J Med Microbiol 55 (2006), 1493-1497; DOI: 10.1099/jmm.0.46683-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Detection of enteroaggregative Escherichia coli in faecal samples from patients in the community with diarrhoea
Claire Jenkins1,
Mathias Tembo2,3,
Henrik Chart1,
Tom Cheasty1,
Geraldine A. Willshaw1,
Alan D. Phillips3,
David Tompkins4 and
Henry Smith1
1 Laboratory of Enteric Pathogens, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK
2 Tropical Diseases Research Centre, Microbiology Unit, PO Box 71769, Ndola, Zambia
3 University Department of Paediatric Gastroenterology, Royal Free Hospital, Pond Street, London NW3 QG, UK
4 Yorkshire and Humber, Leeds Health Protection Agency Laboratory, Bridle Path, York Road, Leeds LS15 7TR, UK
Correspondence
Claire Jenkins
claire.jenkins{at}royalfree.nhs.uk
Received 15 April 2006
Accepted 21 July 2006
The aim of this study was to assess the usefulness of a multiplex PCR assay targeting the aat, aaiA and astA genes for the detection of typical and atypical enteroaggregative Escherichia coli (EAEC) in bacterial cultures from faecal samples from patients with community-acquired diarrhoea. The isolates harbouring these genes were also tested using the HEp-2 cell-adhesion assay to clarify their EAEC status. aat, aai or astA was found in E. coli faecal isolates from 39 (7.8 %) of 500 patients, and 20 of these strains adhered to HEp-2 cells in a pattern characteristic of EAEC. Eight isolates carrying the aai or astA gene but not the aat gene were shown to be HEp-2 cell test positive, although 12 strains with this genotype were HEp-2 cell test negative. Using the HEp-2 adhesion assay as the gold standard, the addition of primers detecting aaiA and astA to the aat PCR increased the number of EAEC isolates detected, but identified strains of E. coli that were not EAEC. The variety of genotypes exhibiting aggregative adherence highlights the problems associated with developing a molecular diagnostic test for EAEC. This PCR assay detects a variety of strains exhibiting characteristics of the EAEC group, making it a useful tool for identifying both typical and atypical EAEC.
Abbreviations: AA, aggregative adherence; DA, diffuse adherence; EAEC, enteroaggregative Escherichia coli; EAST, enteroaggregative heat stable toxin; HPA, UK Health Protection Agency; LA, localized adherence; LEP, HPA Laboratory of Enteric Pathogens.
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INTRODUCTION
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Enteroaggregative Escherichia coli (EAEC) is the most commonly isolated pathogenic E. coli group from patients with diarrhoea in England (Tompkins et al., 1999; Food Standards Agency, 2000; Wilson et al., 2002), and has emerged as an important pathogen in travellers' diarrhoea (Adachi et al., 2001; Wilson et al., 2002), diarrhoea among children (Huang & Dupont, 2004) and in immunocompromised patients (Gassama-Sow et al., 2004). EAEC are defined by their ability to adhere to HEp-2 cells in an aggregative adherence (AA) pattern, described as a ‘stacked-brick’ formation (Nataro et al., 1987; Law & Chart, 1998). However, the HEp-2 cell adhesion assay is unsuitable as a diagnostic tool, as it is labour intensive and requires experienced microbiologists to interpret the results (Nataro, 2005). A PCR method based on the presence of essential virulence factors would improve the diagnosis of EAEC disease.
Certain strains carry a high-molecular-weight plasmid (pAA) associated with AA (Vial et al., 1988; Law & Chart, 1998), on which a number of virulence genes are located, including an antiaggregation protein transporter gene (aat; previously referred to as CVD432 or the AA probe) (Baudry et al., 1990; Nishi et al., 2003) and an enteroaggregative heat stable toxin (EAST) gene (astA) (Savarino et al., 1991; Menard & Dubreuril 2002). Located on the chromosome, on the pheU pathogenicity island, are the gene clusters SciI and SciII. SciII comprises a cluster of ORFs under the control of AggR, designated the AggR-activated island (aai). The contribution of the aai genes to EAEC pathogenesis is currently unknown, but these genes do not appear to play a role in adherence (Harrington et al., 2006).
PCR assays that target genes located on the pAA, including aat, aap and aggR, have been described (Schmidt et al., 1995; Cerna et al., 2003). However, EAEC are a heterogeneous group, and not all strains that adhere to HEp-2 cells in a stacked-brick formation harbour the pAA plasmid (Cobeljic et al., 1996; Itoh et al., 1997; Dudley et al., 2006). Alternative target genes that would identify both pAA-positive and -negative EAEC strains have been sought (Gioppo et al., 2000; Elias et al., 2002), but EAEC-specific genes characteristic of both groups have not been identified. Work carried out in this laboratory has shown that the aaiA and astA genes are potential targets for detecting both groups (Jenkins et al., 2005, 2006).
In this study, a PCR assay targeting the aat, aaiA and astA genes was used to detect EAEC in faecal samples from patients with community-acquired diarrhoea. Strains harbouring one or more of these three genes were assessed for their ability to adhere to HEp-2 cells to confirm their EAEC status. The aim of the study was to assess the usefulness of the PCR for detecting typical and atypical EAEC.
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METHODS
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Faecal samples.
Five hundred faecal samples, one per patient, were collected at the Leeds Health Protection Agency (HPA) Laboratory between May and August 2003. These samples were sent to the laboratory by general practitioners (GPs) in the Leeds area and were from patients with community-acquired diarrhoea who had visited their GPs. Of the 500 patients included in the study, 202 were male and 289 female. Information on the patients' gender was unavailable for nine of the samples. Four hundred and three samples were from adults (18 years old and over), 91 were from children (younger than 18 years old), and the patient's age was not recorded for six of the samples. Details of recent travel abroad (in the last 12 months) were noted for 57 patients, who had visited 33 different destinations. The countries visited most frequently included Spain (7), India (6) and Pakistan (5).
The samples were analysed at the Yorkshire and Humber Leeds HPA Laboratory for routine enteric pathogens. The pathogens detected included Clostridium difficile (6), Campylobacter species (12), Salmonella species (10), Shigella sonnei (2), Shigella flexneri (1), rotavirus (7), small round structured virus (2), Entamoeba coli (1) and Entamoeba histolytica (3). The appearance of each sample was noted and recorded as formed (58), semi-formed (332) or liquid (109). This information was not recorded for one of the samples.
These faecal samples were forwarded to the HPA Laboratory of Enteric Pathogens (LEP) on a daily basis, except on Fridays, to avoid the samples being in the post over the weekend. The samples were packed on ice until processed by LEP staff on the day of receipt.
Bacterial strains.
The control strain used for the PCR reactions was an EAEC of serotype O44 : H18 (042), described by Nataro et al. (1987). The following E. coli strains were used as control strains in the HEp-2 adherence assays: EAEC strain 042 (Nataro et al., 1987) for the enteroaggregative phenotype, enteropathogenic E. coli strain E2348/69 (serotype O127 : H2) (Scotland et al., 1989) for the localized adherence (LA) phenotype, and the diffuse adherence (DA) control was a clinical isolate from a 13-month-old female admitted to hospital in the East End of London with acute diarrhoea, who had no other pathogens detected. E. coli DH5
, a strain of K-12 that does not cause diarrhoea in humans and has no specific pattern in adherence assays, was used as a negative control.
Identification by PCR of bacterial isolates containing aat, aaiA or astA from faecal samples.
The faecal cultures were plated onto MacConkey agar and incubated overnight at 37 °C. Nutrient broths were inoculated with a sweep of mixed colonies from the MacConkey plates, incubated at 37 °C for 2–4 h, and examined for the aat, aaiA and astA genes by multiplex PCR. Two microlitres of broth culture were added to the PCR mix, which consisted of 1x PCR buffer, 200 µM dNTPs, 1 µM each of aat, aaiA and astA forward and reverse primers, and 0.75 U Hotstar Taq (Qiagen). PCR-grade water was added to make a final reaction volume of 25 µl. The primers used in the PCR reactions have been described previously (Schmidt et al., 1995; Elias et al., 2002; Jenkins et al., 2006), and are shown in Table 1
. The amplification conditions were as follows: an initial denaturation of 95 °C for 15 min (to co-activate Hotstar Taq DNA polymerase), then 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min for 30 cycles, and a final extension of 72 °C for 10 min. Each primer set could be run individually using the same PCR amplification conditions.
In samples of mixed colonies with a positive PCR result, a pure-colony pick containing aat, aaiA or astA was obtained from the original MacConkey plate in all cases as follows. Five single colonies from each plate were inoculated into nutrient broth and incubated at 37 °C for 4–6 h. The single colonies were then examined for aat, aaiA and astA genes using the multiplex PCR, as described above.
Characterization of the bacterial strains using the HEp-2 adhesion assay.
All single-colony isolates were examined by the HEp-2 cell-adherence assay described by Nataro et al. (1987), with slight modifications. HEp-2 cells were grown for 24 h to 50–70 % confluence on 13 mm diameter coverslips in 24-well flat-bottomed tissue-culture plates in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % fetal calf serum, 2 % L-glutamate and 1 % non-essential amino acids (Sigma-Aldrich). Prior to inoculation with bacterial strains, the HEp-2 cells were washed and 1 ml fresh DMEM medium with 1 % D-mannose (Sigma-Aldrich) was added. Isolates were recultured from Dorset's egg slopes to MacConkey agar and subcultured into LB broth culture, before overnight incubation without shaking at 37 °C. Twenty microlitres of the overnight LB broth culture were added to each well, and plates were incubated at 37 °C in an atmosphere of 95 % O2/5 % CO2 for 3 h. The HEp-2 cells were washed thoroughly with PBS to remove non-adherent bacteria and fixed with 70 % methanol for 30 min. After fixation, the coverslips were washed five times with PBS, stained with 10 % Giemsa (BDH) for 15 min, rinsed and air-dried, before mounting in DPX mountant (BDH) prior to light-microscopy examination using a x40 objective. The adherence patterns were assessed and recorded (Fig. 1). When the 3 h incubation showed no adherence or an equivocal pattern of adhesion, a 6 h HEp-2 adherence assay was performed.
Serotyping.
Each isolate was biochemically confirmed as E. coli and serotyped by the LEP, using a scheme based on the heat-stable somatic (O) and flagellar (H) antigens (Gross & Rowe, 1985). Strains that could not be serotyped using the LEP serotyping scheme were designated O?, O-rough or H?. Rough strains do not express an O antigen and therefore cannot be typed using a phenotypic serotyping scheme.
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RESULTS AND DISCUSSION
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Strains of E. coli harbouring the aat, aaiA or astA genes were found in faecal samples from 39 (7.8 %) of 500 patients. Sixteen of these isolates had the aat gene, the aaiA gene was found in 14 isolates and 29 strains had the astA gene. Twenty of the 38 strains adhered to the HEp-2 cells in a stacked-brick formation. A wide range of serotypes was detected, although 12 of the 36 strains could not be serogrouped in the current scheme and were designated O?. The details of these patients and the genotypes of their EAEC isolates are shown in Table 2.
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Table 2. Patient details and combinations of aat, aaiA and astA in bacterial strains isolated from patients during this study
Abbreviations: F, female; M, male; N, no travel history; ND no data supplied; D, diarrhoea; V, vomiting; NT, strains not serotyped, as failed to grow on subculture.
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Of the 16 patients that had isolates harbouring the aat gene, 12 also had the aaiA gene, eight carried the astA gene and 12 showed AA to HEp-2 cells in a stacked-brick pattern. Campylobacter species was also isolated from the stool of one of these patients (Table 2
). The aat gene PCR showed good correlation with the HEp-2 adhesion assay, as 75 % of aat-positive isolates were confirmed as EAEC. Baudry et al. (1990) first used the aat gene (referred to as the CVD432 EAEC probe) as a target for the identification of EAEC, and claimed that the probe was 89 % sensitive. Although subsequent studies have shown that the sensitivity varies between 15 and 90 % (Okeke & Nataro, 2001), the aat gene has remained the most popular target in molecular assays for the detection of EAEC (Schmidt et al., 1995; Cerna et al., 2003; Pabst et al., 2003; Amar et al., 2004). In this study, seven patients with aat-positive strains were known to have recently travelled outside the UK to destinations that included Nepal, Pakistan, Egypt, Sri Lanka and Mexico. Other studies have suggested an association between EAEC and travellers' diarrhoea (Adachi et al., 2001; Wilson et al., 2002).
Strains of E. coli harbouring the aaiA gene, but not the aat or astA genes, were isolated from two patients, one of which showed an aggregative phenotype. Twenty-one patients had strains of E. coli that carried the astA gene, but not the aat or aaiA genes. Seven of the 21 (33 %) strains adhered to HEp-2 cells in an aggregative pattern (Table 2). Other pathogens were isolated from four of these 21 faecal samples, including Campylobacter species (3) and rotavirus (1). A DA pattern was observed in two of the isolates that carried the astA gene, and one strain exhibited an LA pattern of adhesion (Table 2). Taking the adhesion assay as the gold standard, the inclusion of the primers detecting the aaiA and astA genes to the aat PCR enabled the assay to identify eight additional EAEC isolates and 12 isolates that were not EAEC.
The astA gene was once considered characteristic of the EAEC group (Savarino et al., 1991), although it has since been found in only a subset of EAEC and has a broad distribution among other pathogenic and non-pathogenic E. coli. (Savarino et al., 1996; Zhou et al., 2002). Other workers have demonstrated that the prevalence of astA-positive E. coli in the stools of adults and children without diarrhoea can be as high as 38 % (Savarino et al., 1996; Vila et al., 1998). However, some E. coli strains isolated from humans with diarrhoea are found to harbour no known virulence factors other than EAST (Paiva de Sousa & Dubreuril, 2001; Nishikawa et al., 1999). We cannot comment on the ability to cause diarrhoea of the strains that carried the aat, aaiA or astA genes but did not exhibit characteristic EAEC adherence, as this study was not designed to assess the role of these putative virulence factors or to determine whether strains harbouring these genes cause disease.
Using the HEp-2 adhesion assay as the gold standard, the addition of primers detecting aaiA and astA to the aat PCR increased the number of EAEC isolates detected, but identified strains of E. coli that were not EAEC. The variety of genotypes exhibiting AA highlights the problems associated with developing a molecular diagnostic test for EAEC. Our PCR assay detects a variety of strains exhibiting characteristics of the EAEC group, making it a useful tool for identifying both typical and atypical EAEC.
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ACKNOWLEDGEMENTS
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We greatly appreciate the help of the healthcare scientists working at Yorkshire and Humber, Leeds HPA Laboratory, especially Jill Perkin and Hannah Armitage. We would also like to thank Dr Edward Dudley and Professor Jim Nataro (Center for Vaccine Development, University of Maryland, Baltimore) for providing us with the primers for the aaiA gene, and for all their help and advice during this study. This study was funded by the Food Standards Agency.
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TOP
INTRODUCTION
METHODS
