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Int. J. Biol. Chem. Sci. 2(3): 272-280, 2008

ISSN 1991-8631

Original Paper http://indexmedicus.afro.who.int

Enteroaggregative Escherichia coli: a public health hazard in Yaoundé,

Cameroon?

Pierre René FOTSING-KWETCHE 1,2, Joseph KAMGNO 3, François MOUCHET 3,

Antoine BANZA 4, Philippe SORLIN 1, Jocelyn THONNON 1, Thomas NJINÉ 2, Michel
BOUSSINESQ 3,5 and Marie-Christine FONKOUA 1*
1
Laboratory of Experimental Bacteriology, Centre Pasteur du Cameroun, P.O. Box 1274, Yaoundé,
Cameroon.
2
Laboratory of General Biology, University of Yaoundé I, Cameroon.
3
Laboratory of Epidemiology, Centre Pasteur du Cameroun, Cameroon.
4
Institut de Formation et de Recherche Démographique (IFORD), Yaoundé, Cameroon.
5
Institut de Recherche pour le Développement, Département Sociétés et Santé, UR 24 Paris, France.
*
Corresponding author, Tel: +237 22 23 10 15, +237 99 94 41 11; Fax: +237 22 23 15 64;
E-mail: [email protected]

ABSTRACT

The prevalence of various pathotypes of Escherichia coli was investigated during a case-control study
conducted in children diarrhoea in Yaoundé. Isolates obtained from the stools samples of children aged 6
months to 5 years were selected on phenotypic basis, and identified by virulence genes detection using
polymerase chain reactions. The most prevalent pathotype was enteroaggregative Escherichia coli (25.8%).
Enteropathogenic Escherichia coli (3.6%), enterotoxigenic Escherichia coli (1%), and enteroinvasive
Escherichia coli (0.2%) followed. No shiga toxin-producing Escherichia coli were identified.
Enteroaggregative Escherichia coli was not associated with diarrhoea (cases 26.1%, controls 25.5%; P=0,887),
unlike enteropathogenic Escherichia coli (cases 6.7%, controls 1%; P=0.003). Investigations into documented
potentials of enteroaggregative Escherichia coli in causing diarrhoea and other related pathologies indicated
that it could be a major public health threat in Cameroon despite the fact that it was not found associated with
clinical diarrhoeal cases in this study.
© 2008 International Formulae Group. All rights reserved.

Keywords: Cameroon, diarrhoeagenic Escherichia coli, public health.

INTRODUCTION cannot be distinguished based on their

Diarrhoea is a major cause of morbidity biochemical properties, not even on their
and mortality throughout the world, especially serotypes as is the case with Salmonella and
among children in countries with limited Shigella. For this reason, it is difficult to
resources, due to poor hygiene and sanitation assess their role in causing diarrhoea. Based
(Adachi et al., 2002; Nguyen et al., 2005; on their virulence and pathophysiology, six
Nguendo Yongsi et al., 2008). It is the third pathotypes of E. coli are known to be involved
most frequent cause of medical attention in in gastroenteritis (Nataro et al., 1998; Nataro
Yaoundé, Cameroon (Fotsing-Kwetché, and Kaper, 1998).
2002). Diarrhoeagenic bacteria routinely
Unlike Salmonella enterica and investigated in Cameroon include Salmonella,
Shigella that are typical pathogens in humans, Shigella (by serotyping) and enteropathogenic
Escherichia coli is made up of pathogenic and E. coli (by serogrouping). Barely 28% of
non-pathogenic strains. These two categories

© 2008 International Formulae Group. All rights reserved.

 P. R. FOTSING-KWETCHE et al. / Int. J. Biol. Chem. Sci. 2(3): 272-280, 2008

diarrhoea episodes are elucidated as caused by within two weeks from the day of sample
these pathogens (Fotsing-Kwetché, 2002). collection. Cases and controls were children
Recent studies conducted in Nigeria of the same age, sex and from the same
(Okeke et al., 2000a, 2000b) and in Gabon neighbourhood. Parents or guardians consent
(Presterl et al., 2003) showed that to participate in this study was obtained. Once
enteroaggregative E. coli (EAEC) was collected, the specimens were kept in a
frequently isolated in this sub-region of refrigerated container, and immediately
Africa. Due to its heterogeneity, research on transported to the laboratory for pathogen
EAEC is very expensive (Weintraud, 2007). screening. The isolates represented the
In developing countries, the high prevalence predominant (more than 70% of the
of EAEC contrasts with the lack of resources population) colony phenotype observed in
for their detection. Efforts to design culture after twenty-four hours at 37 °C. Once
convenient and efficient protocols in the selected, they were cryopreserved at -20 °C in
contexts of resource limitation are, however, a brain-heart infusion made up of 30% (v/v)
being made (Wakimoto et al., 2004; Jenkins et glycerol, for subsequent virulence genes
al., 2006). It was reported (Nataro and Kaper, screening.
1998) that identification approaches based on
aggregative adherence phenotype (the gold Reference strains used
standard) could include both pathogenic and The reference strains used in this study
non-pathogenic strains (sharing factors included strains E2348/69 (Nataro et al.,
conferring a common phenotype, but lacking 1985) for enteropathogenic E. coli (EPEC),
the necessary virulence determinant). 17.2 (Baudry et al., 1990) for
In Cameroon, very little public health enteroaggregative E. coli (EAEC), C600VT2
implications have been identified for (O`Brien et al., 1984) for shiga toxin 2 (stx2)-
diarrhoeagenic E. coli. Subsequent to a case- producing E. coli and H19VT1 for shiga
control study on children with and without toxin 1 (stx1)-producing E. coli (STEC),
diarrhoea, conducted in Yaoundé from EDL1493 (Pasteur Institute of Dakar,
December 2001 through February 2002, and Senegal) for heat stable (ST) and/or heat labile
from April through June 2002, the prevalence (LT) toxin-producing enterotoxigenic E. coli
of E. coli pathotypes known to play a major (ETEC). Strains M90T for enteroinvasive E.
role in diarrhoea was investigated. To coli (EIEC), and Hb101 that served as
characterize EAEC, a glass assay was adapted negative control were kindly provided by the
from the modified cell culture technique Pasteur Institute of Bangui, Central African
(Okeke et al., 2000b). EAEC (identified by Republic.
aggregative phenotype) bearing the virulence
gene astA, were then assumed to be Selection of potential pathogenic strains
potentially diarrhoeagenic. Beyond pathotypes Selection criteria for potential
characterization, whether EAEC could be pathogenic strains of E. coli were phenotypic
regarded as public health worries was also and included serogrouping and mannose-
discussed in this paper. resistant haemagglutination assay, according
to Evans et al. (1979), Zamora et al. (1997)
MATERIALS AND METHODS and Baraduc et al. (2000).
Setting, sampling, and culture methods Serogrouping
This study was conducted in Yaoundé, Colonies harvested from an overnight
during the dry season (December 2001 culture at 37 °C on Mueller Hinton agar
through February 2002), and the rainy season underwent slide agglutination assay, using the
(April through June 2002). Cases and controls classical twelve monovalent antisera (BioRad,
were children aged six months through five France) directed against O-antigens known to
years. Children who defecated three or more occur in EPEC. Tests were carried out
unformed stools in 24 hours or the ones from according to the manufacturer’s instructions.
whom this was observed, but in which Mannose-resistant haemagglutination assay
resolution took place 72 hours earlier were (MRHA)
regarded as cases. Controls included children This was carried out, as a slide agglutination
from whom no such signs were observed
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assay in the presence of 2.5% D-mannose Yamamoto and Echeverria (1996), and all the
according to Okeke et al. (2000b). others from Gassama et al. (2001). The
different tests were conducted according to
Confirmation assays the following considerations:
Selected isolates that showed MRHA- - Reaction volume composition
and/or agglutinated the antisera used The reaction mixture consisted of a 10x
underwent a glass adherence assay, and were reaction buffer, 25mM MgCl2, 10 mM dNTPs,
further tested for the presence of astA, eae, 100mM of each primer, 5U/µl Taq
stx1, stx2, st, lt and ipaH genes by PCR. polymerase, 10µl of the appropriate DNA
Glass adherence testing suspension. This was completed to 50µl with
EAEC is the only pathotype, which sterile water.
adheres on both cell lines and glass with - Amplification parameters
stacked-brick or honeycomb phenotype. Prior Amplification was achieved in a
to screening, a series of twelve duplicated thermal cycler, GeneAmp® PCR System
experimental tests were reproducibly 9700TM, (Applied Biosystem). For all the
performed (using reference strains) on Hep-2 genes, predenaturation was achieved at 94 °C
cells with the modified cell culture technique for five minutes, denaturation at the same
(Okeke et al., 2000b) and on cover slides. This temperature for one minute, polymerization at
technique with slides free from cells was 72°C for one minute, and final extension at 72
carried out using the following procedure: °C for seven minutes. The time for annealing
All bacterial strains were grown overnight in was also the same for all the genes (45
tryptic soy broth without shaking. 250-µl of seconds), but the required temperature varied
Dulbecco’s modified Eagle medium (eae 65 °C; stx1, sta, lt, ipaH 51°C; stx2 50
containing 0.5% D-mannose was transferred °C, and astA 56 °C). Thirty-five cycles were
to each well of a twenty-four well cell culture required to complete the process. The
cluster (Corning Incorporated, USA) amplified product was resolved by a 1.5%
previously equipped with a 8 mm diameter agarose gel electrophoresis stained with
glass slide. Then 10 µl of the bacterial ethidium bromide, completely soaked in a 1x
suspension (0.5 Mcfarland) was added per TBE buffer for 40 minutes at 100 milivolts.
well. The set was incubated for three hours at The gel was then visualised under ultra violet
37 °C and 5% CO2. After incubation, the transillumination. A positive result was
preparation was washed three times with determined by the presence of an expected
phosphate-buffered saline, fixed with 70% size PCR product, with a 100 bp molecular
methanol for five minutes, stained with 10% weight marker.
Giemsa for 15 minutes and examined by oil
immersion light microscopy (magnification, Confirmation criteria
100x). Each isolate was tested in duplicate Confirmation of EAEC was based on
and examined by two bacteriologists. the fact that though the astA gene can be
Adherence to the glass with a stacked-brick or detected in other pathotypes, it is
honeycomb phenotype indicated a positive predominantly associated with attaching and
test. effacing lesions-producing (eae-positive) E.
PCR assays coli, and EAEC identified by Hep-2 cell
- Lysate preparation culture (Nataro and Kaper, 1998; Presterl et
A bacterial suspension prepared from al., 2003).
sterile distilled water and bacterial colonies An exclusion diagnosis was then
harvested from an overnight culture at 37 °C designed accordingly. Isolates that were
was heated for 10 minutes at 100 °C, and, positive for astA, exhibited stacked-brick (or
immediately transferred into ice for 1 minute. honeycomb) adherence and negative for other
After a 10 minutes centrifugation at 10000g, genes were classified as potentially
the supernatant containing the bacterial DNA diarrhoeagenic EAEC. Those that were
was collected for polymerase chain reaction positive for stx (stx1 and/or stx2) genes were
(PCR), using the primers listed on Table 1. regarded as STEC. Those that were positive
Primer sequences for astA were obtained from for eae and negative for other tests were

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Table 1: Primers used in this study.

Designation Primer sequence Target gene Expected amplicon size in Pathotype

base pairs (bp)
Eae1 5’-GGC TCA ATT TGC TGA GAC CAC GGT T-3’
Eae2 5’- GCA AAT TTA GGT GCG GGT CAG CGT T-3’ eae 494 EPEC

East1 5’-CCA TCA ACA CAG TAT ATC CGA-3’

East2 5’-GGT CGC GAG TGA CGG CTT TGT-3’ astA1 111 EAEC

SLTI-1 5’-GAA GAG TCC GTG GGA TTA CG-3’

stx1 130
SLTI-2 5’-AGC GAT GCA GCT ATT AAT AA-3’ STEC
SLTII-1 5’-TTA ACC ACA CCC ACG GCA GT-3’
stx2 346
SLTII-2 5’-GCT CTG GAT GCA TCT CTG GT-3’
Sta 1 5’-CTG TAT TGT CTT TTT CAC TC-3’
sta 182
Sta 2 5’-GCA CCC GGT ACA AGC AGG AT-3’
ETEC
LT 1 5’-GCG ACA AAT TAT ACC GTG CT-3’
lt 707
LT 2 5’-CCG AAT TCT GTT ATA TAT GT-3’
IpaH 1 5’-GCT GGA AAA ACT CAG TGC CT-3’
ipaH 424 EIEC
IpaH 2 5’-CCA GTC CGT AAA TTC ATT CT-3’

Eae: enterocyte attachment/effacement; East: enteroaggregative stable toxin; SLTI: shiga-like toxin I; SLTII: shiga-like toxin II; Sta: stable toxin a; LT: labile toxin; IpaH: invasion plasmid H.

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identified as EPEC. All lt and/or st-positive DISCUSSION

isolates were identified as ETEC, while ipaH- In the present study, EPEC were
positive E. coli were classified as EIEC. significantly associated with diarrhoea (6.7%
Isolates were not classified if they harboured in cases and 1% in controls). It has been
both the eae and astA genes, or exhibited assumed (identified by serogrouping) to be the
aggregative adherence phenotype, but negative most common pathotype of E. coli in
for all the genes targeted in this study. Yaoundé. Its prevalence was then estimated at
26% (Fotsing-Kwetché, 2002).
Statistical analyses This low infection rate reflects a regional
Statistical analyses were conducted tendency. In the case-control study conducted
using the Chi-square (χ2) test, adjusted with among children less than five years in Nigeria
Yates correction when necessary. The (Okeke et al., 2000b), a prevalence rate of
associated likelihoods were accessed at 5% (P 1.8% for EPEC was reported among the
value <0.05 indicated significant difference). cases. During a similar study in Gabon
(Presterl et al., 2003), no EPEC cases were
RESULTS reported in children of eleven years or
Specimens from 384 children were younger. The rate of infection due to EPEC in
screened for diarrhoeagenic Escherichia coli. the central African sub-region is probably
Samples collected during the dry season lower than expected in countries with limited
included specimens from 57 cases, and 63 resources (Baraduc et al., 2000).
controls. During the rainy season, specimens The ETEC, EIEC and STEC were the
from 123 cases and 141 controls were least frequent pathotypes observed in this
examined. study. Their overall frequencies were 1%,
Five hundred and nine strains of E. coli 0.3% and 0%, respectively. They were also
isolated and cryopreserved underwent less frequent in Nigeria (Okeke et al., 2000b),
selection tests for potential virulence (2.4%, 1.2% and 0.6 %, respectively). In
properties. Amongst these isolates, 136 belong Gabon (Presterl et al., 2003), 4.6% were
to an identifiable O-serogroup [the most positive for ETEC. No STEC or EIEC isolates
prevalent serogroups included O119 (25%), were identified in that study.
O111 (17%), O126 (13%), and O127 (11%)], Concerning EAEC, though increasingly
and 197 showed a MRHA phenotype. Two acknowledged as a global pathogen, its role in
hundred and sixty nine isolates (53%) fulfilled causing diarrhoea remains controversial.
at least one of the selection criteria and were AstA-positive EAEC were not
subjected to confirmatory PCR tests. significantly associated with diarrhoea in this
Results obtained after confirmation tests are study (26.1% in cases, 25.5% in controls).
presented on Table 2. These results, in accordance with those
Overall, EAEC was not associated with reported from Nigeria (Okeke et al., 2000b),
diarrhoea (26.1% in cases, and 25.5% in contrasted with those obtained during a
controls; P=0.887). EPEC was significantly similar investigation in Brazil (Zamboni et
associated with diarrhoea (6.7% in cases and al., 2004) and in India (Anvikar et al., 2008).
1% in controls; P=0.003). Of the 136 strains The question whether astA-positive EAEC
that belonged to an identifiable O-serogroup, could be regarded as a health issue in
57 (41.9%) were ultimately identified as Cameroon, in spite of its lack of association
EAEC. Four strains of ETEC were identified, with diarrhoea is then posed.
two of which were isolated from cases. One Nataro et al. (1998) observed that
strain of EIEC was isolated from one case. No EAEC was an emerging global pathogen.
STEC was detected in this study. Regarding Nishikawa et al. (1999) also reported an
adhesion phenotype, all astA-positive E. coli outbreak due to a strain with astA as the
were also positive for adhesion phenotype single virulence determinant, and proposed
(91% of adhering isolates). Seven isolates that diarrhoeal specimens should be examined
presenting aggregative adherence phenotype for the enteroaggregative heat stable toxin 1
were negative for all the genes targeted, while (EAST-1, the transcription product of astA)-
three were positive for both eae and astA. producing E. coli, to determine how these

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Table 2: Distribution of aetiologies with regard to clinical category.

Dry season Rainy season Total

2
P
cases controls cases controls cases controls value value
Pathotype 180 204
57 63 123 141
EAEC 22 38.6% 21 33.3% 25 20.3% 31 22.0% 47 26.1% 52 25.5% 0.02 0.887

EPEC * 2 3.5% 0 0.0% 10 8.1% 2 1.4% 12 6.7% 2 1.0% 8.80 0.003

ETEC 1 1.8% 1 1.6% 1 0.8% 1 0.7% 2 1.1% 2 1.0% 0.14 0.708
EIEC 0 0.0% 0 0.0% 1 0.8% 0 0.0% 1 0.6% 0 0.0% ND

EHEC 0 0.0% 0 0.0% 0 0 0 0.0% 0 0.0% 0 0.0% -

25 22 37 34 62 56
* Significant association with diarrhoea
EAEC: Enteroaggregative E. coli; EPEC: Enteropathogenic E. coli; ETEC: Enterotoxigenic E. coli; EIEC: Enteroinvasive E. coli; EHEC: Enterohemorrhagic E. coli.
ND: not determined

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organisms are distributed worldwide. travellers (Huang et al., 2003), and HIV-
McVeigh et al. (2000) gave evidence of the infected subjects (Gassama et al., 2001;
enterotoxic activity of the EAST-1 protein. Mossoro et al., 2002). Growth retardation and
The EAST-1 enterotoxin was also found to be malnutrition due to EAEC were also reported
associated with intestinal inflammatory in children (Nataro et al., 1998; Nataro and
response (Nataro et al., 1998; Jiang et al., Kaper, 1998). Accordingly, the pathogenic
2002), most frequently in children (Piva et potentials of EAEC are very likely
al., 2003; Zamboni et al., 2004). Several other underestimated when children diarrhoea is
authors (Okeke et al., 2000b; Piva et al., regarded as the sole pathology.
2003) used the astA determinant to identify In fact, up to 41.9% of
EAEC in different settings. seroagglutinating strains were ultimately
In addition, the present study indicates identified as astA-positive EAEC. It is likely
association between the astA gene and the that most strains diagnosed by
adherence phenotype (91% of adhering seroagglutination (the routine procedure in the
isolates are positive for astA). Adhesion and setting) as EPEC belong to this category. The
toxin production are major mechanisms need to better characterize E. coli in the
involved in EAEC diarrhoea. Why strains routine procedure is an urgent research
were not associated with diarrhoea remains a priority in Yaoundé. Works are underway to
puzzle. Genetic and environmental factors identify others potential aetiologies of
interact to produce a phenotype, including childhood diarrhoea.
virulence (Martinez and Baquero, 2002).
The absence of association between Conclusions
astA-positive EAEC and diarrhoea could be The predominant pathotype identified
related to a set of parameters among which in this study was EAEC, followed by EPEC
are: the patient’s general health status, the that was less frequently isolated than
strain involved, the diagnosis techniques used expected. The isolation rates of different
including case definition, and the level of pathotypes were similar to those observed in
endemicity. In fact, it was observed (Adachi et other countries of the sub-region. AstA-
al., 2002) that in the regions where EAEC is positive EAEC could be a major public health
endemic, it is frequent in food. In an earlier danger in Yaoundé. These highlight the need
study (Ndayo et al., 2000), it was reported that to improve the protocol used in this study in
food sold in the streets of Yaoundé and order to better characterize EAEC and
Douala (towns in Cameroon) were strongly implement it in routine process.
contaminated by E. coli. The study did not,
however, investigate the different pathotypes. ACKNOWLEDGMENTS
The genes involved could also influence the Molecular biology studies on E. coli
onset of diarrhoea (strains of this pathotype were supported by a grant from the French
are very heterogeneous as regard virulence ministry of foreign affairs. We are also highly
determinants). Wakimoto et al., (2004) indebted to Professor Patrick Grimont (Institut
highlighted the fact that strains possessing the Pasteur de Paris) and Dr Malika Gouali
transcriptional activator, aggR, exhibited (Institut Pasteur de Madagascar) for assistance
stronger biofilm formation, typical and advice.
aggregative adherence, and were likely more
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