Detection of eae, bfpA, espA Genes on Diarrhoeagenic Strains of Escherichia coli Isolates

The Enteropathogenic Escherichia coli (EPEC) is one of pathogenic strain of diarrheagenic E. coli group in children and infant that occurs in developing countries. The significant virulence factors in pathogenic EPEC are eaeA (E. coli attachingeffacing ), bfpA (bundle-forming pilus A) and espA (encoding secreted protein A) genes. The use of DNA probes to detect the virulence genes in E. coli in Indonesia is not common yet. In this experiment the gene fragments of eae, bfpA, and espA were used as probes to detect the EPEC among E. coli isolates from stool specimensin of diarrheic children attending Public Health Centers in Yogyakarta. The DNA samples were isolated from 49 diarrheagenic E. coli isolates. The DNA probes of eae, bfpA and espA were obtained by amplification of DNA fragment of EPEC O126 using PCR technique. Furthermore, those probes were used to identify the presence of those genes among E. coli isolates using hybridization technique. The results showed that 42 (85.7%) + + isolates were espA , 25 isolates (51%) were eaeA (EPEC strains). Therefore among 25 isolates of EPEC, 20 isolates (80 %) + among EPEC were bfpA (typical EPEC strains).


Introduction
Enterophatogenic Escherichia coli (EPEC), an important paediatric diarrhea pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic 'attaching and effacing' (A/E) lesions on small intestinal enterocytes.EPEC adhesions that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and intimin (Cleary et al., 2004).The genetic determinants for the production of A/E lesion are located on the locus e n t e r o c y t e e f f a c e m e n t ( L E E ) , a pathogenicity islands that contains the genes Corresponding author: Susi Iravati, Department of Microbiology, Gadjah Mada University School of Medicine, Yogyakarta 55281, Indonesia.Telp: +62-274-6492458, Fax: +62-274-581876 membrane, where they form a pore through which other bacterial effector molecules, such as Tir, enter the host cell.Tir is the receptor for intimin, which is translocated via the EspA filament and EspB / EspD pore into the host cell and incorporated into the membrane.As well as interacting with intimin, this protein is also involved in p r o m o t i n g c y t o s k e l e t a l a c t i o n rearrangement in the host cell (Kuhne et al., 2004).
The eae gene, which is located in the 'locus of enterocyte effacement; (LEE) pathogenicity islands, and the bfpA gene, located on a plasmid called the EPEC adherence factor (EAF), have both been used for identification of EPEC and for subdivision of this group of bacteria into typical and atypical EPEC.E. coli strains with + the A/E genotype (eae ) that harbour the EAF + plasmid (bfpA ) are classified as 'typical EPEC'; most of these strains belong to certain O : H serotypes.Strains with the A/E genotype that do not posses the EAF plasmid (bfpA) are classified as 'atypical EPEC' (Nataro and Kaper, 1998).For many years, diagnosis of EPEC was based on O : H serotype identification (Levinde & Edelman, 1984).During the last two decades, the pathogenic mechanism of EPEC infection has been clarified, this has resulted in a change in diagnostic methods from serogrouping to phenotypic and genotypic methods (Afset et al., 2003).
Identification of diarrheagenic E. coli strains requires that these organisms be differentiated from non pathogenic members of the normal flora.Serotypic markers correlate, sometimes very closely, with specific categories of diarrheagenic, however these markers are rarely sufficient in and on themselves to reliably identify a strain as diarrheagenic.In addition to its limited sensitivity and specificity, serotyping is tedious and expensive and is performed reliably only by small number of reference laboratories.Thus, detection of d i a r r h e a g e n i c E .c o l i h a s f o c u s e d increasingly on the identification of characteristic which themselves determine the virulence of these organisms.This may include in vitro phenotypic assays which correlate with the presence virulence factor.Indeed, molecular method remain the most popular and most reliable techniques for differentiating diarrheagenic strains from non pathogenic members of the stool flora and distinguishing one category from another.Substantial progress has been made both in the development of nucleic acidbased probe technologies as well as PCR methods (Bekal et al., 2003).
The use of DNA probes in hybridization technique to detect the gene target on diarrheagenic E. coli in Indonesia has not been used routinely.The aim of this study is to identify the presence of virulence factor genes: eaeA (E. coli attaching-effacing), bfpA (bundle-forming pilus) and espA (encoding secreted proteinA) on E. coli strains among E. coli isolates from stool specimens of diarrheic children attending Public Health c e n t e r s i n Y o g y a k a r t a u s i n g t h e hybridization technique and DNA probes of eae, bfpA and espA .

Samples
Samples (stool specimens) were collected from dirrheic children under 5 years of age attending Public Health Centers in Yogyakarta.EPEC O126 was used as the positif control and E.coli ATCC 10536 non EPEC strain was used as the negative control, both were derived from Biofarma Laboratory, Bandung, Indonesia.Three set of primers espA, bfpA and eae, LB media, M9 (minimal media), assay media, PCR Core kit (Roche), Agarose, Dig DNA Labeling and Detection Kit (Roche).

Culture and identification methods
Culture and biochemical identification of E. coli were done according to standard microbiological methods (Madigan et al., 2002).Polymerase Chain Reaction (PCR) assay The eae, bfpA and espA probes were prepared by PCR amplification of the genes on EPEC O126.The following specific primers were used for PCR : a. Specific primer eae (Aranda et al., 2004), was received from Alpha DNA eae 1 : 5' -CTGAACGGCGATTAC GCGAA -3' eae 2 : 5' -CCAGACGATACGATC CAG -3' b. Specific primer bfpA (Aranda et al., 2004), was received from Alpha DNA bfpA 1 : 5' -AATGGTGCTTGCGCTT GCTGC -3' bfpA 2 : 5' -GCCGCTTTATCCAACCT GGTA -3' c. Specific primer espA (Knutton et al., 1998) was received from Cybergere Ab Forward : 5' -GCG AGT ACT TCG ACA TC -3' Reverse : 5' -TTA TTT ACC AAG GGA TAT -3' The amplification was carried out in volumes of 25 ml according PCR Core Kit m a n u a l w i t h t h e r m o c y c l e r .T h e amplification conditions were as follows:

DNA hybridization
Diarrheagenic E. coli were tested by hybridization with the specific DNA probes eaeA, espA and bfpA.The DNA hybridization reaction was carried out according to Dig Labeling Kit recommendation.The EPEC O126 strain was used as positive control for bfpA, eae, and espA probes, whereas the non EPEC strain (E. coli ATCC 10536) was used for negative control.

Results and Discussion
The eae, bfpA and espA genes amplification The results of eae, bfpA nd espA genes amplication from EPEC O126 strain are shown in Figure 1.The DNA marker was used to detect the size of the amplification products, as follows : the fragment of gene espA gene detected as a band of 579 bp, eaeA gene detected as a band of 917 bp and bfpA gene detected as a band of 326 bp.Those fragments were used as probes on dot blot hybridization The eaeA, bfpA and espA genes detection All 49 E. coli isolates were tested by hybridization technique using specific DNA probes of eaeA, bfpA, and espA as shown on Table 1. and Figure 2, 3, and 4.Among the 49 + E. coli isolates, 42 isolates (85.7 %) were espA  bfpA .This study showed that 51% of E. coli isolates from diarrheic children were identified as EPEC that consist of 20% Typical EPEC and 80% Atypical EPEC.This results was in agreement with the results of other studies which had shown that 30 to 40% of infant diarrhea can be attributed to EPEC in developing countries (Naro and Kaper, 1998).

Conclusion
DNA probes of eae, bfpA and espA could be used for detection of typical and atypical EPEC of diarrheagenic E. coli isolates, instead of using serotyping method.