Development of PCR for screening of enteroaggregative Escherichia coli

Abstract

In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe. A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region. Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene. Pure cultures of E. coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test. Of 50 E. coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR. All 43 of these strains reacted with the EAggEC probe. Six EAggEC strains gave negative results by both molecular techniques. In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E. coli demonstrated a positive PCR result. The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens. Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory.

Public health risks associated with Enteroaggregative Escherichia coli (EAEC) as a food‐borne pathogen

“…In 1995 the first PCR tool was developed based on the sequence of the EcoRI/PstI fragment of pCVD432 plasmid, corresponding to the CVD432 probe (Schmidt et al, 1995). Such a PCR was evaluated against the cell culture adhesion and the hybridization assays and returned a 97.7% concordance.…”

“…In detail, most of the 456 E. coli strains negative to the adhesion assay included in the experimental design were negative to both the PCR and hybridization, while five of the seven strains displaying the aggregative adhesion pattern onto Hep-2 cells monolayers were positive to both the molecular assays. Finally, a few strains negative by the adhesion test were positive when subjected to the molecular approach, lowering its overall specificity (Schmidt et al, 1995). An additional PCR primers pair was designed on the sequence of the CVD432 probe sequence and included into a multiplex assay for the detection of diarrhoeagenic E. coli together with primers specific for the eae, stx, st, lt, ipaH genes (Toma et al, 2003).…”

“…The PCR tool based on the CVD432 probe sequence deployed by Schmidt et al (1995) was used in a study aiming at assessing the importance of EAEC as aetiological agent of acute diarrhoea among children in Calcutta, India, in parallel with the adhesion test conducted with the HeLa cultured cells (Dutta et al, 1999). A blind comparison of the two methods was done using the E. coli strains isolated from 254 children with acute diarrhoea and returned a sensitivity value for the PCR of 78.8% and a specificity of 97.5% when the adhesion test was used as the gold standard assay (Dutta et al, 1999).…”

“…In 2003, EAEC isolates displaying the enteroaggregative pattern of adhesion on cultured HeLa cells were screened by PCR for the presence of a panel of genes present on the large plasmid pCVD432 (Tsai et al, 2003). Specific primers were designed on the sequences of the genes astA, aafA and aggA (Tsai et al, 2003) and used for the screening in addition to the CVD432 primers deployed by Schmidt et al (1995). The study included six E. coli strains with the typical stacked brick adhesion and about 400 negative strains counting either laboratory strains (60 isolates) or isolates from the Taichung Veterans General Hospital, Taichung, Taiwan (337 isolates).…”

“…In many cases most of the plasmid markers used in all the PCR assays described seem to be adequate to correctly identify EAEC (Schmidt et al, 1995;Czeczulin et al, 1999;Dutta et al, 1999;Sheikh et al, 2002;Tsai et al, 2003;Aranda et al, 2007;Brandal et al, 2007;Muller et al, 2007;Cordeiro et al, 2008;Gomez-Duarte et al, 2009;Baranzoni et al, 2014). The variability of the plasmid structure and sequence, and the possibility that this mobile genetic element may be lost has led to the conclusion that chromosomal markers had to be included in the molecular screening assays.…”