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.
collections
- retrospective theses [1604]