They

have also bccn isolatcd frcm brackish, fresh, eqtuarine, marine, chlorinated and un-

chlorinated water supplies worldwide, with highcst numbers achieved in the warmer

months (Seidler et a1 1980, Burke gt a 1984, Van-dar Kooj 1988, and Alonso

1994).

Aeromonas has been isolated from diseased cold and warm blooded animals for

over 100 years and fro~n humans since the early 1950s (Sailarelli 1891, Hill gt a 1954).

Thus, as both non motile and motile aeromonads have caused serious fish disease for a

number of decades, the motile aeromonads have also emerged as a serious microbial

threat to human populations and particularly to the immunocompromised. Although there

have been many reports of acute diarrhoea associated with Aeromonas species in faecal

samples, yet Aeromonas have not fully been generally accepled as enteric pathogens.

This may be because, incorrect laboratory identification has led to failure to isolate

Aeromonas species in patients with diarrhoea, while strains isolated from patients without

diarrhoea have not been tested for enterotoxigenicity. However, epidemiological studies

of the role of enterotoxigenic Aeromonas species in acute diarrhoea have been hampered

by difficulries in isolating the organisms (Trust and Chipman 1979, Burke 4 1982 and

1983)

The present review , attempts to present a concise update of methods of isolation,

identification, virulence Ilctvrs arid antibiotic susceptibility testing of species

isolated from various geographical area of the world.

2.1. TAXONOMY. (a) A landmark in the systematic of Aeromonas occured in 1976 -

when Popoff and Veron used numerical taxonomy to analyse 68 mesophilic aeromonads,

mostly from environmental sources, for 203 morphological, biochemical and

physiological characters at 30°C incubation temperature. Numerical taxonomy had just

been defined by Sneath and Sokal as 'the grouping by numerical methods of taxonornic

units into taxa on the basis of their character state. First, the presence or absence of

selected characters was determined in a group of microorganisms to be classified. This

information was then converted into a binary form that was suitable for numerical

analysis, and then compared using a computer. A minimum of 50 characters was

recommended for analysis that would cover morphological, biochemical, and

physiological properties.

(b) With the publication of the first Edition of Bergev's Manual of Systematic

Bacteriology- in 1984, the genus Aeromonas still resided in the family Vibrionaceae, and

the description by the chapter author (Popoff) included the following species: three motile

species (A hydrophila A sobria and A caviae) and one non motile specie of A.

salmonicida,. Popoff's general description of the genus can be summarized as follows.

Aeromonads occur as straight cells that are rod shaped with round ends to coccoid. Their

size range is 0.3-1 .OI,m in diameter and 1 .O-3 SP in length and they exist singly, in pairs

or short chainss They are Gram-negative, facultalive anaerobes and generally inotilc by a

single polar flagellum, although one species is non-motile. Metabolism of glucose is both

fermentative and respiratory. They are oxidase- and catalase-positive. reduce nitrates to

nitrites, and utilize carbohydrates with the production of acid, and acid with gas. They

are resistant to'the vibriostatic agent 01129 (2.4-diamino-6,7-diiso-propylpteridine) with

an optimum growth temperature of 22-28°C and a mol% G + C content of 57-63 (Bd,

T"J .

More recent awareness of the role of Aeromonas species in human disease notably

diarrhoea, has renewed interest in the taxonomy of the genus, which has been in a state of'

confusion. The only generally accepted species is Aeromonas salmonicida, the b

aetiolkical agent of furunculosis in salmonid fish, and has never been implicated in

. human disease. Numerical taxonomy and studies of polynucleotide relatedness, have

established the existence of at least three other species or biotypes namely, A hydrophila

A. sobria and A caviae. Further sub-groups within each species have been established --

(Popoff and Veron 1976; Popoff a 198 1).

Phenotypic and hybridization groupings based on genetic characterization have further

differentiated A~ornonas into thirteen species, or hybridization groups (FIGS) as shown

below (Altwegg a 1990 a and b).

Mesophilic group. The mesophilic group of motile species are considered to he

potemid human pathogens; A salmonicida the only member of the psychrophiiic group

has not been recovered from humans. The role of Aeroinonas species as potential agents

of human gastrointestinal disease has still not been resolved. However, identification of

the three commonly accepted species (A hydrophila, A sobria, A cavae) is usually made

in a clinical laboratory. The differentiation of Hybridization groups within phenotypic

species requires methods which are not in common use or genetic methods (Kampfer a al

1992; Altwegg @ 1990; Abbott a 1992) D.N.A. hybridization groups as modified

from Altwegg (1990 a and b) is shown below.

DNA HYBRIDIZATION GROlJPS

DNA Hybridization Group (Genospecies) Phenotyp~c Group (Phenons) Nanird Species .~ --

1 ' - A hydrophil;~ - A. hytlrophila

A caviae --

A sohria --

A caviae (A. puncrata)* --

A media -- A media -- A eucrermphila - A sohria --

A veronii* -- A* - A Veronii* --

A V e r w d ~ k c - 12 t A w1udwr111 --

* A .punctata has been shown to be identical to A caviae

* DNA groups 8 and 10 have been shown to be identical

2.2 ISOLATION. Aerorrlvnas survive in glycerol-buffered phosphate transport

medium for only 5 days (Morgan 4 1984). Cary-Blair medium and Stuart's transport

medium have also been used successfully by other researchers as transport media (Janda

2.2.1 CULTURE MEDIA. Although direct plating may be used when Aeromonas is

present in large numbers in stools, recovery is improved by enrichment. A number of

enrichment media have been used including alkaline peptone water, tryptone soy broth,

tryptone soy broth plus sodium chloride and tryptone soy broth supplemented with

ampicillin (Okrend gt 1987). Tryptone soy broth supplemented with ampicillin is most

widely used, although alkaline peptone water is preferred by some workers and has the

additional advantage of recovering occasional ampicillin sensitive strains.

Although a number of sclective agar media and enrichment broths have been

devised for the recovery of Aeromonas species (Von Graevenitz and Bucher 1983), the

use of a selective agar for the recovery of Aeromonas species as part of a routine gastro

imestinal work up may be costly and time coinsuming (Desmond and Janda 1986).

Holmberg and Farmer (1984), suggested that the isolation of Aeromonas species from

stools be reported only when moderate to heavy growth occurs on primary plating.

However, moderate-to-heavy growth of Aeromonas species from stools can be cxpecled

only if the media used are not inhibitory to the growth if Aeromonas. For this reason

Desmond and Janda (1986) decided to evaluate thc growth of Aeromonas on enteric

media widely used for the isolation of salmonella, shigella and other cntcric pathogcns, CIA;(

found that aedia such as Eosin methylene BIue (E M B), BriIliant Green and Bismuth

sulphite are generally unsatisfactory for the recovery of Aeromonas species, owing to the

' poor plating efficiency and relatively smaller colony size of Aeromonas species on these

media. Part of their findings also showed other common enteric media such as Hektoen

enteric and Xylose-lysine-desoxycholate (XLD) as being unsuitable because they contain

sucrose. Aeromonas species that are generally sucrose positive (85 %), are ovcrlookcd on

these media. Desmond and Janda (1986) recommended the use of at Ieast two different b

agars when the isolation of Aeromonas species is being attempted. Recent researchers

recommended the routine use of Blood' agar in the detection of Aeromonas species from

faeces (Gracey gt A 1984, Janda gt 4 1983, Janda gJ 1984a and Robinson 1984).

Although Desmond and Janda (1986) supported this recommendation; because swarming

Proteus species on blood agar can prevent the isolation of Aeromonas species, they

recommended the enteric agar media as an adjunct to the use of blood agar. Owing to

cost.constraint, the variety of media that can be used for the work up of faecal specimens

m y be limited and as a result, many laboratories will continue to rely on blood agar and

enteric media for the isolation of Aeromonas species. Among the enteric media used for

this purpose; desoxycholate citrate agar (DCA) appears to be the least inhibitory for

Acromonas spccies. It is, thcrcforc, appropriate that enteric agar medium selected for-

routine use in the work up o f gastro intestinal specimens sliould not only bc able lo isolate

Salmonella and Shigella spccics satisfactorily, but be flexible enough to recover other

potential enteric pathogens of n~cdical importance.

A wide variety ol' differential or selective media have been designed for the

isolation of Aeromonas species from environmental and faecal specimens (Joseph gt 4

1988). No single medium has received general acceptance. However, blood agar

containing Ampicillin lOmg/litre and cefsulodin-irgasan-novobiocin agar (CIN)

originally developed for the isolation of Yersinia enterocolitica are suitable for the

recovery 'of Aerornonas from faeces, and are most widely used (Kay gt 4 1985). Direct

oxidase testing is no1 rccommcndcd however, on cefsulodin-irgasan-novobiocin agar

(CIN) because mannitol fermentation results in a low PH and possibly false negative

oxidase reaction (Hunt a 4 1981). Thiosulfate-citrate Bile salts - sucrose agar is

inhibitory for Aeromonas species and therefore should not be used (Kay a 4 1985).

Isolation frequencies of Aeromonas species can, however, be increased by using

enrichment broths described earlier.

2:2:2 CULTURAL CHARACTERISTICS, Aeromonas species are facultative

anaerobes that grow well at 35°C 'and produce colonjes 1-3mnl in diameter, circular,

convex wilh a smuoth edge, white to grey and LranslucenL on a variety of media within 24

hrs. Members o f the genus Acn~rnonas are asporogenous Gram regative rods 1-4/,m long

and 0 . 4 p - lpln wide: Except for Aeromonas salrnoilicida which is non motile, the

aerornonads possess polar, usually monotrichous flagella similar to Pseudomonas species.

The clinically important species viz. Aeromonas hydrophila, A sobria and A caviae are

motile.

Most strains of A hydrophila and A sobria show large zones of beta haemolysis on

blood agar, whereas, strains of A caviae are usually non haemolytic. Several types of

haemolysis may occur around single colonies. These include a broad zone of' beta-

haemolysis, a double zone of partial haemolysis or a narrow zone of alpha haemolysis.

Some strains show a marked tendency to become mucoid, but never to the same degree

as Klebsiella. Although originally described as non capsulate (Popoff and Veron 1976),

later studies have shown that many strains do have a capsulc (Kuijper 1989).

In pure culture on MacConkey agar strains of A hvdro~hila and A sobria group

usually appear as non-lactose fermenters. However, up to 40% of isolates of A caviae

may be lactose fermenting. Aeromonads grow on more inhibitory "enteric" media such as

desoxycholate citrate agar (DCA), although colonies may be lrnrn or less. They do not

usually grow on thiosulphate citrate bile salts agar (TC BS). (von Graevenitz and Altwegg

1991). In the routine clinical laboratory, the most important characteristics that should

lead to a presumptive diagnosis of Aeromonas species are, a positive oxidase reaction,

growth on MxConkcy agar and fermentation o f carbohydrates.

2:3 IDENTIFICATION.

Since the division of mesophilic strains into three groups, later known as A

hydrophila, A caviae and A sobria by Popoff and Veron in 1976, several studies of 100

strains with a majority of clinical isolates have shown three major clusters identified by

biochemical testing (Kuijper gt 4 1989; Altwegg gt 4 1990 Carnahan and Joseph 1993).

Depending on the tests used, additional groups could be identified either within the

clusters +or as further small clusters. It was also recognized that the A hydrophila, A

caviae and A sobria phenotypes might be related to difl'erences in disease production and

elaboration of toxins (Janda et a1 1984; Turnbull a a 1984; and Barer et a1 198

However, early DNA hybridization studies placed the previously designated species

appropriate hybridization groups and identified several new groups. Since 1983 six new

species have been proposed (Hickman-Brenner gt 4 1987; and Cannahan et a1 1991),

although some hybridization groups are still unnamed because there are too few known

representatives to undertake a proper study. Several attempts have been made to delineate

a biochemical testing scheme which will accurately identify Aeromonas to the geno

species level (Altwegg et a1 1990; ~annahan et a1 1993). Generally, thus for clinical

laboratories, a simple approach is division of the inesophilic aeromnnads into one of these

three complexes formally known as the phenotypes A hydrophila, A caviae and A sobria

The identification of bacterial colonies as Aeromonas species in primary cultures is

not generally difficult. Occasionally problems may be encountered when using an oxidase

test to screen colonies on primary plates. To the casual observer, the mesophilic species

may be mistaken for pseudomonads (Millership 1996). Aeromonas species are

cytochroine oxidase positive and can be easily excluded from thc Enterobacteriaeceae by

performing an Oxidase test. They are differentiated from Pseudomonas species by

fermentative utilization of glucose as pseudomonads utilize glucose oxidatively. Also

Aeromonas species are Indole positive while Pseudomonas are negative. Separation from

other members of the family, Vibrionaceae may be more difficult in the light of the

multitude of new Vibrio species (von-Graevenitz and Altwegg 1991). Key differential

features are; resistance to the Vibriostatic compound 01129 (2.4-diamino-6-7-diiso

propylpteridine), no growth in 6% sodium chloride and absence of ornithine .

decarboxylase, except in A Veronii, Von Graevenitz and Altwegg (1991) found some of

the commercial identification kits reported so far, as being reliable for the identification

of members of the Vibrionaceae,but with two problems (i) Aeromonas caviae may be inis

identified as Vibrio - tluvialis. The two organisms can easily be differentiated by testing

the ability to grow in Nutrient broth without added NaCl or with 6% NaC1.

(ii) To dale, most data bases contain "A m p t i i l a " which ~ncludes A hydrophila sensu

scricto, A caviae and A sobria. It can also bc anticipated that strains of recently knvwn

species viz. A vcronii and schubertii would be mis identified unless additional tests are

perfomed. A number of workers havc sought a simplilied means of differentiating

between Aeromonas species. Tests for a CAMP- like factor have been used to

differentiate beween A hvdro~hila A caviae and A sobria. (Figura and Gugliel~nentti

1987) while cephalothin sensitivity has been proposed as a marker for A sobria. Neither

of these approaches has been fully successful and subsequently Namdari and Bollone

(1989) have devised a simple means of differentiation based on the "Suicide"

phenomenon, acrogenicity, and aesculin hydrolysis. The presence of glucose in a growth

medium is associated with a suicide phenomenon. The basis of this phenomenon is the

suppression of the tricarboxylic acid cycle when glucose is supplied in the growth

medium, this leads to accumulation of acetate and cell death (Namdari and Cabelli,

2.4 TYPING OF AEROMONAS. In comparison with other enteropathogenic bacteria

the typing of Aeromonas is in its infancy. It is considered that the development of a

simple suitable discriminatory typing scheme would he of cansidcrable value in enharicing

the understanding of the epidemiology of Aeromorias and its role in human disease.

2.4.1 SEROTYPING. Although serotyping has been applied lo A l~ydro~l~i la

pathogenic to fish, relatively little attention has been paid to hul~~aii pathogenic

strains.However, a serotyping scheme for A hvdro~hila, A sobria and A caviae based on

lipopolysaccharide antigens was developed by Fricker(l987).He was able to type 46% of

strains isolated from human faeces, with 16 antisera. Subsequently an extended scheme

was developed (Thomas 1990), which recognised .52 provisional serotypes and

which should permit the typing of mesophilic aeromonads from a wide range'of sources;

Some investigators have attempted to develop typing schemes bascd on other

characteristics. Elbashir and Millership (1989) developed a typing scheme based or1 the

haemagglunating activity of Aeromonas from different sources, but this schemc was of

b

little value.

*2.4.2 POLYACRYLAMIDE GEL ELECROPHORESIS. (Typing by PAGE)

A more promising approach was the use of sodium dodecyl sulphate-polyacrylamide gel . .

electrophoresis to produce protein finger prints .Radio labelling was used originally to

visualise the proteins (Stephenson gt 1987) but silver staining was found to be more

rapid and convenient (Millership and Want 1989).Each strain of Aeromonas typed,

appeared to have a uni,que finger print, thus offering condiderable value in

epidemiological typing. Protein finger printing is not suitable for use in non-specialist

laboratories, where the use of radio-isotopes is avoided.

2.4.3. MOI ,LSCULAR TYP1NG:lOTHER SPECIAL METIIODS.

Almost every available technique has been cxa~nincd as a taxonomic tool for

Aeromonas species Although unlikely to be used in most laboratories at present, these

methods may be useful fhr reference laboratories i l l the identification of atypical strains.

In the future, these methods may also provide an alternative to conventional biochemical

characters for automated identification systems.(Millership 1996). The majority of

studies have divided strains into A hvdrophila A cavix and A sobria rather than into

hybridization groups. Shaw and Hodder (1978) showed that the core lipopolysaccharides

of what was effectively the three phenotypes, A hydrophila A sobria and A-caviae were

different and could be used as an aid to classification.

A decade later, Canonica and Pisano (1988) showed that fatty acid profiles of b

. Aeromonas species were also different using gas-liquid chromatograpy (GLC). Following

this, other workers have confirmed the usefulness of f'atty acids (Hansen et al 1991,

Kampfer a 1994, and Huys et a1 1995.) and other cellular components such as

quinones as chemotaxonomic markers of Aeromonas pheno and geno species.

2.4.4 BIOCHEMICAL/HYBRIDIZATION TYPING.

The g e m Aeromonas contains two well separated groups (Popoff 1984). The first

group consists of a single psychrotrophic non motile species A salmonicida which is a

strict parasile under natural conditions and is the causative agent of the economically

important filrunculosis disease of salmon and trout but is non pathogenic to man.

The second group consists uf rnesophilic motile strains of which only three were

described by Popoff a gJ (1981) A. hydrophila, A. sobria and A. caviae. Subsequent

DNA hybridization studies have recogniscd at least 11 species of mesophilic

aeromonads.(see paragraph 2.1) Most human pathogenic strains fall into hybridization

groups. 1(A hydrophila) 4(A caviae) and 8 (A sobria). Other species have also been

associated with diarrhoea1 disease including A veronii and Aschubertii(hybridisati0n

group X) (Hickman-Brenner at a1 1988).

Although motile aeromonads may be clearly differentiated into groups by DNA

hybridisation techni8ues there is no simple means of differentiating these groups by

biochemical reactions. Alabi and Odugbemi (1990) devised a simple biochemical means

of. identification in laboratories with the limited resources often found in developing . .

countries (fig, 1)

2.5, EPIDEMIOLOGY OF AEROMONAS INFECTIONS.

Mesophilic aeromonads are an ubiquitous group of Gram-negative bacilli that are

widely distributed in the environment and pathogenic for both warm and'cold blooded

animals. In humans, they cause a wide range of extra intestinal infections and are also

considered to be a cause of diarrhoea1 illnesses. Gastroenteritis-associated strains are

readily found in a wide variety of foods (Kirov 1993). Some of these strains are able to , . . a c J ~

grow even in refrigerated foods (Beuchat, 1991). \ #

-y B W

Although water is considered the main source of Aeromonas infection, such strains

have the potential to be food borne pathogens.(Wadstrorn and Ljunghl991). There are

a few published cases in which Aeroinonas species have been strongly suspected as a

cause of food borne gastroenteritis. Suspect foods have included oysters, shrimps, edible

land snails, fish and sashim (Kirov, 1993). In human infections, though all age groups are

susceptible to infections with Aeromonas, children are at greatest risk ( ~ s a v i s and

Washingtonl986, Megrud 1986). Most cases occur between six months and two years of

age and the frequency falls markedly among children older than five years(Varnam and Evans

1991). In adults, Aeromonas infections are most common in people over 60 years of age.

2.5.1 HOST RISK FACTORS. Formula fed children or those with altered gastrointestinal

tract flora as a consequence of disease or antibiotic adminisiration may be at enhanced risk of b

infection with A caviae, this organisin being favoured by the elevated intestinal pH

value (greater than 7.5) found in such children (Namdari and Bottone 1990)

Disseminating infection is largely a disease of the immunocompromised, particularly in ,

individuals suffi.ring from leukaemia or cirrhosis. Males account for over 80% of people

with underlying cancer who suffer Aeromonas infections (Harris gt 1985). In common

with many other Gram negative enteropathogens, illness due to Aeromonas. is more

common where general standards of hygiene are poor (Varnam and Evans 1991).

Aeromonas have been associated with traveller's diarrhoea in Asia (Echeverria

al 1984, Gracey g 4 1984) and may also be a cause of this syndrome in Latin America

and Africa (Black 1986) Depending on the geographical location, however, either A

hvdro~hila arid A sobria (Australia, Thaila~ld and Bangladesh) or A caviae (Europe and

United States) is more often found in such diarhoeal patients (Janda and Duffcy 1988).

Also unusual is the wide variation of carrier rate between continents (0.2-27410)

(Pitarangsi a al 1982).These carrier strains may also exhibit virulence factors. It is not

known whether the development of some kind of immunity adds to this phenomenon.

Unusual however, for a bacterial diarrhoea is the lack of secondary spread (no epidemics)

and the inability to elicit symptoms in volunteers after feeding (Morrun et a1 1985 and

Holmberg et: 1986).

A number of studies indicate a summer peak of infection (Burke 1984b, Agger gt

1985, Nashikawa and Kishi 1988, Wilcox gt 1992). Strains involved usually possess

virulencr associated properties such as the ability to produce enterotoxins, cytotoxins,

haemolysins and an array of proteases and or iilvasive ability. However, the relative

importance of the various virulence-associated factors are still not well understood.

Not all strains with the above properties may be virulent and combinations of

bacterial virulence factors, as well as host pre-dihposing factors may be necessary to

result in diarrhoea. Predisposing risk factors identified include; hospitalization,

antimicrobial therapy, neutralization of gastric acid or inhibition of acid secretion, hepatic

diseases. and underlying enteric conditions e.g. gastric and colonic surgery, colon cancer,

gastrointestinal tract bleeding and inflmunatory bowel disease (George et a11985 and

Moycr 1987).

There is no readily used animal model that reproduces Aeromonas associated

diarrhoea. A trial with healthy human volunteers was largely unsuccessful and the

infectious dose range of bacteria is not known. Only two of fifty seven healthy volunteers

developed diarrhoea, following ingestio~l of up to 10~%ells of Aeromonas (Morgan L&

7 985). However in this trial, no data was given regarding the adherence properties of the

strains used or the immune status of the adult volunteers.

2.5.2. WATER AS MAIN SOURCE OF INFECTION. The main source of infection is

thought to be water. Aeromonads can be found in high numbers in virtually all waters,

even chlorinated drinking water (Hazen a 1978, Burke .g l984b; schubert 199 1). In

Australia, increased levels of Aeromonads in drinking water (> lo2 per 100ml) have been

reported to coincide with the increased incidence of Aeromonas associated gastro enteritis -

in the 'summer months (Burke et a1 1984b,c) and drinking untreated water has been

identified a; a significant risk factor in the USA (Moyer 1987).

A recent investigation in the Netherlands in which Aeroinonas strains from human

diarrhoea1 stools and from drinking water were typed by three different neth hods

(biotyping, serotyping and gas-liquid 'chromatography of cell wall fatty-acid methyl

esters) reported little over-all similarity between Aeroinonas strains from these sources

(Havelaar et a1 1992) .It was therefore concluded that Aeromonas strains which were able

to infect the human gastrointestinal tract represent a selection from a great variety of

environmental strains and that such strains are rare in drinking water in thc Nether-lands.

Strains with in vitro putative virulence properties of diarrhoea-associated

in (> 70%) the warmer water of'

1986, Kirov and Hayward 1993).

main land Australia (Burke a 1984a, Kirov Q

Most epidemiological studies have shown Aeromonas

species in stools to be significantly more often associated with diarrhoea than with- the

carrier state (Salk gt 1988). Association with the consumption of untreated water was

also conspicuous (Holmberg gt 4 1986).

2.5.3 FOOD AS A SOURCE OF INFlECTION.

Aeromonas species are also common in a wide variety of foods (Callister

and Agger, 1987, Okrend gt 4 1987, Abeyta and WekeIl 1988, Nishikawa and Kishi

1988 and Palumbo gt & 1989). They may be introduced from water, animal faeces

containing organisms or food handlers, symptomatic or asymptomatic. The ubiquitous

occurrence of the organism, means that it has the potential to be a food borne pathogen,to

which reviews over the last few years have drawn attention. ,(Buchanan and Paluinbo

1985, Morgan and Wood 1988; Waldstrom and Ljungh 1991). Aeronwnas has also been

isolated from snails, fishes and other food materials in Nigeria (Obi and Nzeako 1980,

Okafor and Nzeako 1985).

2.5.4 CLINICAL FEATURES OF AEROMONAS INFECTION. Gastrq-

intesrinal infe&ions causcd by Aeromnnas spccies are of two distinct types. The first type . which accounls fur at least 75% of cases, is a cholcra - like illness characterized by

watery stools without fcver ur with r r~~ld Scvel-. Thore may be vomiting in childrcn aged

less than two years and in paticnts of any age: diarrhoea may hc accompanied' by

abdominal pain or cramps. The second type of infection is dysentery - like, and is

characterzed by bloody, mucoid stools. Vomiting is rare but there may be abdominal pain

in this type oS infection.

Gastroenteritis due to Aeromonas species is usually mild and self limiting

(Holmberg and Farmer, 1984), but severe and life threatening cases of both types of

infection have been reported (Champsaur a A, 1982). In a severe case of cholera - like

infectionathe symptoms resemble those of cholera pravis and differential diagnosis is not

possible in the absence of the bacteriological examination of stools. Such infections may

be fatal. The duration of symptoms in such severe cases of dysentery - like infection may

be prolonged for a month or more, although complete recovery is expected after

appropriate antibiotic therapy. Acute self limited diarrhoea is more frequent in ybung

children; in older patients, chronic enterncolitis with or without predisposing conditions

may also be observed (George 1985). Fever, vomiting and faecal leucocytes or

erythrocytes may or may not be present (George 1985, Holmberf gt a 1986) . .

2.5.5. SYSTEMIC SPREAD.

Significant systemic spread usually occurs only in the immuno-compromiscd host.

Very little is known of the pathogenesis of such infections (Stelma 1988) but it is possible

to demonstrate marked differences in the behaviour of the three species A. sobria, A.

hydrophila and A. caviae. A, sobria is considered to be highly virulent with respect to

bacteraemia; A hydrophila to be of intermediate virulence and A caviae to be avirulent

and to be involved only in rare polymicrobic infections (Janda gt a 1983). Despite these

observed differences it has not been possible to correlate virulence in disemminating

infections with enterotoxin production, haemolysis or cytotoxin production (landa et a1

1984). The only factor that may be related to differences between species is the generally

greater invasiveness of A sobria (Watson 1985). Later work with A hydrophila has

raised the possibility that an extra surface protein layer the "S" layer is involved in

systematic infections (Dooley gt 4 1988; Murray gt a 1988).

2.6.0 MECHANISM OF PATHOGENICITY:

Motile aeromonads have been reported to cause various infections in man, being

most commonly implicated in diarrhoea, wound infections and septicaemia involving

gram negative organisms. Aeromonasqecies also produces a wide range of extracellular

toxins and enzymes. The multiplicity of extracellular products of motile Aeromonas

specks, has to disagreement about their properties, as well as about their direct

involvement in the enleropathogenicity of species of this organism. This has resulted in a

great deal of controversy and confusion. However, such extracellular biologically active

factors as enterotoxins, cytotoxins and hi@$molysins have been reported to be produced by

motile aeromonads and have been studied in relation to their pathogenicity (Donta a a 1978, Cumberbatch a 1979, Johnson and Lior 198 1 and Stelma 4 1986). Relatively

little is known about the relationship between enterotoxin, haemolysins and cytotoxin

produced by Aeroinonas isolates. However, several reports (Donta gt 4 1978,

Cumberbatch a 4 1979) suggest a parallel activity of these three factors in Aeromonas

species.

2 -6.1 ADHERENCEIINVASIVENESS.

Motile Aeromonas speicies have other propertics that have been reported to be

associated with virulence, such as adherence and invasiveness. The attacll~nent of enteric

pathogens to the intestinal mucosa is an essential step in the pathogenesis of

gastrointestinal infection. A dose association of bacteria to mucosal surfaces in required

for their virulence. This attachment allows for iniximal effect of any toxin that the

organism may produce and is a pre-requisite for successful invasion (Sanyal a A 1983)

Attachment of enteropathogenic bacteria to cells of the gastrointestinal track has been

described by Freter a a (1976), and Moon a (1979). Lack of intiinate contact

resulting in reduction of virulence has also been observed in case of vihrio chrllel-ae

(Cliitnis a 4 1982). Hence, the attachment cnteropathogens to thc intestinal mucosa is

very csscntial in the pathogenesis of gastro-intestinal infections . .

It has been documented that adhcsins are important hctors for Esch'erichia L

(Klcmm 1985) and strains of enterotoxigenic E. coli lacking these factors do not causc

diarrhoea in animals (Burrows gt 1976) or when l'cd to human volunteers (Satter white

et 1978). Carrello g 4 (1988) studied the ability of Aeromonas species to adhere to -

HEp-2 cells. An association between diarrhoea and a high level of adherence was

obscsved in the majority of the faecal isolates and in none of the environmental isolates.

The haemagglutination (HA) assay i:e agglutination of erythrocytes by bacteria is the

commonly used procedure to determine the ability of micro-organisms to adhere to

eukaryotic cells and thus has becn used as a tool to study the mechanism of their

attachment to human cells. (Majeed and Macrae 1994). Majeed and Macrae (1994)

showed in their work that erythrocytes from different animal species may be used and

1

that further information on the nature and specificity of the receptors on the eucaryotic

cell surface can be obtained by testing various sugars for their ability to inhibit the

haemagglutination assay (HA).

It has also been reported that enterotoxigenic diarrhoeal isolates of A hvdrophila

show haemagglutination activity, but no such lrclivity was observed with non-toxigenie

strains of A. caviae commonly isolated from non diarrhoeal infections or the environinent

(Burke 1 1984a).

The lnechanism of agglutination of human '0' group erythrocytes by cholcrae

was found Lo be similar to that involved in the adherence of Aeromonas species to brush

bwders of' intestinal epithelial cells (Jones and Freter 1976).

Sarlyal gt al (1983) reported that most of the enterotoxigcnic strains of' A

hvdrophila possess properties of agglutinating erylhrocytes of human and different

animals; in this respect, no difference exists among isolates from diarrhoea cases, normal

healthy individuals and en~~ironmental sources. They also observed that

haemagglutination by their own strains could not be inhibited by D-inannose. Similar

mannose resistant haemagglutination properly has been observed in other enterotoxigenic

bacteria, that could be correlated with adherence of the organisms to intestinal epithelial

cells. (Duguid 1979 and Evans and Hoa 1977). Strains of cholerae, A lisuefaciens

and certain other entero- bacteria showed mannose resistant haemagglutination and

presence of fimbriae (Tweedy gt 19%).

According to Sanyal a al (1983), the fimbriae of A hydrophila strains

morphologically resemble those of A liquefaciens, being short, straight and numerous in

numbers like the common fimbrae of Escherichia a. Most of the strains gave strong

haemagglutination with human 0 group and guinea pig erythrocytes, a phenomenon

usually observed with enterotoxigenic E. coli strains. They found that the agglutinins did

not bind to D-mannose, D-glucose, D-galactose or D-fructose indicating that these strains

probably possess different sugar combining sites that mediate adherence.

Similar observations were made by Atkinson and Trust (1980) with two observed

mannose sensitive haernagglutinations with certain strains of A hydrophila isolated in

other parts of the world. This may be indicative of the fact that strains isolated from

different geographic regions may differ in their adherence specificities. Duguid et a1

(1979) noted that the haemagglutinins of A hydrophila are heat labile and are absent in

alcohol preparations of supernates indicating the possibility of their being protejn in

" . nature, and are also cell bound, as no activity could be demonstrated in the supernates.

Althougb rnannose - resistant haemagglutination which is often considered to be a

reliable indicator of the presence of pili (Archer and Kvenberg 1988), it does not appear

to correlate with diarrhoea and adherence/colonisation in Aeromonas (Stelma, 1988).

Direct evidence however has been presented by Gqrrello a al (1988), who were able to b

demonstrater an association between diarrhoea and high level adherence.

2.6.2 PRODUCTION OF ENTEROTOXIN.

Individual species of Aeromonas have been associated with particular clinical

syndromes, the frequency of which varies geographically and seasonally (Champsaur A

1982, Gracey gt 1982); however, reports on individual species remain sparse.

Aeromonas species have been found to produce a variety of extracellular products

including cnterotoxins; (Sanyal al 1975), and this factor is reported to be highly

associated with certain biochemical characteristics (Burke et a1 1982; Turnbull gt 4

1984). At least three different enterotoxin product have been identifkd. (a) a cholera-like .

enterotoxin (Acrolysin) (b) a cytotonic enterotoxin not neutralised by cholera antitoxin

and stable at 56°C and (c) a cytotoxic enterototoxin with haemolytic activity, unstable at

56°C (Notermans & 1986).

2.6.3. AEROLYSIN.

The production of a cholera like enterotoxin by an Aeromonas species was demonstrated

by Shimada _et (1984). These authors used passive latex agglutination and Enzyme-linker-

Irnmuno-sorbent assays to test 179 strains of Aeromonas hydrophila, for thc production of

cholera-like enterotoxin. A strain was considered to produce cholera-like toxin, if culture fluid

gavi positive reactions in both assays and if the reactivity was neutralized by the addition of

cholera-like antitoxin to the sample. The production of cholera-like enterotoxin was demonstrated

in culture'filtrates of 8(4.5%) out of the 179 strains tested.

Cholera-like enterotoxin (Aerolysin) is a beta-haemolysin and consists of a single

poiypcptide o f MW52,OOO that possesses haemolytic, cnterotoxic and cytotoxic activity

(Rose gt A 1987). The toxic mechanism is similar to that of the alpha toxin of

Sta~hvlococcus aurens. Two precursor forms, bind to the eukaryotic cells and aggregate

to form holes of approximately 3nm diameter which lead to destruction of the membrane '

permeability barrier and osmotic lysis (Howard gt 4 1987).

Although aerolysins produced by different isolatcs are all biologically similar,

sig~iificant chemical and iirununological differences may cxist (Rose @ 1989a). In all . .

cases, thc cyloly~ic and enterotoxic activitics arc likely to contribute significanlly lo the

palllogenesis of &omonas infcclions (Rose et 1989h).

2.6.2.2. CY TOTONIC, ENl'EKO1'OXIN.

A cytotonic enterotoxin produced by Aeromonas was first described fully by

Ljungh gt (1981, 1982). The toxin is of MW 50,000 and is stable to heating for 10

minutes at 56°C. The toxin was considered initially to be antigenically distinct from

cholera toxin (Ljungh gt 1982: Chakraborty 4 1981). But this contention was not

supported by Potomski gt A, (1987a, b), who described a cytotonic enterotoxin which

cross-reacted with anti cytotonic (CT) serum. The relationship between the Aeromonas

cytotonic enterotoxin and cholera toxin was defined by use of a synthetic oligonucleotide

probe, t~ show relatedness between the two toxins (Schultz and McCerdell 1988).

Evidence currently available suggests that the cytotonic enterotoxin fulfils

requirements for a role in diarrhoea1 disease. However, the toxin has only been isolated

from a low percenlage of Aeromonas strains (Stelma 1988).

A further 44,000 molecular weight cytotonic enterotoxin was described by Chopra

and Houston (1989). Although this enterotoxin was not cross reactive with cholera toxin,

it induced elevated levels of cyclic w i n Chinese hamster ovary cells and thus, may 'act

by a similar mechanism to cholera toxin and Eschericha & labile toxin.

2.6.2.3 CYTOTOXIC ENTEHOTOXIN.

Cytotoxins generally give rise to cell damagc or death and may produce dysentery-

like illness. Graccy 11 (1982) reported ~ t ~ t 20% of gastrointestinal infections due to

Aeromonas specics are of the dysenteric type. Furthermore, marly workers have reported

that majority. of the clinical isolates of Aromonas species produce cyloloxins

(Cumbernatch gt 4 1979; Johnson and Lior, 198 1 ; Janda gt a 1983).

Most reports of animal studies have noted the heat liability of enterotoxin (Burke

gt al 1981; Johnson and Lior, 1981: Turnbull gt al 1984) and it is quite likely that these

assays have detected [he cytotoxin. Further support for this view is provided by the work

of Asao (1984) who purified a haemolysin with a molecular weight, corresponding

to the beta-haemolysin reported in the work of Ljungh gt 4 (1981). This substance was

reported to have cytotoxic activity in Vero cells and is enterotoxic in ileal loop tests. A

second haemolysin, alpha, has been described and only little work has been done on it. b

(Ljungh gt 198 1).

Indirect evidence for the production of a cytotoxic enterotoxin was presented by

Cumbernatch gt 4 (1979) and Johnson and Lior (1981). The first direct evidence was that

of Asao 4 (1984) who showed the toxin to be a protein of molecular weight of 50,000

which was inactivated by heating for 5 minutes at 56°C. There was a strong correlation

between the cytotoxin and haemolysin activities (Burke A 1983) and the haemolysin

was subsequently shown to be a beta-haemolysin. Although some workers such as Steln~a

et (1988), contend that the beta haemolysin alone can cause diarrhoea, others have - found no correlation between cytotcixin production and gastroenteritis. The toxin is

unrelated to either cholera toxin Tirnmis @ d 1984 ) or shiga toxin ( Kindschuch a 4

1987).

2.6.3 HAEMOLYSINS.

Aeromona hydronhila and A. sobria have been reportcd to produce extra-cellular

products such as haemolysin among other toxins and enzymes (Barer g 1986, Monfort

and Balleux 1988; Singh and Sanyal 1992b). It has also been reported that entqotoxic

strains of Aeromonas species are Beta-haemolytic (Burke gt 1982 and 1983) and most

of these B-haemolytic strains were either A hydrophila or A sobria but rarely A caviae.

The findings however were at variance with the work of Singh and Sanyal (199%) who

found that the majority of the strains studied, produced beta haemolysis and that this

property was not exclusive to only A. hydrophila and A. sobria, but was also applicable

to A. caviae in almost equal proportion. Their investigations explained that this beta

haemolysis may be due to the genetic evidence that sequences, homologous to the b-

haemolysin gene are present in all species of Aeronlonas including A caviae.

Furthermore, 3-haemolysin in Aeromonas has been suggested to be a cyfotoxin

(Barer a al 1986: Husslein 1988) A recent report on the cytotoxicity of A caviae

strains indicate that most of them possess this property and the failure by earlier'workers

to detect this haemolytic property, was probably due to thc use of nicdiuni containing

higher level of glucose which has deleterious effect on this haemolytic property.

(Naindari _e_t 4 1989).

In 'the findings of Singh and Sanyal (1992) a signitlcant number of bcta-

haemolytic strains (35%) failed to show any entcrotoxic activity wlicn tested. In addition,

two of the eight alpha haernolytic and 10 out of the 31 non haemolytic strains of A sobria

and A caviae also produced e~lterotoxin in the initial tests. However, the beta-hac~nolytic

strains showed significantly more enterotoxic activity than the alpha and non haeinolytic

strains, independently of their species designation. Figura a (1986) and Eko and

Utsalo (1989) also encountered a few enterotoxic non haemolytic and non toxic beta-

haemolytic strains in their studies. Thus, these studies clearly demonstrate that the

capacity for enterotoxin production in Aeroinonas species is not confined only to thc beta-

haemolytic strains but that the alpha and non-haemolytic isolates also possess this

property although to a lesser extent (Singh and Sanyal 1992). They also observed .:. - -

) _ - . alpha and non production of Beta-haemolysis, after one to three consccutive

passages through rabbit ileal loops, along with initiation of fluid secretion. This indicates

that this process may influence the control of Beta-haemolysin and toxin production.

f ore-over, the titres of haemolysin elaborated in the culture filtrate increased with each

passage.

These observations indicate that a repression and de-repression phenomena may

also operate in the case of the Beta-haemolysin gene and that the rabbit ileal loop provides

a micro-environment conducive to its expression. This is confirmed by reversion of these

strains to their original alpha and non haemolytic characters. These data also go to show

that all Aeromonas strains irrespective of their species designation and. source of

isolation, possess a Beta-haemolysin gene and may elicit a secretory response in the gut.

Passage through the gut of a susceptihle host probably controls the expression of Beta-

haemolysin and enterotoxin production.

2.6.4. ENDOPROTEASES . Among the extracellular enzymes produced by Aeromonas species are the

endoproteases. Aeromonas hydrophila produces two endoproteases: proteinase I and 11,

as well as an aminopeptidase (Pansome fl al, 1986). Ljungh and Wadstrom (1983) had

suggested a pathogenic role for the protcases produccd by Acrolnonas spccics. 1 Iowcvcr,

the author concluded that proteases while contributing to pathogenicity, are unlikcly 10

play a major role.

2.6.5. SODIUM CHANNEL INHIBITOR.

Another extra cellular compound produced by Aeromonas is a tetrodotoxin-like

Sodium channel inhilitor that was isolated from strains of A hvdrophila (Tamplin 4 1987) It has been postulated that this may have an important and hitherto unrecognised

role in the pathogenicity of Aeromonas infections (Stelma 1988).

2.6.6. GENETIC CONTROL OF VIRULENCE.

Although there has been considerable interests in the virulcnce factors of

Aeromonas, there remain, many aspects of the pathogenicity of the organism which are

unknown, or only poorly understood.

There is no evidence of an involvement of plasmids in the control of virulence:

Production of the cholera toxin-like cytotonic entcrotoxin is known to be under

chromosomal control, but firm information is lacking for other virulence factors

(Chakraborty a 1984). Temperature - dependent alterations in growth kinetics and

protein profiles have been reported (Shattner a al 1988) but the significance is uncertain.

2.7.0. ANTIBIOTIC SUSCEPTIBILITY.

Gastro-intestinal infections with Aeromonas species are generally self limiting, but

antimicrobial treatment may be necessary in serious infections particularly systemic

infection in irnmunu-compromised patients.

Most workers have rcportcd some sin~ilarities in the antimicrobial susceptibility

patterns observcd in strains isolated in Britain, Canada and the Unilcd States-ol' America

(Smith 1980; Fainstein et a1 1982; Gray 1984 and Gosling 1986). Acromonas spccics

have been found by many workcrs to be susceptible to Chloramphenicol Tetracycline and

the Aminoglycosides, particularly Gentamicin. Kanamycin, Amikacin and Tobramycin.

(Gluskin gt 1992 and Reinhardt a 1985). Most workers, however, failed to find

significant differences in antibiotic sensitivity pattern among the three species of

Aeromonas (p > 0.5) (Gluskin gt al 1992). Although most Aeromonas infections are self

limiting, and specific treatment is not required, rehydration and supportive therapy may

be necessary where diarrhoea is severe. Antimicrobial therapy is indicated where

symptoms are prolonged and in systemic infections. The drugs of choice are

Chloramphenicol, Tetracycline and Cotrimoxazole (Janda and Dufi'ey 1988). .

2.7.1. ' ANTIBIOTIC RESISTANCE.

Most Aeromonas strains are resistant to the broad spectrum penicillins ('ampicillin,

amoxycillins carbenicillin etc) but susceptible to the second and third generation

cephalospvrins (Motyl a 1985 and Reinhardt and George 1985). Development of

resistance to these drugs, however, has been observed, and Von Graevenitz (1991) noted

that there are at least four Betalactamase in these species. Two o f them are susceptible to

clavulanic acid, which however does not reduce the Minimum Inhibitory Concentration

(MIC) of Ampicillin to the susceptible range. (Bakken g 1988).

. Aerornonas hydronhila and A caviae are often resistant to Cephalothin and to

ccfanandole than A sobria, whereas A caviae is more often susceptible tu Mezlocillin

than are the othcr two s p e w s (Motyl 1985). Daily gl (1981) detected

betalactamase activity in 13 of 16 ampicillin and carbenicillin resistant strains either prc-

or post- exposure to ampicillin, they however failed to detect betalactamase from three strains

that did not produce detectable levels of Betalactainase even after 1 hour of pre-assay exposure to

2OPg of ampicillin per ml. The findings of Daily gt (1981) suggest that resistance to both drugs

(ampicillin and carbenicillin) may be due to a secondary mechanism to penicillin resistance

Environmental isolates of Aeromonas rarely show significant drug resistance, but human isolates

are not in frequently resistant to Clrluramplienicol and tetracycline. Gosling 1986 found that

nearly all the strains used in his work were resistant to concentrations of amoxycillin, and that the

resistance were probably due to the activities of betalactainases produced by the. Aeromonas - species. .

>

CHAPTER 3

MATERIALS AND METHODS

ISOLATION OF AEROMONAS

3.1.1. COLLECTION OF STOOL SPECIMENS (FAECES). A total of 1150 faecal

samples were collected from neonates, infants, children and adults vvcr a period of 4

years, between mid February 1992 to. end of January 1996. Most (764) of the subjects

were patients of diarrhoea (patients with complaint of more than 3 loose stools per day)

attending various hospitals and clinics in Enugu State. The remaining subjects included

normal healthy staff and student members of the University of Nigeria Nsukka Campus.

The latter category were referred to as normal for the purpose of this study.

3.1.2 SOURCE OF ENVIRONMENTAL SAMPLES. Five hundred and fifty samples

from the environments of Enugu State were also collected in a wide mouthed

plastic containers or universal bottles as follows.

Sources of samples

Ysukka township market

University of Nigeria

Nsukkia campus

Enugu-ezike and environs

Eslio stream Nsukka

Obukpa market

Udi market

Adada river ,

Ovoko Town

Nsukka town

Nike lake

Obollo- Afor

Number screened Sample type

Mainly vegetable and water

samples

Water samples

Mainly vegetables and water

samples.

Water samples

Mainly water and garden

egg-solanium species.

Vegetables (lettucelggrden

eggs-solanium species.

Water san~ples

Water stored in local pots

Water samples

Water samples

Water stored in local potslin

restaurants.

Waterlassorted vegetables.

3.1.3. METHOD O F COLLECTION (Faeces). Stool specimens of the infants,

children and adults were collected in universal bottles, while stool specimens from neonates were

collected with the aid of rectal swabs. Most of the swabs were commercially made, while some

were made in the laboratory. Essentially, all the specimens were collected in the mornings from

+ various hospitals and clinics and were cultured on appropriate media, on the same evening.

Information regarding the age, sex, and clinical condition (i.e whether the patient was diarrhoea1 or

not) was obtained.

3J.4. COLLECTION OF ENVIRONMENTAL SAMPLES. Water from the various

environmental areas were collected into sterile conical flasks (50ml) or sterile 50ml medical flat

bottles and occasionally sterile universal bottles (25ml) were also used for collection. For the

vegetables and garden eggs, (solarium species) these were bought in the open markets within the

area mentioned in chapter 3.1.1.

3.2. ISOLATION METHODS. (FAECES) Loopfuls from each of the faecal specimens were

inoculated into a selenite 'f broth and Alkaline peptone water and also streaked on plates of

MacConkey, Desoxycholate citrate Agar (DCA), Brain, Heart Infusion Blood Agar with and

without ampicillin supplement. Emulsions of each feacal specimen was made in saline (wet

mount) on a slide and examined for the presence of ova or cysts of protozoa.

3.2.1. (CULTURE O F ENVIRONMENTAL SAMPLES) With the environmental water

samples, ~25mls of each water sample was membrane filtered into a bottle. After filtration the

membrane pad was carefully removed with the aid of sterile forceps onto

blood agar containing 15\,g per inillilitre of ampicillin. Tla same was repeated for a plain

blood agar without ampicillin.

3.2.2. (VEGETABLES) lgm of each batch of vegetables or gardern eggs (Solanium

species) was. homogenised in a sterile mortar containing 9mls of sterile distilled water

with the aid of a sterile pestle. he homogenised vegetables or garden eggs ( 1 : l O wiv)

was made into a paste. A loopful o f this paste was plated on blood agar plates as

described earlier, and also on selenite "F" and Alkaline petone water broths, MacConkey

and DCA plates. All the plates were incubated overnight in a 37.C incubator. The Brain

Heart Infusion Blood agar plates with and without ampicillin were placed in a carbon-

dioxide exlinction jar before incubating at 3742 to providc a microaerophilic

environment.

The plates were then examined on the next day, for typical morphological

characteristics. The selenite broth and the Alkaline peptone water were sub cultured into

another fresh desoxycholate citrate agar plate and incubated again overnight at 37.C.

The methods for preparation of all media and test reagents etc are given in the appendix.

3.2.3. COLONIAL MORPHOLOGY. Most suspicious colonies showed large zones of

beta heamolysis on blood agar; however, non-haemolytic colonies were also observed.

The colonies were circular with smooth edges and about 1.5-3mm in diamete;. The

majority of the suspicious colonies appeared as non-lactose fermenting colonies on cntcric

agars.i:e (MacConkey and DCA). . a smaller number produced lactose fermenting

colonies.

3.3. IDENTIFICATION. Suspicious colonies were subjected to the following tests. .

3.3.1 OXIDASE- Test. This was the first preliminary test that was carried out on any

colony suspected to be one of the mesophilic aeromonads on blood agar. The oxidase

reagent (a 1 70 aqueous solution of dihydrochoride of tetra-methyl-para-phenylene-

diaminc) is colourless when freshly prepared, but is rapidly oxidised to a purple-coloured

derivative by organisms that produce both peroxidase and hydrogen peruxide.

A speck ol' the suspic;ious colony was pickcd on onc cdgc ol' a clc;~n glass slidc and

smeared on a piece of filtcr paper already soaked in the oxidase reagent. The appearance

of a deep blue colour indicates the presence of oxidase. A strongly oxidasc positive-

organism gives a deep purple colour on the paper within 10 seconds; a weakly positive

one takes 10 - 60 seconds. The development of a blue colour after more than one minute

was ignored.

3.3.2: GRAM REACTION. All the suspected colonies were Gram stained and they all

appeared as Gram negative rods, measuring about 1-4 urn long and 0.5 to 1.0,m wide.

3.3.3. MOTILITY TEST. A loopful of an overnight culture in peptone water, was

. transferred to the centre of a clean cover ship. This coverslip with the liquid culture was

picked up with a glass slide, on which there was a plasticine ring, such that the drop was

directly in the centre of the plasticine ring. This preparation was smoothly but quickly

inverted, so that the droplet of culture hangs below the coverslip, but is prevented from

'

touching the slide by the depth of the plasticine at the corners (HANGING DROP

METHOD). This was then observed undcr thc rnicroscopc for true motility.

3.4. BIOCHEMICAL I'ISS'I'S:

3.4.1 Sugar Fermentation Test. The suspected colonies of each isolate were lightly

inoculated into the freshly prepared carbohydrate media viz. glucose Lactose, Mannitol,

Inositol, Xylose, Dulcitol Arabinose, Adonitol and Salicin.

Before incubation, the Durham' s tubes were completely filled with the culture

medium in the inverted position in the screw capped bijou bottles. The sugars were

incubated for 18-24hours, (overnight ) in a 37oC incubator. After incubation, the next

day, the sugars were examined for the production of ACID and GAS, by change in

colour of the indicator from pale-yellow to pink. Gas production was detected by the

development of bubbles in the closed upper-end of the durham tube.

3.4.2 U ~ E A HYDROLYSIS. (UREASE TEST).

Christensen's urea agar slopes were inculated heavily (stab inoculation) and

incubated at 37oC overnight. Change of colour of the medium from yellow to red within

4 hours indicated strong urease activity. This change if observed after 24-48 hours

incubation was considered a weak ureasc activity.

3.4.3 PRODUCTION OF HYDROGEN SULPHIDE Pruleus inirabilis served as a

positive control (H,S Production). Test cultures were stab-inoculated into an agar

medium containing iron citrate - KLIGLER IRON AGAR and incubated at 37oC.

Blackening of the medium after 24 hours incubation was considered indicative of H,S

production.

3.4.4. AESCULIN HYDROLYSIS.

Aesculin agar slopes were stab inoculated with the test organisms and incubated

overnight at 37.C. A positive test was indicated by a colour change from grey to black,

while the agar slope remained unchanged in a negative reaction.

3.4.5. METHYL RED TEST. A 5ml quantity of MRVP medium was inoculated with

two, loopfuls of a pure culture of the test organism and incubated at 37oC for 1-3 days.

The culthre was then divided into two equal portions.

To one portion was added five drops of 0.04% methyl red solution. A change in

ar to a megenta red colour indicated a positive result. A change to a yellow colour

indicated a negative result. Escherichia coli served as a positive control.

3.4.6. VOGES PROSKAUER (VP) TEST. To another portion of culture, was

added 1.5ml of a 5 % solution of alpha naphthol in ethanol and 0.5ml of a 40% aqueous

solution of potassium hydroxide. The culture bottle was then shaken vigorously. A

positive reaction developed within 5 minutes and this was indicated by the appearance of

a red colour which later deepened to magenta. (A faint pink colour developed late and

was ignored).

3.4.7. Idcntilication wax carried out according Lo the criteria of Popoff and Vcron (1976)

and Popoff (1984) and a simple scheme by Alabi and Odugbemi (1990) (fig. 1) The

Biochemical idcntification and differenlialion of Aeromonas species was carried out using

table 1.

TABLE 1 Biochemical Identification/differentiation of Aeromonas Species.

AEROMONAS SPECIES

Tests

Oxidase

Motility

Haernoly sis (5 % Human blood)

Gas from Glucclsc

Acid from Arabimse

Acid from Mannitol

Acid from Irlosilol

Acid from Salicin

Acid from* Sucrose

Acid from Lactose

Methyl red test

Voges Proskauer Test

Aesculin hydrolysis

Citrate utilization

A hydrophila -

t

+ +

A caviae --

+ + -ve

-ve

t

+ -ve

+ +

V(4O % + ve)

+ ve

-ve

+ -Ve

Identification of a few representative isolates was further confinlied by the use of API

20E (Analytical profile index 20E)

3.5.0 HAEMAGGLUTINATION. (HA) TEST.

3.5.1. COLLECTION AND PREPARATION OR RBCS. The method of Atkinson -

and Trust ( I 980) was followed. *

Human group '0 ' blood obtained from volunteer donors, horse and sheep blood

collected from the abattoir at Obollo-Afor Markel in Udenu Local Government Area,

while healthy rabbits bought from the local dealers were bled, through vene puncture.

The various hlood samples were collected in an anticoagulant (sodiuni cilrate). The

various erythrocytes were washed as indicated earlier, and prcparcd as a suspcnsion of

3 % V/V in phosphate buffered saline (PBS).

3.5.2. PREPARATION OF' BACTERIAL SUSPENSION:

Bacterial suspensions were made by adding two loopfuls from fresh (18-24 hours)

cultures on blood agar to 0.5ml of phosphate buffered saline (PBS), to provide a

concentration of approximately 10" organisms per ml visual nephclmeter standard.

3.5.3. TEST PRO~EDURE. The tests were carried out at room temperature, by

mixing 20ul of each bacterial suspension with an equal volume of 3% washed

erythrocytes of human group 0 blood. The tests were repeated for the sheep, hqrse and

rabbit erythrocytes respectively, on a white porcelain tile. The bacterial arid erythrocytes

suspensions were thoroughly mixed on the tile with the aid ol'a tooth pick.

A control consisting of equal volumes of each washed erythrocyte suspension and

phosphate buffered saline (PBS) was included on each tile.

Aeromonas strain was considered to be positive, if haemagglutination reaction was

immediate and complete and this was scored as 2+ (+ + strong), or if the reaction was

incomplete or not instantaneous, bul occurred within 5 minutes; this was scored as1 +. No agglutination or any haernagglulinatioi occurring after 5 minutes was regarded as

negative.

3.5.4. HAEMAGGLUTINATION INHIBITION (HAI) Test.

3.5.4.1. Preparation of sugars. One percent solutions of three sugars were used namely;-

Glucose, Galactose and Mannose. The sugars were prepared in phosphate buffered salilie

(PBS) as a 1 % w\v solution.

3.5.4.2. Test procedure;- This was carried out by mixing 20C,l of 3% red blood cell

suspension washed as previously described with an equal quantity of a test 1 % sugar

solution and bacterial suspension (i:e 20P1 01: 3% rbcs, 20P1 of 1 % sugar and 20P1 of

bacterial suspension). The three suspensions were mixed together on a white tile with the

aid of a tooth pick. b

Reactions were compared with positive controls consisting of equal volumes of bacterial

suspension (HA positive) and red cell suspension; and a negative control which consisted

of one volume (2OP1) phosphate buffered saline

and 20ml of red cell suspension.

Inhibition was recorded if previously positive Haemagglutination became negative

or if strong HA (2+or + +) became weak (1 +or +) in the presence of sugar solutions:

3.6.0. HAEMOLYTlC ACTIVITY.

3.6.1. I'REPARATIUN OF CELL-FREE CULTURE FILTRATE (SUPERNATE).

A loopl'ul of' each o f h e pure isolates of the Aeromonas spccios was inoculated

into 5ml ol Brrri~l I I cm Inlusion (BHI) broth and incubated at 37.C for 18-24hours. 'The

broth cultures were thcn aseptically centrifuged at 3,000 revolutions pcr minute (rpm) for

15 minutes. The supernates were collected and later filtered through 0.45L,m pore size

membrane filters (Gelman Ann. Arbor MI USA) to remove any remaining ccllular

debris. The cell free filtrates were then collected into sterile bottles and stored in the .

freezer for use within 3 weeks. The cell free filtrate (supernate) constituted the crude

toxin.

3.6.2. PREPARATION OF RED BLOOD CELL SUSI'ENSION.

Suspensions of horse, sheep and human blood were repeatedly washed by mixing

aliquot amounts of each blood with an equal volume of phosphate buffer in a centrifuge

tube. *These were centrifuged at 3000 rpin for 10 minutes. The supernatants were

,a discarded and the deposits were,suspended with phosphate buffer mixed again with

phosphate buffer and centrifuged a second time. The process was repeated until the

supernatants became clear. The supernatants were discarded and the cell deposits diluted

to give a final 1 % erythrocytes suspension.

3.6.3. FIAEMOLYTIC ASSAY. The cell free filtrate (supernate) of each strain was

{ested for haernolytic activity in microtitrc Plates (Linbro Hamden Conn.); by incubating

1WPl of two fold scrial$dilutions of each ccll free filtrate (supernate) with an equal

volume of each of the following 1 % washed erythrocytes, sheep, horse and' human

respectively. The trays were sealcd and left at 374' to incubate for one hour, followed

by another 1 hour incubation at 4oC and thereafter examined for haernolytic activity.

Culture supernates causing greater than 50% lysis on visual examination were

scored as being haenlolytic and the greatest dilution of h e culture supcrnates causing

greater than 50% lysis of the respective erythrocytes was then taken as the. haenwlytic

titre. One haemolytic unit per millilitre (HlJIrnl) therefore was defined as the reciprocal

value of the highest dilution of culture supernates which lysed a1 least 50% of the

respective erythrocytes (1 %vol/vol). Each culture supernatc was assayed in duplicate

with positive and negative controls. The positive control was made up of 200P1 of each

washed erythrocytes with few crystals of saponin (100% lysis). The negative coi'trol

containkd 10OP1 of phosphate buffered saline instead of the culture supernate and an equal

volume of the respective washed 1 %rbcs.

3.7.0 DETECTION OF CYTOTOXIN

The experiments were carried out at the National Veterinary Research Institute

Vom, in Plateau state of Nigeria, where tissue culture facilities exists. All samples were

transported to this location, chilled in a cold box. The lack of Tissue culture facilities at

the University of Nigeria Nsukka campus, necessitated the transportation of these culture

supernates to Vom, where Tissue cultures are carried out routinely and regularly. Thc

detection and assay of cytotoxins werc carried out by the "Tissue Culture Technique"

using Afiican Green Monkcy Kidncy (VERO CELLS) as cell lints.

3.7.1. PREPAKA'I'ION OF CELL LINES (MONO1 ,AY ISK)

A total count of Vero cells was carried out using a Neubaeur counting chamber

and staining with 1 % trypan blue. Dead cclls picked up the stain, while viable cells

remained opaque, thus allowing for differentiation. Having determined the total cells per

ml, the cells were then diluted to contain 100000 (10') cells per ml, with the growth

medium. The growth medium used was EARLE'S MODIFIED EAGLES MEDIUM with

25ml HEPES.(Whittakcr M.A. Bioproducts Walkersville, MD) supplemented with fetal

bovine serum 10% VIV., 2mM glutamine, penicillin solution to contain 100 international

unitslml. and streptomycin to contain 100l,g/ml. 1.0ml volume of fungazone was'also

added to'prevent fungal growth. The cells were then finally seeded either in l00ml

amounts in Roux bottles or in 50ml amounts in large medical flat bottles or in l0ml

amounts in small plastic bottles called FALCONS. The bottles were then incubated at

37oC (laid flat in the incubator under carbon dioxide (co,) so that the cells can adhere to

large flat surface area) for 24 hours; and checked 8 hourly under the microscope, until

there was good confluent growth. This now constitutes the PRIMARY CELL LINE also

known as the PRIMARY MONOLAYER CELL-LINE.

3.7.2.. METHOD OF TOXIN DETECTION (Tissue culture Technique) The culture

plate was checked for purity and good confluent growth, undcr the microscope before

use.The growth medium was decanted or aspirated off and trypsin added to separate and

detach the cells before incubation for few minutes. After incubation, the trypsin solution

was poured off and cells counted fresh growth medium was added to the bottle to make

a total concentration of 10" cells per ml. The cells were observed under the inicroscope to

confirm that they were still confluent. The cells were dispensed into a 96 multi-well

plastic tissue culture plates (FALCONS-Linbro chemical Co., Inc. conn. U.S.A) in 10OP1

amounts. The plates were incubated at 37oc for 24 hours carbon-dioxide (co,) and

checked for confluent growth 8 hourly. The growth medium was poured off, once there . .

was good confluent growth.

Preliminary tests on the culture filtrates were carried out by the addition of 50L,1

(micru litres) of each culture filtrate into duplicate wells. Also 50P1 of the maintenance

meclium (HANKS-E. MEM) was als0 added into the wells from a multi channel pipette,

thereby making a two fold dilution. The multi well plates were then incubated under co,

at 37oC and checked 8 hourly, for 24 hours for any ACTINOMORPHIC cell changes or

CYTOPATHIC EFFECT. This indicates the presence of cytotoxin, thus a cytotoxin

Positive culture supernate (plate 1).

3.7.4 CYTOTOXIN ASSAYS. Culture filtrate (supernate) showing cytopathic effect

with Vcro cells wcrc further assayed by carrying out doubling dilutions in the growth

medium in 5Vp1 anwunts, as shown in table (16) From each dilution, 50P1 was ren~oved

and added to a corresponding well, containing nlonolayer cells, after pouring off the

growth medium in the well. The plates were then incubated at 37oc in a co,. gassed

incubator Two controls wells were set up

(a) An uninoculated monolayer cell well and a well containing plain filtered 1:2 dilution

of Brain Heart Infusion Broth. The titrations were left in the incubator at 37oc under co,

The titrations were then examined aftcr 24hours incubation for cytopathic effect or

actinomorphic changes. Titres were read as the highest dilution of the culture supernate,

to exhibit greater than 50% cytopathic effect i;e to cause the rounding and detachment of

at least 50% of the monolayer cells (exhibition of intracellular vacuolisation or b

actinomorphic changes).

Great care was taken to distinguish the cytopathic effect from any degenerative

changes obs~rved in the negative control.The 1:2 dilution of the Brain Heart infusion

Broth in the second control well, excludes any extraneous toxic effect of the medium used

in the extraction of the culture supernates from the organism. The first control well,

which is the monolayer cell control shows any degenerative changes due to ageing

monolayer

cells tend

rnonolayer

cells, that could be confused with a true cytopathic effect. Ageing monolayer

to exhibit Salsc cytopalhic 4ccts which arc degenerahc cllailges of the

cells that tend to mimic true cytopathic effect.

3.8.0. THE RABBIT INTESTINAL LOOP TEST.(ILEAL LOOP TEST

3.8.1. Preparation of Animals (~abbits1;- The rabbits used for this test werc young adult

rabbits weighing between 1.5-2.5 kilogrames, bought at Ibagwa market in .Igbo- Eze

Local Gcwermnent area of Nsukka. The rabbits were kept for 3 days in cages in the

laboratory, in order to get acclimatized to the room temperature and the laboratory

environment. They wcre well fed and given plenty of water supply. However, the rabbits

were deprived of food but not water during 24 hours, prior to the opening of their

stomachs.

The gastric region of each rabbit was clean shaved and swabbed with methanol

befbre incision. Each rabbit was anaesthesised by injecting lml of KETAMINE-

HYDROCHLORIDE (Quads Pharmaceutical Inc. W. Germany) by intramuscular route.

The gastric region was incised with a sterile scapel blade and opened to expose the

intest~nes. Beginning near the ileocaecal junction, loops were tied with a sterile silk

thread, at intervals of approximately 3cm. Usually 8-10 loops were made in each rabbit

arid two rabbits were used for each supernate assay.

3.8.2. 'The lest proccclure (ILEAL LOOP TEST).

Each culture filtrate (supernate) was injected into each loop in duplicate rahhits, in

lrnl amounts using a sterile 21111 syringe. Loops were tied tightly once again at the site of

injection o f the culturc supernatc to further prevent the supernate from oozing out.

A negative control consisted of linl sterile filtrate of Brain Heart Infusion (BHI)

broth injected into one of the loops in each rabbit. The gut was replaced into its cavity . .

and the rabbit stitched up. The two rabbits were carefully laid in their cages and left for

16 hours.

After 16 hours, the rabbits were opened up again after killing them with an over

dose of the Ketamine-hydrochloride anaesthesia. The reactions in the loops were noted

with particular reference to the accumulation of tluid and distention . The length of each

loop and the volume of fluid content were measured to determine the tluid volume per

unit 1ength.Fluid accumulation in any of the gut segments between loops (spcial loops)

or in the negative control loop invalidated the results.

However, the presence of toxin was indicated by the prescnce of tluid

accumulation in the loops equal to or greater than 1 .Oml/cm.

The DILATATION index (DI) therefore is expressed as a ratio of the fluid

accumulated and the length of the loop (i.e volume + length ratio). This is used as the

index of enterogenicity (Ljungh and Kronevi 1982).

3.8.3 'I'HE SUCKLING MOUSE TEST 1WK ENTEKOTOXIN.

Each culture free tiltratc was tested in 2 (2 - 6 days old) mice for enterotoxin.

of methylene blue dye (0.5gn1) was dissolvcd in l01nl of each filtrate (0.05%). Aliquots .

of 10OP1 of dye hltcrate mixture was given to each of the two suckling mice

intragastrically with a tuberculin syringe A negative control consisting of 1001,1 sterile

Brain Heart Infusion broth containing 0.05% dye mixture was also innoculated into two

mice. The mice were placed on a sheet of blotting paper in a partitioned metal cage,

with not more than two mice in each compartment and incubated at room temperature

(22-24"~) for 3 hours, following the administration of the dye-filtrate mixture.

After incubation, the inice were killed by cervical dislocation. Gastric distension

and'the presence of dye in the small intestine were noted, before the removal of the entire

gut. Intestinal weight (IW) and the remaining body weight (BW) were measured for each

group of mice. The ratio of 1W)BW > .085 is regarded as positive. (Champsaur gt a

1982).

3.9.0. GEL FILTRATION ANALYSIS

3.9.1. CONCENTRATION OF THE CULTURE SUI'ERNATES: A 25ml of each

culture supernate was carefully poured into a dialysis bag and left in a 4M sucrose

solution overnight, to concentrate the proteins. The dialysis bags were then cut

opcn a1 one end and [he conlcnts poured into a sterile bottle. The concentrated

supernates were also tested for activities in rabbits.

3.9.2. GEL SEPARATION. Having confirmed their activities in the rabbit ileal loop

model they were then ready for fractionation by column chromatography, using sephadex

G-50 of dimension, 1.5 x 19cm. The material was eluted with 0.02M phosphate buffer,

pH7.0, maintaining a flow rate of 20mllhr and collectirrg 3-ml fractions.

Buffer used in eluting protein = -0.02m phosphate buffer pH 7.0.

Column dimension - - 1.5 x 19cm.

Gel used = Sephadex G - 50

Flow rate

Fraction size - - 3mlltube.

3.9.3. DETERMINATION OF PROTEIN CONCENTRATION. The concentration of

each protein fraction was determined using Bradford (1976) method, with bovine serum

albumin (BSA) as a standard.

Bradford reagent was prepared by dissolving 100 mg of Serva blue (Coomasie

Brilliant Blue G - 250) in 50 ml of 95% ethanol). The solution was mixed with 1001111 of

85% phosphoric acid and made up Lo 1 litre with distilled water. The solution was filtered

with filter paper before storage in an ainher bottle at room temperature.

Bovine serum albumin (USA) at a concentration of 1mg.inl. in distilled water was

used as a standard. A standard curve was prepared in thc concentration range of

To test for protein, 5inl of the Bradford reagent was added to 0.11~11 of sample and .

mixed gently. A reagent blank containing distilled water was also prepared. The

absorbanceuf the samples was measured spectrophotometricaIly a1 595 nm within 2 mins

, to 1 hr of mixing.

3.9.4. ESTIMATION OF MOLECULAR WEIGHT.

The relative molecular weight of the toxin was estimated by gel fil'tration

chromatograph on a column (1.5 x 19 cm) equilibrated with 0.2M phosphate bull'er (pH

6.8.) This was determined by comparing the toxin elution volume with those of series of

proteins oC known molecular weights.

Protein markers consisted of Myoglobin (15,850), Trypsin (31,600) Bovine serum

albumin (63,000), Lactate-dehydrogenase (1 58,500) Pyruvate Kinase (25 1, 158).

The protein markers and the purified toxin were eluted with 0.2M phosphate

buffer (pH 6.8) in which 3 ml fractions were collected at the same flow rate. Protein

markers were dctected by measuring the absorbance at 280 nm. with a

spectrophotomcter. The molecular wcight of the purificd toxin was calculated from plot

of elution volume against logarithms of the molecular weight of the protein markers.

3.10. ANTIBIOTIC SUSCEPTIBILITY TESI'S.

A Modification of the disc diffusion method of Kirby- Bauer &a\. (1966) was employed.

The discs of the drugs used and their concentrations are given below.

(1) Ampicillin 10k,g (2)Amoxycillin 10Pg (3)Ampicillin + Sulbactam (Unasyn)

30mg.(4) Gentamicin 1 OPg (5) Cotrimoxazolr 25L,g. (6) Chloramphenicol 10Pg (7)

Amoxicillin +clavulanic acid( Augmentin) 30Pg. (8) Te~racyline 10Pg

(9) Cefuroxime (10) Ciprofloxacin (Cipro~in)5~g (11) mfloxacin (Tarivid) (12)

Tobramycin (5@.

The strains tested were defined or classified as SUSCEPTIBLE or RESISTANT,as

recommended by Barry and Thornsberry (1985). Mueller Hinton agar was used as the

medium for the diffusion test.

3.10.1: Test PROCEDURE. Three to four pure colonies of an overnight culture were . .

picked and emulsified into about 5 ml of peptone water. A sterile clinical -swab was

soaked in this peptone water culture; and lightly and uniformly streaked on a Mueller

Hinlon agar plate all over the plate. With the aid of a sterile forcep, each disc of the 12

antibiotics was picked and carefully placed on the plate, being careful not to crowd them

together. The plates were incubated at 37.C overnight.

3.10;2 DETECTlON OF BETALACTAMASE ACTIVITY. The prcsencc of

behlactamase activity was detected according to the modified mcthvd of Escainilla

(1976). as follows:

3.10.2.1 Preparation o f Buffered potassium penicillin G-phenol red. (indicator solution);-

This was prepared by adding 10 ml of sterile distilled water and 1.5 in1 of 0.5%

phenol red solution, to a vial containing 50,000 units of penicillin G (buffered potassium

penicillin G). Sodium hydroxide (1 ml) was added drop wise (approximately 0.5 ml) until

the solution just turned violet (pH 8.5). This then became the stock solution. The stock

solution was dispensed in tubes and if not immediately used, were stored at -20.C.

3.10.3. Test procedure.

Modified Tube test (Escamilla 1976). The test was carried out by dispensing 0.5

ml of Buffered potassium penicillin G phenol-red indicator solution in small test tubes in -

a rack. Using a sterile wire loop, a loopful of each of the test strains from blood agar

plate was emulsified in the 0.5 ml solution, contained to the test tubes and incubated at

room temperature for 1 hour. Strong betalactamase producers gave an instantaneous

YELLOW colour reaction. Some gave in 5-20 minutes while weak reactions took up to

30-60 minutes, before Yellow colour was produced.

('HAWER 4

RESULTS

4.0. PREVALENCE OF AEROMONAS IN FAECAL SAMPLES RESULTS:

Out of 1150 specimens of stools from diarrhoea1 and non diarrhoea1 subjects examined,

157 (13.6 %) yielded Aeromonas species

hydrophila (7.7%) was the commonest,

(2.2 %) respectively (Table 3).

(Table 2). Of the 157 Aeromonas species, A

followed by A sobria (3.8%) and A caviae

SPECIES IN THE ENVIRONMENT.

Out of 550 environmental samples examined, 50(9%) A(wmonas species were isolated

(Table 2). Of the 50 strains isolated 25(50%) were A. hydrophila 20(40%) were A.

sobria

and 5(10%) A. caviae. (Table 3).

The distribution from Aeromonas species from the environmental samples is shown in

table 3.

4.2 AGE DISTRIBUTION OF AEROMONAS FROM HUMAN STOOLS:

(DIARRI IOEAL AND NON-DIARRHOEAL)

Out of 764 specimens of diarrhoeal stools examined, 137 (17.9%) Aeromonas species

were isolated (Table 4). And of the 386 non diarrhocal stools examined, 20 (5.2%)

Aeroirionas species were isolated (Table 5). There is a highly significant difference

(FSLD = P = 0.01) in the prevalcrice of Aeromonas in diarrhoeic (mean 1 8 , b . ~ )

compared to (mean 6.8%) prevalence in non diarrhoeic subjects. Similarly the results of

the statistical analysis showed a significant (P=O.05) drop in the prevalence of

Acromonas from 18.6% ill subjects less than or~e yea] old to 4,8 % in sub.jccts of 20 years

and above. Children under 3 years old had thc highest prevalencc ol Acro~iiolias, both in

the diarrheal and no11 diarrhoea1 groups (Tables 4 and 5 ) . The prevalence ol' Aeromonas

in diarrfloeic stools of neonates and infants (less than lyr. old) was highest Ihllowcd by

that in children 2-3 years old.

In tht: non diarrl~oeic stools children between 2-3 years old had- the highest

prevalence (12.7%) followed by those in thc agc group 4-10 and 11-20 ycars old (1.9%)

respectively (Table 5 ) .

4.3 SEX DISTKIBUTION OF AEROMONAS IN DIARRHOEICI NON

DIARRHOEIC STOOIS.

Out of 400 diarrhoeic males examined, 80 (20%) yielded Aeroinonas species while

with their feinale counter parts 57 (15.7%) yielded Aeromonas species out of 364

diarrhoeic female subjects examined.. In the non diarrhoeic group, out of 200 males

examined, 12 (6%) yielded Aeromonas species and 8 (4.3 %) out of 186 female subjects

whosc stools wcrc examined, also yieldcd Aeromonas species (Table 6).

4.4. DISTRIBUTION OF FAECAL ISOLATES OF AEROMONAS SPECIES

WITHIN THE 4 YR. PERIOD (1992 - 1995).

The yearly distribution of Aeromonas species based on the frequency of isolation

from stool specimens as recorded over a period of 4 years 1992 and 1995 respectikly is

shown in fig.2.

4 .5 . SEASONAL VARIATION OF AEROMONAS SPECIES (1992 - 1995)

The seasonal distribution of Aeromonas for the period (1992 - 1995) under

investigation is shown in fig. 3. As it is apparent, the isolation of Acromonas species .

fjyd- increased progressively from the month of January to the highest or peak level inAand

d e & e d - to the lowest level in the month of December; i.e. the isolation

rate 'wasahighest during the period of mid rainy season (June to August) to early dry

season (fig. 3)

4.6. AGE AND SEX DISTRIBUTION OF AEROMONAS FROM DIARRHOEIC

STOOLS:

Males in the age group 10-20 years old had the highest prevalence rate of 22

(16%) out of 137 isolates while their female counter parts in the same age range had only

15 (1 1 %) isolates. Stools of diarrhoeic rnalcs >20 yrs old had the least prevalence rate of

6 (4.4%) out of 137 while their female counter parts had only 3 (2.2%) isolates (fig. 4).

4.7. OTHER ENTERIC PATHOGENS ISOLATED FROM SAME SPECINIENS

DURING THE PERIOD.

Aeromonas had the second higliesl frequency of recovery as a sole pathogen from

68 (5.9%) out of 1150 stool specimen, whilc Escherichia coli had the highest as ,a sole

pathogen and was isolated from 79(6.9%) out of 1150 stool specimens examined (Table

7). Salmonell+ and Shigella species were also recovered as sole pathogens in 61 (5.3%)

and 2 1 (1.8 %) specimens respectively. The occurrence of the other enteropathogens are

listed in table 7. The current study reports the occurrence of Aeromonas-associated -

gastro-intestinal disease to be fairly more common than that of Salmonella or Shirrella..

Table 2

Isolation of Aeromonas from I w m l specimens and environmenlal sa~nples.

Source of specimen No of samples No positive for Percentage

examined Aeromonas occurrence

Faeces 1150

Environmental 550 v

Total 1700

Table 3

The percentage occurrence of Aeromonas species in Faecal specimens and Environmental

samples.

-- -- -- - -- - -.

Source and specimen No of Isolates Percentage occurrence of different

species of Aerornonas. (%)

A hvdrophila - -- A sobria -- A caviae

faeces (1 150) 157 (13.7) 88 (56.1)

Environiriental (550) 50 (9.1) 25 (50) 20 (40) 5 (10)

Total 1700 207 (12.2) 96 (46.4)

Table 4

Preyalence of Aeromonas spp in diarrhoeic stools o f various age groups.

Age groups in yr. No of'diarrhoeic stools No of isolates % prevalence exanlincd

- -

< I 89

2-3 151

4- 10 190

1 1-20 230

2 21 1 04

Total 764

Table 5 -- --

Prevalence of Aeromonas in Non diarrhoeic stools of various age groups.

Age groups in yr. No of non diarrhoeic stools No of isolates % prevalence

-. examined.

L

< I 48 6 12.5

> 21 . .

105

Total 386

'I'ablc 6

. . Sex distribution of Aeromonas isolates in diarrhoeicINon diarrhoeic stools.

DIARRHOEIC -- -

NON DIARRHOEIC

Male . Female Male Female

No of Subjects 400 364 200 186 examined

No Aeromonas 80 57 12 8 Positive

% occurrence 20 15.7 6 4.3

Fig. 1 A simple scheme for the preliminary identification and diffcrcntiation of Aeromonas from Plesiomonas.

Suspicious isolate of Aeromonas or Plesiomonas

Test for Oxidase

-ve

Discard t-----------

Tesl for

( ( 1 ) Greenisli pig~mc~itntion 011 NA.

+ VC: { (2) Growth in 6.5% Nud broth.

Discard 4- { ( 3 ) Acid from Xylose, dulcitol and adnitol

(4) 1 IZS or IJrease Production

Test for acid from ( I ) Manitol (2) Innosirol

Mannitol +ve Inositol -ve

Mannitol --ve Inositol +ve

Aeromonas species Plesiomonas shigelloides

UJA. hydrophila

H A sobria

, DA. cavia

1992 1993 1994 1995

Year

Fig. 2: Distribution'of isolates of ~eromonas for the 4yr period (1992-1995)

Jah Feb Mar Apr May Jun Jul Aug Sep Oct Nov

Months

Dec

Fig 3: Mean monthly distribution of Aeromonas spp. isolated from human faeces during the period (1 992-1 995)

UD female

1 male

Year Fig. 4 :Distribution of Aeromonas isolates from diarrhoea stools according to age and sex

Table 7 Enteric pathogens isolated from 1150 faecal specimens examined during the period (1991-1995).

Species isolated Total No Percentage No of isolates No of isolates

of OCCLI rre11ce recovered as sole (%)recovered as

isolates pathogens. ( % ) mixed

pathogens.

Escherichia coli 182 14.7 79 (6.9) 103 (9%)

Aeromonas sp . 157 13.6 6s (5.9) 89 (7.7)

Salmonella sp. 7 8 6.8 61 (5.3). 17 (1.5)

gella la sp. 37. 3.2 21 (1.8) 16 (1.4)

Entamoeba 106 9.2 83 (7.2). 23 (2)

llistolytica

Entamoeba coli 63 5.5. 41 (3.6). , 22 (1.9)

.J Girdia lamblia

A 39 3.4 24 (2.10). 15 (1.3).

Cwptosporidium 3 1 - 2.7. 23 (2) 8 (0.7)

Hookworm 2 8 2.4. 17 (1.5) 17 (1.5)

Ascaris lumbricoides 15 1.3 6 (0.52). 9 (0.8).

4.8. PREVALENCE OF AEROMONAS AS SOLE ENTER0 - PATHOGENS.

Out of 1150 subjects whose stool speci~r~c~is were exarni~lcd 68 (5.9%) yiclded

Aeromonas species as cnteropathogens. Of the 68 Aeromonas isolated 45 (66.1 %) were

A hydrophila, 2 1 (30.9%) we1 e A. sobria and 2 (2.9%) A. caviae respectively (Table 8). -

4,.9 DISTRIBUTION OF AEROMONAS SPECIES IN DIAKRHOEIC AND NON

DIARRHOEIC SUBJECTS.

Out of the 137 isolates obtained from diarrhoeic stools, 81 (10.6%) were A

hvdrophila, 31 (4.0%) were A sobria while 25 (3.3%) were A caviae. While in the non

diarrhoeic stools, the distribution of the various species was viz. A hydrophila 7 (1.8%),

A sobria 13 (3.4%) and non for A caviae (Table 9). --

4.10. HAEMAGGLUTINATION ACTIVITY OF AEROMONAS SPECIES FROM a

DIARRHOEIC I NON DIARRHOEIC STOOLS

Th6 haernagglutination propertics of the Aero~nonas isolates from both diarrhoeic

and non diarrhoeic stools are shown in tables 10 and 11.

Table 8

Distribution of Aeromonas species isolated as the wle pathogen from the stools of

patients.

No iso1ated.a~ sole pathogen % Occurrence

A. caviae -- 2

Total 68

Table 9

I

Aerornonn~ species isolated from diarrhocic and normal healthy subject (Non diarrhoeic) ---

'Type of subject - A. hvdrophila -- A. sobria -- A , caviae

Diarrheic (11 =764) 81 (106) 31 (4.0) 25 (3.3)

Non diarrhoeic (n = 386) 7 (1.8) . 13 (3.4) wo)

'Total 1 150 88 (7.7) 44 (3.8) 25. (2.2)

Haemagglutination diarrhoeic stools.

of human group

Table 10

0 red blood cells by Aeromonas -- isolates from

-- --

Aeronmias species No of isolates tcstcd HA poslc~ve Nu Percentage positive - . -- - -- -. .

8 1 A hydrophila 5 1 0 3

A. sobria -- 3 1 15 48.4

A. caviae -- 25 8 3 2

Total 137 74 54

Table 11

Haenlagglutination (HA) of human group 0 red blood cells by Aeromonas isolates from Non diarrhoea1 stools .

Aeromonas spccies No of isolates tested No IIA positive Pcrcentagc positive % - -- -

A. hvdrophila -- 7 3 42.9

A. sobria -- 11 5 45.5

A . caviae -- 2 1 50.0

Total 20 9 45 .O

Out of 137 diarrhoeic isolates tested 74 (54%) were HA positive. 111 the non diarrhoeic

only 9 (45%) out of 20 wcre HA positive (P<0.05).

4.11. HAEMAGGLUTINATION OF ENVIRONMENTAL ISOLATES OF

AEROMONAS SPECIES.

Out of a total o f 50 isolatcs tcslcd Ibr IIaemagglutinalion, 30(60%) isolates

exhibited haelnagglutination properties. A hydrophila was HA positive in 16(64%), out of

25 isolates. A sobria was HA positive in 10(62.5%) out of 16 isolates, while A caviae

isolates was HA positive in 4(44.4%) out of 9 (Table 12).

4.12. HAEMAGGLUTINATION PATTERNS OF AEKOMONAS WITH

VARIOUS ANIMAL ERYTHROCYTES.

Figure 5 shows the HA patterns of Aeromonas with 4 different eryth~ocytes viz.

(1) Human (2) Horse (3) ,Sheep (4) Rabbit.

There were no obvious diffcrcnces in the haemagglutinating propcrties, of these

erythroates although human and horse erythrocytes even though human and rabbit

erythrocytes were slightly higher than Rabbit and sheep erythrocytes.

4.13. HAEMAGGLUTINATION INHIBITION ACTIVITY OF AEROMONAS

SPECIES.

It was observed that the haelnagglutination of Aeromonas isolates from both

diarrheoic and non diarrhoeic stools was inhibited by Glucose, Mannose and Galactose

i.c.were sensitive to h e named sugars.

Table 12

Haemagglutirlativn (HA) activity of environmental isolates of Aeromo~m.

Aeromonas spp. Testcd No. of isolatcs tested No. MA positivc % posilive

--- -- - - - - - - -- - -

A. hydmpll-i 12 2 5 16 64

A. sobria 16 10 62.5

A. caviae 9 4 44.4

Total 50 30 60

4.14 HAEMOLYTIC ACTIVITY 011' AEROMONAS ISOLATES FROM

DIARRIIOEIC STOOLS ON HUMAN CELLS

Out of 137 diarrhoeic isolates tested, 107 (78.1 %) exhibited Beta haemolysis, 2

(1.4%) exhibited alpha-haemolysis and 28 (20.4%) exhibited no liaemolysis. All the

'isulates of Mrophila 81 (100%) and a significant number of A sobria 26 (83.9%)

showcd beta hacrnolysis on blood agar, while none of the A caviae isotates exhibited bela

haeinolysis (Table 13). Howevcr 5 (16 1 % I ) isolates of A sobria wcre found to be non

haemolylic: on blood agar; Only 2 A caviae werc alpha haemolytic, the rest were non

haeinolytic on blood agar. When the haeniolysins were quantified A l ~ p l l i l a had ~ i ~ r e s

ranging from 64 - 512 Hacmolytic units while A sobria and the 2 alpha l~ae~iiolyric A

caviae had tilres ranging from 8 - 512 and neat 1 - 8 haemolytic units respectively (Table

4.15. HAEMOLYTIC ACTIVITY OF AEROMONAS ISOLATES FROM NON

DIARRNOEIC STOOLS ON HUMAN CELLS.

Beta Hacmolytic activity was exhibited in 5 (71.4%) of the of the 7 A hvdrophila and no

haemoly tic ac ivity in 2 (28.6%). The haeniolytic titres of A hydrophila in the non

diarrhoeic stools ranged between 4 - 128 haemolytic units. A sobria had 6 (46.1 %) beta

haemolytic isolates, and 7 (53.8%) lion haemolytic isolates on human blood agar. A

caviae was not isolated from the stools of non diarrhoeic subjects (Table 14).

Haemolytic activity of Aeromonas isolalcs from diarlllocic: s~uols, on llu111a11 rccl blood cells.

Acromorias &of b.-(%) (NU.) % No. (%I Haemol specles isolates showmg. alpha- haemolys~s

tested betahaemolysis haeinolysis 8:; - -- --

range ---

A. hydrophila - 8 1 81 (100) 0 0 64-5 12

A. sobs 3 1 26 (83.9) 0 5 (16.1) 8-5 12

A. caviae 25 0 2 (8.0) 23 (92) 1-8

Total 137 107 (78.1) 2 (1.4)

Table 14

Hamolytic activity of Aeromonas isolates fiom Non-diarrhoeic stools on human blood cells.

Aeromonas No. of No./(%) No. (76) Haemolytic No. (%) species isolates showing alpha- titre range

haemol ysis Non ksted beta- haemol ytic

haemolysis

A. caviae 0 '0 0

Total 20 11 (55) 0

4.16. HAEMOLYTIC ACTIVITY OF ENVIRONMENTAL ISOLATES -OF

AEROMONAS SPECIES ON HUMAN RED BLOOD CELLS.

Of the 50 isolates obtained from the environmental sources 39(78%) displayed bet:\,

haemolysis on blood agar 6(30%) displayed alpha haeniolysis and 5(10%) showed no . .

haemolytic properties. (Table 15). All the A hydrophila (100%) dispIayed beta

haernolysis, 14(70%) out of 20 A sobria isolales were beta haemolytic on blood agai-,

while thc remaining 6(30%) isolatcs displaycd alpha haemolytic activity. No~~to l ' Llie

5(0%) isolalcs of A caviae isolated exhibited any hacmolytic properties. When lhc

liaemolysis displayed by A hydrophila and A sobria were quantified, titres ranged from

4-128 HU (Haemolytic Units) (Table 15).

4.17. HAEMOLYTIC TITRES OF AEROMONAS SPECIES WITH VARIOUS

ERYTHROCYTES

The haemolytic titres of isolates of A e r o m o n ~ with different erythrocytea are shown in

figure 6. Whereas both human and horse erythrocytes responded almost similarly to

Aeromonas haemolysin, sheep and rabbit erythrocytes appeared to be lcss sensitive (fig.

Plate 1

Aeromorias hydrophila showing beta haemolysis on blood agar plate a

Table 15

Haemolytic activity of Environmental isolates of Aeromonas species.

No(%) showing

-- - - -- --- - --

Aeromonas No of isolates B-haemolys~s alpha hacmolysis No l~acmolys~s

spccics. lesled Titre range. . A . hydrophila 25 25 ( 100 %,). 0 0 4- 128

A . sobria --

A. caviae.

Total

Human cells Horse cells Sheep cells Rabbit cells Different RBCs Fig 5: Haemagglutination patterns of Aeromonas species with different types of erythrocytes.

' A hydrophlla

M A sobrra

O A cavlae

Horse Rbcs Human Rbcs Rabbit Rbcs Sheep Rbcs

Different animal Rbcs

Fig 6: Haemolytic titres of Aeromonas culture filtrate with erythrocytes of different animal species

4.18. CYTOTOM@ ASSAY OF CULTURE SUPERNATES ON VERO CELLS-

The cytotoxin assay was carried out on the culture free filtrates (culture supernates) as

shown in the protocol m. The result showed that 94 (68.6%) out of 137 from the

diarrhoeic isolates produced cytotoxin i.e. displayed cytopathic effect on Vero cells (plate 2

and table 17).

4.19. DISTRIBUTION OF CYTOTOXIN PRODUCING AEROMONAS SPECIES

IN DIARRHOEIC STOOLS.

Of the 81 diarrhoeic isolates of - A hydrophila tested for cytotoxin production, 72 (88.9%)

were positive i.e. displayed cytotoxin properties or showed actinornorphic changes on Vero

cells, with a titre range between 4 - 128 cytotoxin units.

A sobria and A caviae produced actinomorphic changes in 20 (64.5%) out of 31 and 2 -- --

(8 %) out of 25 isolates respectively. -- A sobria had titre ranges between 2 - 64 and A caviae

had titres from 1 to 8 cytotoxin units (Table 17).

4.20. DISTRIBUTION OF CYTOTOXIN PRODUCING AEROMONAS SPECIES

IN NON DIARRHOEIC STOOLS.

Out of 20 isolates from non diarrhoeic stools 10 (50%) showed cytotoxic activities (Table

18). A. hvdrophila had 4 (57.1 %) positive with a cytotoxic titre range between 2-32. A

sobria had 6 (46.2 %) cytotoxin producing isolates out of 13. There was no A. caviae

isolated from the non diarrhoel stools.

4.21. DISTRIBUTION OF CYTOTOXlN PRODUCING STRAINS FROM THE

ENVIRONMENT.

Cytoroxic effects were produced by 3 l(62 %) out of 50 Aeromonas species isolated from the

environment. A hydrophila displayed cytotoxic effects in 19(76%) out of 25 isolates, with

titre range between 2 - 680 cytoxin units. A. sobria exhibited cytotoxic effects in 1 l(55 %)

out of 20 with titre ranges between 1 to 160 cytotoxin units; while A. caviae exhibited

cytotoxin effects only in 1(20%) and with titre range between 1 - 160 cytotoxin units with

a titre of 40 cytotoxin units (Table 19).

4.22. ENTEROTOXIN PRODUCTION BY CULTURE FREE SUPERNATES

FROM DIARRHOEIC STOOLS: IN THE RABBIT ILEAL LOOP.

Out of the 137 culture filtrates of isolates from diarrhoeic stools tested for enterotoxin

production, 97 (70.8 %) showed enterotoxic activities i:e fluid accumulation in the distended

ligated ileal loop (plate 3). A hydrophila had 70 (86.4%) positive strains out of 81 isolates,

A . sobgia had 25 (80.6 %) positive out of 3 1 isolates, while 2 (8 %) out of 25 A caviae

culture filtrate were positive for enterotoxin production. A positive enterotoxin (Table 20)

production occurs when there is fluid accumulation in the ligated ileal loop > lml/cm

(volume/length ratio). or dilatation Index = length of the ileal loop in animal I and I1 over

volume of fluid accumulation I and II > 0.5.

4.23. ENTEROTOXIN PRODUCTION BY CULTURE FILTRATES

(SUPERNATES) OF NON DIARRHOEIC ISOLATES:

Of the 20 non diarrhoeic isolates, culture frltrate of 13 (65 %) produced enterotoxin i:e fluid

accumulation distended the ligated ilea loop. Out of 7 A hvdrophila tested 3 (42.9%)

produced enterotoxin, while 10 (76.9 %) out of 13 A sobria, exhibited fluid accumulation

(table 21) A caviae was not isolated in the non diarrhoeic stool. Enterotoxin production =

Fluid accumulation > 1 rnlcm (Volumellength ratio).

PLATE: 2

Showing uninoculated Vero ccll line

Plate 3

Showing greater than 50% cythopathic effect on Vero cell line, by cytoxin positive

supernate: of Aeromonas species.

Table 14

Cytotoxic activity of culture free filtrates of Aeromonas spp. from diarrhoeic stools. '

Aeronionas species

N o tested for N o cytoroxin positive % positive 'I'ILI-c range. cytolox~n

-- - -- ---- - -

A caviae 2 5 2.0 8.0 1-8

Total 137. 94 68.6 -

On the whole, 94 (68.6%) of the 137 diarrhoeic isolates exhibited cytotoxicity.

Table 17

Cytotoxic activity of culture free filtrates (supernates) of Aeromonas spp. from stools of

. Nor~Iiarrl~ocic subjects.

Aeromonas species

No tested for cytotoxin

No cylotoxin positive

% positive Titre range.

A caviae

Total 20 10 50.0 0 '

A hvdrophila had 4 (57.1 %) positive with a cytotoxic titre range between 2 - 32. A sibria had 6 (46.2%) cytotoxin pmducing isolates out of 13. There was no A caviae isolated in the non diarrhoel stool.

Table 18

Cytotoxic effects of culture free supernatcs of environmental strains of Aeroinoilas on Vero cells.

Environmental No tested for No positive for % positive Cytotoxin titre strains. cytotoxin cytotoxin range.

. p--.--pp--,

A hvdro~hila 2 5 19 76. 2-640

A sobria -- 20 1 1 5 5 1-160

A cavaie --- - 5 1 stdl 40

Total. 50 3 1 62 -

Table 19

Enterotoxic activity of culture frcc I'iltratcs (supcrnatcs) o f diarrhoeic stools on rabbit ileal loop.

species of, Aeromonas

No of strains examined

- - - - - - - - -

No Enterotoxin Percentage positive occurrence.

A sobria

A caviae

Total.

Foot note: Volume of fluid accumulation in animal I and IT, over length of thc ileal loop in animal I and 11 > 1 = positive, i.e fluid accumulation > /ml/cm (volume-length ratio) = Dilatation index.

Plate 4 Showing fluid accuinulation and distention of the ileal loop (Positive reaction).

Table 20 Enterotoxin activities of culture free filtrates (supernatcs) of Acromonas spp. isolates from non diarrhoeic stools on rabbit ileal loop.

Aeromonas species. No of strains tesled No Entcro~oxin Percentage posilivc. occun-encc

A hvdrophila - 7 3 42.9

A. sobria 13 10 76.9

A. caviae. - 0 0 0

Total 20 13. 65 Fluid accumulation > lml/cin (volume/length ratio) = positive.

4.24 ENTEROTOXIN PRODUCTION BY CULTU

(SUPERNATES) OF ENVIRONMENTAL ISOLATES.

Of the 50 culture supcrnates of the enviroimental isolates tc

RE FILTRATES

. .

:sted for enterotoxin

production, 33(66%) showed positivc results by exibiting fluid accumulation that

distended the ligated ileal loop.(Table 22). 18(72%) out 25 isolates of A hvdl-ophila were

positive lor cytotoxin production. A sobria showed positive cytotoxin production in

14(70%) out of 20 culture supernates tested. while 160%) out of 5 isolate of A caviae,

showed tluid accumulation i.e enterotoxic activity. (Table 22).

Enterotoxic activities of culture free filtrates (supernates) of environmental Aeroinonas isolates. on rabbit ileal loop.

-- -- -

Acron~onas species. No of strains teslcd No Enterotoxin Percentage positive.

-- occurrence.

A hydrophila - 25 18 72

A. sobria 20 14 70 --

A. caviae. - 5 1 20

Total 50 33 66

Fluid accumulation 2 lnlllcm (volume/length ratio) = posilive

4.25 FLUID ACCUMULATION IN RABBIT ILEAL LOOPS BY CULTURE

FREE FILTRATES (SUPERNATES) OF AEROMONAS SPECIES.

Out of the 68 isolates obtained as sole enteropathogens in diarrhoeic subjects, 45(66.l%)

were A hvdrophila. Of these 45 isolates, 35(77.7%) produced fluid accumulation with'a

volume between 1-2mls and 10(22.2%) produced greater than 2mls of fluid. A sobria

produced 1-2rnl fluid accumulation in 15(71.4%) out of 21 isolates and 6(28.6%)

produced greater than 2mls of fluid. All the 2(100%) A caviea isolates produced fluid

within 1-2ml volume range (table23).

Enterotoxic activity of Aeromonas rabbit ileal loop method.

Table 22

species recovered as SOLE enteropathogen using

Aeromonas species No of isolated tested No Enterotoxin I'ercentage positive occurrence

- " " " ...... " ..... " .. . "" ...

A. caviae.

Total

4.26. ENTEROTOXIN PRODUCTION BY TIIE SOLE ENTERTC PATIIOGENS

USING THE SUCKLING MOUSE TECHNIQUE.

The 68 Aeromonas species isolated as sole entero pathogens from the diarrhoeic stools

were also tested for enterotoxin production using the suckling mouse technique. 01' these,

66(97%) produced enterotoxin with ratio of intestinal weight of mouse, to remaining

body weight >0.085, which is taken as positivc. (Chanq~saur ct 211 1982).

All the 15(100%) A hydrophila isolated as sole entcl-opathogen produced enterotoxin.hy

this method. Out of 21 A sobria, 20(95.2%) also produced enterotoxin by this method,

while 1(50%) out of 2 A carviae produced enterotoxin (table 24).

Table 23

Fluid accuinulation in the rabbit ileal loop as a result of the culture 1'1.ee filtrates of Aeromonas isolates recovered as SOLE pathogens from stools.

Species of Aeromoncrs No ~c\ted for ei~terotoxiil Fluid accumulation (Volume)

vOl.(l-21111) VO! (> 2-2.51111)

A. hvdrophila - 45 .

A. sobria -- 2 1

A. taviae. 2

Total 68

Vol = Volume 1 + Vo1. 2 = Mean volume of accumulation in the ileal loop 2 (> lml)

Vol. 1 and Vul. 2 refer to the accumulated volumes in the ileal loops of the two rabbits mted for each culture filtrate.

4.27. ENTEROTOXIN PRODUCTION BY THE POTENTIAL SOLE ENTERIC

PATHOGENS USING TIIE RABBIT IIXAI, 1,OOP METHOD.

All the AEROMONAS species isolated as sole enteric pathogens from the diarrhoeic

stools, produced enterotoxins as detected by the rabbit ileal loop method; i:e distention of

the ligated loop or the accuinulation of fluid> lmllc~n (volume1 length ratio). This result

shows that enterotoxin production like the cytotoxin and haemolysin production are

produced more in diarrlloeic stools P > 0.05 (table 23).

4.28 RELATIONSHIP BETWEEN THE VIRULENCE DETERMINANTS OF

AEROMONAS SPI'. FROM DIARRHOEIC\NON DlARRHOEIC FAECAL

ISOLATES.

The haemolytic activities of the diarrhoeic isolates showed similar pattern with the non

diarrhoeic isolates (Table 26). However, the haemolytic titres were higher with the

diarrhoeic isolates(titre of 512 as against 6 4 ~ e ) . Similarly, the cytotoxin production

were slightly higher in the diarrhoeic group than in the non diarrhoeic. A h~drophila in

the diarrhoeic group produced cytotoxin in 72(88.9%) as against 5(71.4%) strains in the non-

diarrhoeic group. The frequency of cytotoxic activity of A sobria in the diarrhoeic strains

(64.5 % and 69.2 % respect/vely P > 0.05) (tables 23 and 26).The enterotoxin

production was higher in the diarrhoeic group 86.4% for A hvdrophila, 80.6% fo; A

sobria, as against 42.9% and 69.2% for A hydrophila and A sobria respectively in the

non diarrhoeic isolates. The haemagglutination activities were also higher with tlic

diarrhoeic isolates (63%,48.4% and32% for a A hvdrophila, A sobria and A caviae

respectively) than with the non diarrhoeic isolates (42.9% and 46% for A hvdrophila, A

sobria respectively) .(tables 25 and 26)

4.29 THE VIRULENCE DETERMINANTS OF AEROMONAS SPP. ISOLATED

AS SOLE ENTEROPATHOGENS.

The results of the present study show that Aero,onas species isolated as sole

enteropathogens expressed higher percentage of virulence factors. Hae~uolysiri was

produced by all the A hydrophila (100%) and A sobria (100%) isolated in diarrhoeic

stools as sole enteropathogens. Cytotoxin production was similar to the hacmolysin

production. The two species (A hydrophila and A sobria) isolutecl as sole enteropathogens

produced cytotoxin. (Table 27 and 28). All the species isolated as potential sole

enteropathogens also produced enterotoxin.

Table 24

Enterotoxic activity of the culture filtrates of Acromonas isolated as SOLE pathogens in faeces, using the suckling mouse technique.

Aeromonas species No of isolated tested No Enterotoxin Percentage positive occurrence:

-- - - -.

A hydrophila - 45 45 1 00

A. sobria 21 20 95.2

A. caviae. - 2 1 50.0

Total 68 66 97.0

Intestinal wt of mouse > 0.085 = Positive. Remaining body wt

Table 25: Frequency of the virulance determinants of Aeromonas species isolated from Diarrhoeic and Non-diarrhoeic stools. Aeromonas spp Characteristics Aromonas isolates from:

Diarrhoeic Non- Diarrhoeic

(n = 137)

A hydrophila - No. of isolates 8 1

No. haemolytic(%) 81 (100.0)

Haemolytic units range (mean) 6-5 12 (64)

No. cytotoxigenic (%) 72 (88.9)

Cytotoxin titre range (mean) 4-128 (32)

No. Haemagglutinating (%) 51 (63.0)

A sobria -- No. of isolates 3 1

No. haemolytic(%) 26 (83.9)

Haemolytic units range (mean) 8-5 12 (64)

No, cytotoxigenic (%) 20 (64.5)

Cytotoxin titre range (mean) 2-64 (16)

No. Enterotoxigenic ( %) 25 (80.6)

No. Haenlagglutinating (%) 15 (48.4)

A. caviae -- No. of isolates 25 0

No. haemolytic(%) 2 (8.0) 0

Haemolytic units range (mean) 2-8 0

No. cytotoxigenic (%) 2 (8.0) 0

Cytotoxin titre range (mean) 1-8 0

No. Haemagglutinating (%) 8 (32.0) 0

Table 26

The virulence determinants of Aeronionas spp isolated as sole pathogens from 68 diarrhoea] stools, (%).

Strains (%)

Tests. - A hydrophila A. sobria -- A. caviae.

Isolation. No (70) isolated 45 (66.1). 21 (30.9) 2 (2.9).

No (%) with haemolytic activity 45 (100) 21 (100) 2 (100).

Range of haeniolytic titre

Cytotoxin activity

Range of cytotoxin titre

Enterotoxin activity

No with volume of fluid accumulation (> 1) Dilatation index

No (%) with haemagglutination activity

4.30 RESULT OF THE GEL, FILTRATION ANALYSIS:

The elution of the protein fractions present in each conccnlralcd culture I'rce

supernate showed the presence 01' 34 protein fractions, collected in 3 ml volumes and at a

flow rate of 20 ml per hour. The absorbance readings of' the various fractions at OD 595

nm are given in appendixes XIV & XV.

However, the results of the absorbance reading, the diastatic index i.e

volume/length ratio and the elution volumes of the more active fractions are in tables 29

and 30 for A hvdrophila and A sobria respectively.

Actual absorbance reading = Absorbance reading of fraction minus the absorbance

reading of the blank.

4.31. STANDARD MAKKER PROTEINS: Marker proteins i.e Myoglobin, Trypsin,

Bovine serum albumin, Lactate dehydrogenase and Pyruvate Kinase, together witli their

various elution volumes and the logarithms of their molecular weights are shown in table

4.32. MOLECULAR WEIGHTS OF PEAK PROTEIN FRACTION IN TEST

SAMPLES:

s The logarithms of the marker protein" were plotted against their elution volumes and a straight

line graph was obtained, which was used in the estiniation of the molecular weights of the peak

protein fractions in our two isolates (culture supernates). (fig.7).

Table 27

Absorbance reading at OD 595111n, the Diastatic Index and the Elution volumes of Active

fractions of sample A. (A hydrophila)

Fraction No

Blank 8 9 10 11 12 13 14 15 16 17 18 Control

Absorbance Reading

Actual Aborban ce Reading

Av. Volume of fluid accumulate d

Av . Length of loop.

Diastatic Index i.e volume/le nght ratio

Elution vol.

Note: Actual absorbance reading '= absorbance reading minus blank absorbance

reading.

Table 28

Absorbance reading, the Diastatic Index and the elution volumes of the Active fractions of sample B (A sobria).

Fraction No.

Blank 8 9 10 11 12 13 14 15 16 17 18 Control

Absorban ce reading

Actual Absorbance reading

0.359 0.370 0.341 0.426 0.914, 0.381 0.355 0.331 0.161 0.131 0.077

Average volume of fluid accumulat ed

Average length of loop in cn1.

Diastatic Index i.e volume.leng th ratio in cm.

Elution vol.

Note: Actual absorbance reading = absorbance reading minus blank absorbance reading.

4.4 4.8

Img. hlol. Wt.

Table 29

Standard (Marker) proteins with logarithms of their molecular weights (MW). and their elution volumes, on Gel - filtration using Bio-Gel P-4.

Protein Log Mol Wt. Elution Volume (rnl)

1. Myoglobin 4.2 62

2. Trypsin 4.4 58

3. Bovine serum albumin 4.8 50

4. Lactate dehydrogenase 5.2 , 42

.5. Pyruvate Kinase 5.4 37.5

THE ELUTION OF PROTEIN FRACTIONSIACTIVITY FROM SAMPLES

A AND B.

Figs 8 and 9 show the elution profile of protein fractions in saniples A and B (A

hydrophila and A sobria) including their enterotoxic activities i.e distention and

accumulation of fluid in the rabbit ileal loop > - lmllcn~. It was observed that there was a

single peak protein fraction between tiactions 10 and 12 for samplc A i.e A hydro~hila.

The enterotoxic activity was vcry similar with the same peak activity between fraction5

10 and 12 as was with the protein profile. A sobria had peak activity between fractions 11

and 13, for protein and enterotoxic activities respectively (fig. 10).

The molecular weight of the peak fraction for sample (A hvdrophila) A = 11

(peak fraction) x eluted 3ml volume = 33 = log 5.64. Antilog = 43,700 approximately.

Sample B = 12(peak fraction) x 3ml = 36 = log 5.48 = Antilog - 30.200 -

approximately. .

b

Protein absork&

fraction tube number Fig 8: Elution profile of protein fractions of A. hydrophila and

their enterotoxins on Sephadex G-50

+a- Protein Absorbance

+ Enterotoxin Activity

8 9 10 11 12 13 14 15 16 17 18

Fraction tube number

Fig. 9: Elution profile of protein fractions of A. sobria and their enterotoxins on sephadex G-50.

Plate 5 Showing Enterotoxic activities of some active fractions of A hvdrophila

S = Sample

4.34 SUSCEPTIBILITY PATTERNS OF AEROMONAS SPECIES ISOLATED

AS POTENTIAL SOLE ENTEROPATHOCENS.

Aeromonas species isolated as sole enteropathogens were subjected to the disc

* diffusion method of susceptibility testing. The results show that all the species were

susceptible LO gentankin, ccfuroxime, lloxacin and ciprofloxacin and are all resistant to

Ampicillin and Amoxycillin (Fig. 10 ). The susceptibility results for the 3 Aeromonas

species were very similar. The species were highly susceptible to Augmentin and Unasyl

but moderately to chloranlphenicol Co-trirnoxazole and Tctracylinc. (Table 32).

4.35 BETALACTAMASE PRODUCTION BY AEROMONAS ISOLATED AS

POTENTIAL ENTEROPATHOGENS.

The results of this test showed Aeromonas as a potent betalactamase producer. The

isolates produced betalactamase in 65(95.6%) out of 68 ampicillin resistant strains and

66(97%) out of 68 amoxycillin resistant strains. (Table 33)

However, 5 strains that were resistant to Unasyn and 2 to Augmentin failed to

produce betalactamase (Table 33)

Table 30

Antimicrobial susceptibility profile of 68 Aeromonas strains isolated as sole

entrbpathogens.

Antimicrobial agents. Disc concentration in (pg/ml). No %Susceptibility .

Amp." 10 0 (0)

Am. 30 0 (0)

Aug 201 10 66 (97.0)

Tob 10 68 (100)

Cef 10 68 (100)

Cipr 5 68 ( 100)

V m p = ampicillin; Am = amoxycillin, Te = tetracycline; S x T = Cotrimoxazole; C =

Chloramphenicol; Aug = augmentin (amoxycillin + clavulanic acid); Un = Unasyn (ampicillin + Sulbactam), CN = gentamicin; Tob = tobramycin; Cef = Cefirroxime, Of1 = Ofloxacid (Tarivid), Cipr = ciprofloxacin.

Table 31

Betalactamase production by the Ampicilin- and Amoxycillin- resistant strains of

Strains Resistant to No tested No (%) Betactamase positive.

Ampicillin 68 65 (95.6)

Amoxycillin

Unasyn

CHAITER 5

DISCUSSION

Motile aeromonads have been reported to cause various infections in man, being

most commonly implicated in diarrhoea, wound infections and septicaemia (Majeed and

Macrae, 1994). They were earlier considered by some authors as opportunistic pathogens

in irnmunocompromised patients ( Schoch and Cunha 1984; Holmberg a 4 1984), but

currently they are recognised as causes of a spectrum of gastrointestinal disease ranging

from self limiting diarrhoea, to acute persistent dysentery (Golik gt 4 1990; Gluskin gt 4

1992). They have been isolated from children with acute diarrhoea and from adult with

travellers diarrhoea (Echeverria a 198 1 ; Burke a a 1983).

The present study has yielded valuable information on some epidemiological

aspects of human infection due to Aeromonas species and certain virulence factors

associated with these organisms. Varying rates (1 % - 31 %) of isolation of Aeromonas

from diarrhoeal stool samples have been reported from different parts of the world

(Pitarangsi et a1 1982; Mikhail 1990; Deodhar et a1 1991). An unusually high

prevalence of Aeromom species (52.4%) in diarrhoeal children was reported from

Peruvia (Pazzaglia a 4 1991). 111 the present study, Aeromona species were recovered

from the faeces of 137 (17.9%) out of 764 diarrhoea1 and 20 (5.2%) out of 386 non

diarrhoea1 subjects. The difference was statistically significant (p < 0.05).

Regarding the relative distribution of different species of Aeromonas,' A

hydrophila was the commonest species recovered from faeces in this study. This is in

agreement with reports from India, Australia, Thailand and Bangladesh (Pitarangsi 4

1982; Janda and Duffey 1988, Deodhar gj 4 1991). In the United States and Europe,

however, A caviae is the most predwninanr spccics isola~cd Sl-on1 children Icss ~lla11 5

years of age (Figura a al 1986; Altwegg and Johl 1987; Janda and DufSey 1988). It

seems likely that the unhygienic environments and particularly the lack of properly

treated drinking water in the developing countries including Nigeria may account for the

predominance of A hydrophila. Besides this study support the view of Altwegg and Geis

(1989) that Aeromonas species predominantly involved in diarrhoea may vary

geographically.

Information on the occurrence of Aeromonas species in environmcntal sources in

hiigeria has been lacking. lIowcver, thcre havc been a few studies on Aeromonas

hydrophila associated with some animal foods used for human consumption. Obi and

Nzeako (1980) recovered A hydrophila from (71.8%) of specimens of the edible snail,

Achatina achatina a popular protein source in local diets. Nzeako and Okafor (1985) and

Nzeako (i990) identified Aeromonas hydrophila as an important and frequent pathogen of

the fish which like the edible snail Achatina achatina is also incorporated in the local diets

as a popular protein source. These findings may possibly be of public health significance

in the spread of Aeromonas associated gastroenteritis in Nigeria.

There is no previous record of isolation of Aeromonas from water or plant sources

in Nigeria. In the present study, A e r o m o n ~ was recovered from 9 % of environmental

samples i.e drinking water from borelloles, storage tanks, streams and water taps,

including some vegetables such as garden eggs or "Aiiara" in Igbo vernacular, botanically

referred to as solanium species. Garden eggs are regularly eaten in this part of Nigeria

and it is customarily offered to visitors as ("Kola") an expression of welcome. The

recovery of Aeromonas species from these environmental sources, have a far-reaching

public health significance in the spread of these organisms to man, since Aeromonas

species have been strongly associated with diarrhoea in this study.

In a recent hospital based study in Nigeria (Eko and Utsalo 1990), Aeromonas

species were isolated froin 10 (2.5%) out of 400 diarrhoeic patients and 2(1.0%) out of

200 non diarrhoeic. Thus, they found no significant difference in the relative frequency of

isolation from this organism from diarrhoeic and non diarrhoeic stools. The present study

showed a significant difference between the percentage prevalence of Aeromonas in

diarrhoeic samples of' stools (17.9%) and that in no11 diarrhoeic saniples (5.2%). This

defines an aetiologic role for Aeromonas in diarrhoea diseases. The study of Eko and

Utsalo (1990) may not have encountered many cases of Aeromonas diarrhoea probably

because their study was limited to a particular hospital i l l one locality; whereas in the

present study, stool samples were obtained from both children and adults from several

hospitals and medical centres in both urban and rural areas.

Several other investigators have also found significant difference in the prevalence

- of Aeromonas species in diarrhoeic .and non diarrhoeic stools (Echeverria g al 1981 ;

Gracey 1982; Burke a 4 1983; Agger a 1985). The isolation rate of Aeromonas

from diarrhoeic children recorded by different workers vary from 4 % to 13 % ( ~ u r k e gt

a1 1983; Gluskin 1992). In the present study, the prevalence in diarrhoeic children up to

10 years old was 21.2%, which may be a reflection of the poor state of personal and

environmental hygiene in the study area.

The higher frequency of isolation of Aeromonas from diarrhoeic infants thin from

faeces of older children and adults is consistent with the observation of several other

investigators '(George al 1985; Gluskin a 1992;). Aeromonas was isolated from 55

(22.9%) of diarrhoeic infants and 13 (12.6%) of non diarrhoeic infants and these rates

gradually declined with advance in age up to 20 years and above. The decline of .

Aeromonas may be due to the attainment of the stability of the faecal flora with age.

with age. There is lack of microbial competition in infants and children which probably

favours colonization of the intestine by Aeroinoilas species. The isolation of Aeromonas

(8.6%) from diarrhoeic subjects older than 20 years in this study, suggests that

Aeromonas- associated diarrhoea, may not be limited to infants and children alone but

may also affect adults.

.The present study has high lighted mean monthly distribution of Aeromonas

species isolated from human faeces between 1992-1995. This distribution attained its peak

bctween the months of July and August, falling gradually to'the lowest in the month of

December.Some investigators have also observed a seasonal trend in the prevalence of

Aeromonas species. Studies in soine countries have reported peak isolation rates in the

summer and autumn (Burke 1983:l Millership et a1 1983; Agger et a1 1985; Gluskin

et a1 1992). In Nigeria, between the months of July through August to Septembei-, there is -

usually abundance of fish foods fresh water and vegetables as compared to the dry and %

hot weather in the ~nonths November and December, when vegetables and most water

' taps often go dry.

A few workers have compared the sex distribution of Aero~nonas in stools. In the cqrrent

study, the higher frequency of isolation of Aeromonas species from males (33.3%) than

from females (10.3%) is not easily explained. It may probably result l i o ~ n the greater b

involvement of males in out door environment-related occupations and their limited

choice of drinking from environmental water sources, contaminated by Aeromonas.

However, the sex distribution rates in the current study are comparatively close to those

repolled by Gluskin (1992) who isolated Aeromonas from 81 patients of which 54

were boys while 27 were girls.

Although we concentrated on Aeromonas in this study, a check was made for

salmonella and other enteric bacterial pathogens. It is interesting to note that Aeromonas

species were more frequently isolated (5.9%) in diarrhoea1 diseases than salmonella

(5.3%). A similar observation has been reported inAustralia by Kirov (1984) who

isolated Aeromonas in 5(4.7%) of 107 patients auld salmonella in 4(3.7%) patients.

Several virulent factors have been emphasized to be of importance in the pathogenesis of

Aeromonas associated diarrhoea. The attachment of entero pathogenic bacteria to cells of --

the gastro intestinal tract has been described by Freter gt d(1976) and Moon a A (1979).

Lack of intimate contact, resulting in reduction of virulence, has also been observed by

some workers (Chitnis et a1 1982) Hae~nagglutination property is considered as an

adhesive and virulence factor for bacterial cnteropathogen including Aeromonas,

(Atkinson a 4 1980: Burke gt 1984; Watson 1985; Burke at a1 1986; Singh and

Sanyal 1393). A close association of bacteria to r r u m a l surfaces is required for their

virulence. This attachment allows for maximal effect of any toxin that the organism may

produce and is a pre-requisite for successful invasion. Sanyal et al (1983) reported the

haemagglutinin properties of A hvdrophila and clearly indicated that most of the

enterotoxigenic strains possess properties of agglutinating erythrocytes of human and

different animals; although they found no significant differences between isolates 01'

"* diarrhoea cases or healthy individuals and their environment. In the current study, greater

prevalence of haemagglutination property was found in the diarrhoeal isolates (54%) than

in the non diarrhoeal (45%) and in the environmental isolates (27.8%) respectively.

Majority of the strains of Aeromonas in this study produced beta-haemolysis and this

property was limited only to A hydrophila and A sobria and rarely A caviae. It has been

reported that enterotoxic strains of Aeromonas species are beta-haemolytic and tha-t most

of these beta haemolytic strains were either A hydrophila or A sobria but rarely A caviae

(Barer et al f986; Kuijper et a1 1989; Majeed and Macrae 1989; Eko et al 1989). This

finding was supported by the observa!ions from the present study, where almost all the

beta haemolysins were produced by A hydrophila and A sobria. None of the isolates of A

caviae produced beta haemolysin though two of thein from diarrlioeic subjects showed

alpha hacmolysis and thc percentage of beta haeinolysin producing strains was greater

with &hydrophila than with A sobria. Indeed all the isolates of A hydrophila isolated in

this study from diarrhoeic stools produced beta-haemolysin. . .

In the present study, a greater number of the strains of Aeromonas produced

cytotoxin regardless of their source. Diarrhoeic stools produce more cytotoxin isolates

(68.6%) than non diarrhoeic .(50%) and environmental (61.10%) isolates respectively.

* Some differences in the occurrence of cytotoxin isolates were also observed amongst the

three species of Aeromonas. A hvdrophila exhibited the highest frequency of cytotoxin

production (88.9%) in the diarrhoeic, 57.1 % in the non dial-rhocic and 61.1 70 in the

environmental isolates respectively. This was closely followed by A sobria 64.5%,

46.2% and 50% for diarrh~eic, non diarrhoeic and environmental isolates respectively. It

is noteworthy that cytotoxin was also detected in 2 (8%) of 25 A caviae isolates from

diarrhoeic stools and # @o%) out of such isolates from thenvironmental sources. There

was no cytotoxic A caviae isolated from the non diarrhoeic stools. There was no

cytotoxigenic A. caviae isolated from the non diarrhoeic stools. It was therefore obvious

that cytotoxic enterotoxin was produced by majority of strains of A. hvdrophila and A.

sobria in the current study. Asao gt (1984) has shown evidence for the production of a

cytotoxic. enjerotoxin and has further shown that this cytotoxin possess a molecular

weight of 50, 000 and play an active role in diarrhoea caused by Aeromonas species.

Regarding the relative distribution of different species of Aeromonas, A

hydrophila was the commonest species recovered from faeces in this study. This is in

agreement with reports from India, Australia, Thailand and Bangladesh (Pitarangsi gt

1982). In the United states and Europe, however, A caviae is the most predominant

species isolated from children less than 5 years of age (Figura gt 1986; Altwegg and

Johl 1987; Janda and Duffey 1988). It seems likely therefore that the unhygienic

environments and particularly the lack of properly treated drinking water in the

developing countries including Nigeria may account for the predominance of A

hvdrophila.. Regional differences in the distribution of the different Aeromonas species

probably accounts for the differences seen in the species associated with gastro entritis in

different parts of the world and within a given country Kirov (1993).

Many authors have noted the production of exotoxins (haemolysin, cytotoxin and

enterotoxin)by Aeromonas .However, this is largely confined to A hvdrophila and A

sobria (Gosling 1986; Figura el al 1986; Megraud 1986; and Tricker et a1 1989). This

was also confirmed in the present study, where the n~ajority of A hvdrophila and A sobria

strains, but only a few strains of A caviae were able to produce cytotoxin, and cytotoxins

produced by these few strains of A caviae were comparatively of very low titre. These

findings support previous observations that Aeromonas gastroenteritis is a spectrum of

species related diseases, being associated mostly with A hydrophila and A sobria. Besides

the cytotoxic titre ranges of these two Aeromonas species were higher than that in the A .

caviae. Enterotoxin production by Aeromonas has been considered a major attribute in

the mechanism of diarrhoea causation. Some investigators have employed rabbit ileal

loop' (Sanyal and Sing 1975; Wberbatch et al 1979; Singh and Sanyal 1992), while

some others have used suckling mouse for the detection of this toxin in ~efmonas .

(Annapurma and Sanyal 1976; Burke et a1 1981 ; Shiinada at al 1984; Noternlans et al

1986), Obi and Nzeako (1980) using the suckling mouse technique detected 58.3%)

enterotoxin production from isolates of A hydrophila obtained from the snail Achatina

achatina in Nigeria. It is significant to observe in this study that the production of

enterotoxins was more common in the diarrhoeic isolates of A hvdrophila (86.4%) and A

sobria (80.6). With thc A caviae isolates, enterotoxin productio~i was observed only in 2

(8%) out of 25 isolates from diarrhoeic stools. No enterotoxin was detected in the non . diarrhoeic A caviae strains.1t is however observed honl the results of this study, that

there was good correlation between the cytotoxin and the enterotoxin production by A b

hydrophila isolated from diarrhoeic stools

The previous notion that A caviae does not produce an enterotoxin, (Burke

a11983; Figura et a1 1986;) and thus not considered to be a human pathogen may be -

questioned in the light of the results of this study, where 2 A caviae isolates produced

eliterotoxin. This finding supports the report of Altwegg (1985) who have found this

species to be a cause of gahro enteritis. This study has also linked A caviae with gastro

enteritis. More-over a recent study by Sing11 and Sanyal (1992) has demostrated that

strains of all three Aeromonas species (A hydrophila, A sobria and A caviae, are

potentially enterotoxigenic (rabbit ileal loop assay) regardless of source; and strains that

showed little or no fluid accumulation in initial experiments became enterotoxin

producers after one to three passages through rabbit ileal loops. Therefore, it is essential

that species identification within the genus Aero1non~1~ may have diagnostic and

prognostic value in gastro enteritis. s

It is noteworthy that all (100%) the A hydrophila and A sobria isolates recovered

as sole potential enteric pathogens in diarrhoeic stools, elaborated beta haemolysin,

cytotoxin and enterotoxic activities, which indicated good correlation in the expression of

virulence factors in cases of diarrhoea, where only Aeromonas species are isolated.

Perhaps it is possible that the expressions of haemolysins, cytotoxins and enterotoxins

may be a condition for Aeromonas associated diarrhoea to occur.

The recovery of Aeromonas species from solanium species and drinking water

sources have a far reaching public health significance in the spread of these organisms to

man and may readily serve as sources of infection transmission resulting in diarrhoea

cases associated with Aeromonas species. Although Aeromonas may be isolated from a

wide range of vegetables and other food materials at the retail level and from portable

water; there is some disagreement over the most important source of human infection.

b - Holmfrg a d(1986) considered water to be the major source, while Agger and Callister

(1987) contended that foods and vegetables were more commonly involved.

Aeromonas species have been shown to produce both a heat stable cytotonic

enterotoxin of molecular weight 50.0kd and a heat labile cytotoxic enterotoxin of 50.0kd . molecular weight as well. ( Asao g 4 1984) Column chromatographic analysis of culture

supernates in the current study using sephadex G-50 revealed 34 protein fractions each

with a peak of enterotoxic activity at fractions 11 and 12 and with molecular weight of

43.7kd and 30.2kd respectively for A hvdropliila and A sobria. Species of Aeromonas

produce a wide range of putative toxins, but the relationship of these products either

singly or in combination with pathogenicity in man remains largely unresolved. It is

likely therefore, that the combined activities of these toxins may contribute significantly

to the entaropathogenic effects of Aeromonas in gastroenteritis.

The recovery of toxigenic A hydrophila and A sobria in faecal specimens appears

to correlate with diarrhoeal disease. Both species are considered environmental pathogens

(Cumberbtch 1979) and are frequently found in water sources. Their expression of

virulence f a c ~ ~ r s that characterise diarrkagenic pathogens strengthens the impression

that the isolation of A hydrophila and A sobria from diarrhoeal stools may have been

- preceded by the ingestion of contaminated water and perhaps along with another -

diarrhoeal pathogen. Volunteer studies may be required definitely to establish the

potentials of these two Acromonas species as a gastro- intestinal pathogen.

Regarding the in vitro susceptibility tests, the two members of the aminoglycoside . . . .

0 group i.e gentamicin and tobramycin, as well as the,tloxacin were very effective, thus

suggesting the therapeutic potentials oi' these drugs. A large percenlage ol' the isolalcs

produced beta-lactamases and this may account for their resistance to the penicillins

(ampicillin and amoxycillin)~ There were no significant differences observed in the

susceptibility patterns among the three species of Aromonas. The few penicillin resistant

strains that failed to produce betalactamase, may suggest a secondary mechanism of

penicillin resistance.

CONCLUSION.

The present study has yielded important infor~nation relating to several aspects of

epidemiology and p;lthogel-$is of Aerornonas associated diarrhoea viz. h

A. ' Aeromonas species could be isolated from the faeces of Nigerians, regardless of

their age. Diarrhoea associated with Aeromonas species more often affects infants

between less than a year old and 3 years than all the other age groups.

B. The organism may be carried asymptomatically in the Gools by a small proportion

of normal subjects. A hydrophila and A sobria are more prevalent than A caviae in the

faeces of diarrhoeic cases. Strains carried by normal healthy people may also have the

ability to produce virulent I'actors associated with strains f'rom diarrhoea cases. Not all

Aerornonas species appear to be pathogenic as some were isolated from perfectly healthy -

individuals.

C. The recovery of ~eromonas species particularly the exotoxin producing strains

from several water sources and in Garden eggs (solanium species) has a far- reaching

effect on the epidemiology and public health significance of Aeromonas associated gastro

, enteritis and further implicates drinking water as a possible source of infection. Our

findings wggest that drinking untreated water which is prevqlent in the study area is a

risk factor for acquisition of these organisms. From the few interviews conducted, it was

obvious that enteric symptoms in affected persons which tend to be severer in children,

couplcd with the fact that diarrhoea1 isolates were highly enterotoxic, suggests that

enterotoxigenicity ofAeromonas species in this study cause disease by enterotoxin

production.

It however remains to be determined whether the elaboration of' enterotoxin in

some strains of A hydrophila and A sobria is a genetic character of chromosomal origin

or from plasmids acquired from some other bacterial enteropathogen present in the

human intestinal milieu. . .

Greater awareness of this organism as a potential enteropathogen is warranted and

hospital and other laboratories should attempt to identify these bacteria by testing colonies

on blood agar for oxidase production.

D. Human volunteer studies or intestinal biopsies of patients with diarrhoea, from

whom Aeromom species are isolated, will probably be required to definitely establish

whether mesophilic Aeromonas species are gastro-intestinal pathogens. Nevertheless,

based on the present findings we recommend the routine search for Aeroinonas species,

in cases n f gastro-enteritis, in all clinical laboratories in Nigeria, particularly in

diarrhoea11 episodes involving both children and adults.

Brain heart infusion solids.

Proteose peptone

Dextrose

Sodium chloride

Disodium Phosphate

PH 7.4+0.2.

Method of preparation.

APPENDIX I

BRAIN HEART INFUSION (OXOID CM 225). BROTH.

Composition. Calf brain infusion solids 12.5gm

5.0 gm

10.0 gin

2.0 gm

5.0 gm

2.5 gnl

37gm of dehydrated medium was added to 1 litre of distilled

water. This was mixed properly and distributed into 20ml amounts in universal bottles.

The bottles were sterilized by autoclaring at 12 1oC for 15 minutes.

13RAIN HEART INFUSION AGAR

Composition. same as BHI (oxoid cm 225) with the addition of lOgm Agar Nol .

Method of preparation: 23.5 gm of the dehydrated medium, was suspended in 500ml of

distilled water, put to boil, to sterilized by autoclaving at 1210 for 15 minutes. .

Fer the preparation of Brain Heart Infusion Blood Agar (BHIBA) expired sterile

human blood was added at a concentration of 5 % into the sterile medium held at 50.C

Brain Heart infusion Blood Agar with Ampicillin as supplement.

Ampicillin solution. Dissolve 1 capsule of 250mg Ampicillin in sterile 25 ml distilled

water

250mg/25nd = lOmg/ml

= 10,000 mg/nd.

conc. required in SOOml blood agar at 151,g/inl is.

15xS00mg/ml = 750OPg/ml

.+oQQ ==4

7588 5 . e 3mls of 10,OOOCLg/ml Ampicillin solution, makc up to 4ml with lml sterile

distilled water. Add this 4ml Ampicillin solution to the 500ml Brain Heart infusion blood

agar = Ampicillin Blood Agar containing 15mg/ml Ampicillin. Pour this blood agar

solution into sterile petridishes in 20ml amounts, allow to solidify and incubate overnight

as a sterility check. The plates are ready for use if after 24hr incubation, there was no

vosible bacterial growth on the plats. The plates are stored in the refrigerator at 40C until

ready for use.

APPENDIX I1

MAcCONKEY AGAR ( 0 x 0 1 ~ CM7)

Composition. Peptone.

Lactose.

Bile salts.

Sodium chloride.

Neutral red.

Agar

PH 7.4 + 0.2.

Method od preparation. 52 gm powder was suspended in a ltre of distilled water,

brought to the boil, to dissolve completely.

The solution was sterilized by autoclaving at 121.C for 15 minutes, poured in .

20ml amounts into sterile petri dishes and allowed to solidify. The petri dishes (agar

plats) are incubated overnight at 37oC to check for sterility. The plats are then ready for

use if after overnight incubation at 37oC, there was no bacterial growth on each plate.

They are then stored in a refrigerator at 40C until used.

APPENDIX I11

NUTRIENT AGAR (slants and plates)

(CM3). .

Composition.

Lab-lemco powder. 1 gm/litre

Yeast extract 2 g m ,,

Peptone 5 , , ,,

Sodium chloride 5 , , ,,

Agar 15 , ,,

Method of preparation. 28 gm of powder was suspended in a ltre of distilled water

and boiled to dissolve completely. This was sterilized by autoclaving at 121.C for 15min

and poured in 20ml amounts into sterile tubes (slants). The plates and slants are'then

treated as stated earlier.

APPENDIX IV

DESOXYCHOLATE CITRATE AGAR (HYNES) Oxoid cm 227.

Composition. Lab-Lemco powder 5 gin/litre

Peptone 5gm ,,

Lactose 1 0 , ,,

Sodium citrate 8.5 ,,

Sodium thiosulphate 4.5 , ,

Ferric citrate 1.0 , ,

Sodium desoxycholate 5.0 , ,

Neutral red 0.02 ,,

Agar 12.0 ,,

PH 7.3+ 0.2.

Method of preparation. 52 gm of dehydrated medium was suspended in a litre of

distilled water and brought to boil, to dissolve completely. The ~nixture was. agitated to

prevent charring. The agar surface should be allowed to dry before use.

APPENDIX V

MUELLEK-HINTON AGAR

(Oxoid cm 337).

Composition.

Meat infusion. 6.0 gmllitre

Casein hydrohysate. 17.5 , , , ,

Starch. 1.5 ,, ,,

Agar no 1. 10.0 ,, ,,

PH 7.4 k 0 . 2 -

Method of preparation. 35 gm of dehydrated medium was suspended in a litre of - distilled water. The mixture was brought to the boil, to dissolve completely.

The mixture was then sterilized by autoclaving at 121.C for 15 min and poured in

20ml amounts into sterile petridishes to solidity. When solidified are incubated at 37oC

overnight. When certified sterile as described earlier, they were then stored in the

refrigerator at 4oC , until ready for use.

APPENDIX VI

SELENITE BROTH BASE (Oxoid cm 395)

Composition. Bacteriological Peptone. 5.0 gmllitre

Sodium phosphate 10.0 ,, , ,

Sodium Biselenite 4 ,, ,,

Method ol'preparation. 4 gm of sodium bisetenite was dissolved in a litrc of dislillcd

water to which is added 19 gm of dehydrated medium powder. The mixture was warmed

to dissolve and dispensed in 5ml amounts into bijou bottles. The bottles with their

contents were sterilizer in a boiling water bath or Autoclaring destroys lhc broth.'

AI'I'ENDIX VII

PEPTONE WA'l'ER SUGARS

Composition. Peptone water

Peptone. 1 Ogm

Sodium chloride 5gm.

Method. 15gm of dehydrated medium was added to a litre of distilled water. Mixed

very well and distrubuted in 5ml amounts in bijou bottles.

The bijou bottles and their contents were sterilized by autoclaving at 121.C for 15

minutes.

SUGARS.

One percent solutions of different sugars viz.

1 Glucose, (2) Arabinose, (3) Manitol (4) Inositol (5)' Sucrose (6) Salicin were

prepared in peptone water and dispensed in 31111 quantities into bijou bottles containing

durham's tubes. The bottles and. their contents are sterilized by autoclaving at 1016

pressure for 10 minutes. The sugars were allowed to cool and incubated at 37.C

overnightyor sterility.

If the sugar solutions remained clear after incubation, indicats sterility. If they

.

appeared turbid, means that the sugar solutions were contaminated and discarded. Once

the sugars were tested for sterility, were then ready for use.

APPENDIX VIII

UREA AGAR BASE (Oxoid crn 53).

Composit ion. Peptone 1.0 gmllitre

Dextrose 1.0 ,, ,,

Sodium chloride 5.0 ,, ,,

Disodium phosphate 1.2 ,, ,,

Potassium dihydrogen phosphate 0.8 , , , ,

Phenol red 0.012 ,,

Agar 15 gmllitre.

Met hod. A 2.4 gm quantity of the medium was mexed with 95ml of distilled water,

boiled to dissolve completely. The mixture was sterilized by autoclaving at 11542 for 20

min, cooled to 50.C; and aseptically 5ml of' sterile 40% urea solution (SR 20) was added

to the mixture. After mixing propeily, was distributed in lOml amounts into 402 bottles

and allowed to set in the slope position. When solidified the bottles with their contents

were incuhated at 37oC overnight to check for sterility as stated earlier. The absence of

bacterial growth and change of colour of the slopes indicated satisfactory preparation.

APPENDIX IX

KLIGLER IRON AGAR (Oxoid cm 33).

Composition. Lab-Lemco powder. 3. 0 gmllitre

Yeast extract 3 . 0 ,, ,,

Peptone 20. 0 ,, ,,

Sodium chloride 5 . 0 , ,,

Lactose 10. 0 , ,,

Dextrose 1. 0 , ,,

Ferric citrate . 0. 3 , ,,

Sodium thiosulphate 0. 3 , ,,

Phenol red 0.05 , ,,

Agar 12.0 , ,,

PH 7.4 t 0.2.

Method of preparation. 55 gin clchydratcd medium was suspended in a lctre of

distilled water, and brought to boil to dissolve co~npletely. The mixture was well mixed

and distributed into bottles, sterilized by autoclaving at 121oC for 15 mi11 and allowed to-

set as slopes. The medium is tested for sterility as described earlier

APPENDIX X

METHYL RED VOCES-PROSKAUER MEDIUM (MRVP). (OXOID CM 43)

Corn~osition Pepton P. 5 gmllitre

Dextrose 5 ,, ,,

Phosphate buffer 5 ,, ,,

PH 7.5 0.2.

Method. 15 gm of dehydrated powder was added to a litre of distilled water.

The mixture was distributed in 51nl amou~~ts into final containers and sterilized by

autoclaving at 121oC for 15 minutes.

AESCULIN AGAR SLOPE.

omp position. 10 gm of proteose peptone (oxoid)

1 gm of ferric Aminoniuin citrate

2 gm of Aesculin

15 gm of Agar powder.

These were dissolved in a litre of distilled water, PH being adjusted to 7.2. The solution

was then dispensed in 10ml amounts in bijou bottles and sterilized by autoclaving at

121.C for 15 minutes. They were sloped to solidify.

APPENDIX XI

Percentage prevalence of Aeromonas sp. in diarrhoeic and non diarrhoeic subjects

of varying ages.

SUMMARY TABLE

Age in yrs Diarrhoeic Non diarrhoeic Mean

20 and above 8.65 0.95 4.80

Mean 18.05 6.75

LSO (P=0.05) for comparing diarrhoeic and non diarrhoeic, Mean = 0.73 15

APPENDIX XI1

Sex distribution of Aeromonas isolates in diarrhoeic and Non diarrhoeic stools.

DIARRHOEIC (A)

Age in Yr Male Female Total Means

20 and above 5 3 8 4

(B) NON DIARRHOEIC

1 - < 3 4 3 7 3.5

3 - < l o 3 1 4 2

10 - <20 1 1 2 1

20 and above 1 1 0 . 5

Z 12 8

X . 2.4 1.6

Mean for^ and B (15.8 + 2.4),+ 2 = 6.6 ( = 9.1)

Appendix XV

Protocol of cytotoxin assay of the culture supernates (Culture filtrates) on African monkey kidney (Vero) cells. Rowofwells. 1 2 3 4 5 6 7 8 9 10 maintenance 1 2 3 4 5 6 7 8 9 10

(m199) medium (pl)lOO 100]100] 1001 1001 1001 1001 1001 1001 1001.

Culture supenate 100 100 100 100 100 100 100 100 100 100Discard.Dihitionof (culture 3 - 3 - 3 - 3 - 3- 3- 3- 3- 3- 3- filtrate) 112 114 118 1116 1132 1164 11128 11256 11512 111024. Monolayer 3 - 4 3 - 3 - 3 - 3 - 3 . 3- 3- 3- cells (Vero) 10Op1 100pl 100pl 100pl 100pl 100pl 100pl 100pl 100pl 100pl.

Growth medium poured off before the addition of lOOml of each dilution

Control I Control I1

An ~Illnoculated 10Op1 of 1 :2 dilution

monolayer cell control

with 1 OOpl maintenance

of BHI broth supernatant

filtrate (Bacterial growth medium

medium only. into a well of monolayer cells

All microtitre plates were incubated at 37oc under co, for 24 hours.

Titre. The highest dilution of the culture supernate to exhibit greater than 50% cytopdthic

effect i:e rounding and detachment of the monolayer cells.

NIB, Vero cells were maintained in m199 supplemented with fetal calf serum 2%vv

APPENDIX XI11

Cytotoxin 94 10 1 I 115 38.33

Enterotoxin 97 13 13 123 41.00

Total ' 372 43 4 1 456

Mean 9 3 10.75 10.25 -

F- SLD (P = 0.05) 11.89 for comparing condltion, effect and means.

APPENDIX XIV ' Protein Absorbance (sample A) A hydrophila OD 595nm.

Fraction Nu. Blank reading 0.41 I Ac~ual ~ w d i n g

34. 0.41 1

Note Actual reading = Absorbance reading minus blank reading in 3ml fractions.

APPENDIX XV Protein Absorbance (sample B) A sobria OD 595nm.

Fraction No. Absorbancr QD Actual OD Blank reading 0.41 1

1. 0.410

2. 0.41 1

3. 0.415 0.004

4. 0.441 0.030

5. 0.457 0.W6

34. 0.432 0.021

Note Actual reading = Absorbance reading minus Mank reading in 31111 fractions. 7

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