Ecology of pathogenic Escherichia coli in water : survival and molecular detection

Abstract

Recently there has been a surge of interest in the ability of enterohemmorhagic Escherichia coli (EHEC) to survive in aquatic environments. It has been speculated a correlation may exist between the genotype of an E. coli strain and its ability to persist in an aquatic environment. One of the objectives of this study was to determine if any correlation could be made between the persistence of EHEC strains in natural well water and their ERIC-genotype. Sixteen strains of EHEC were monitored, individually, in untreated well water microcosms incubated at 10°C and 22°C for 56 days. The strains were selected from three serogroups (026, 0111 and 0157) and represented six distinct genotypes determined by ERIC-PCR. The microcosms were prepared, in triplicate, each sample having a final cell concentration of approximately 10^ cfu/ml well water. Levine Eosin Methylene Blue agar was used to determine the cell density of culturable E. co//cells. At 10°C, cell density declined depending on the strains by 1.78 to 6.07 log units in 8 weeks, with six strains falling below the detection limit of 0.8 log cfu/ml by day 56. Of the ten persisting strains, four showed superior survival with cell density decreasing to an average of approximately 5 log cfu/ml while the remaining six strains showed moderate levels of survival, decreasing to a average cell density of approximately 3.5 log cfu/ml. At 22°C all strains eventually dropped by 4.87 to 6.52 log in 56 days with 14 strains dropping below the detection limit. The 16 strains demonstrated highly variable levels of survival with no correlation between ERIC-genotype and the strain’s ability to persist being evident. The second objective of the study was to develop a molecular detection method for pathogenic E. coli employing multiplex-PCR and DIG-labeled DNA probes. The molecular detection of EHEC and other pathogenic £. coli of virotypes, entertoxigenic (ETEC), enteropathogenic (EPEC) and enteroinvasive (EIEC), has been the topic of numerous studies. A multiplex PCR-DNA probing assay to detect the four major E. coli virotypes was developed. Six highly specific PCR primer sets and DIG-labeled chemiluminescent probes were designed to target the shiga-like toxin I and II genes {sitl and sitll) of EHEC, heat stable and heat labile toxin genes (hs and hl) of ETEC, EPECadherence factor gene (eaf) of EPEC and the invasiveness plasmid (iai) of EIEC. The primer sets generate amplicons 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers were tested for specificity against 31 pathogenic £. coli strains, various non-pathogenic £. coli strains and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity for strains from their corresponding virotypes and minimal non-specific reaction with the non-target E. coli and control bacterial strains. To improve the specificity of the multiplex-PCR amplification, six DIG-labeled oligonucleotide probes were designed to hybridize with the six multiplex-PCR amplicons. Chemiluminescent detection of probe hybridization against multiplex-PCR dot blots revealed high degrees of specificity of probes for the target amplicons. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major E. coli virotypes.