€¦ · lw." h! .- .. studies on aeromonas species isolated from '# r 4 diarrhoeal and...
TRANSCRIPT
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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