RT-PCR: a potential solution to the detection of viable-but-non-culturable Escherichia Coli 0157: H7 in the environment

Despite evidence that selective enrichment inhibits the recovery of stressed E. coli O157,
the nature of the effect of selective agents on the pathogen has not been identified to date.
Initial studies were completed to elucidate the nature of the response of E. coli O157: H7 12900 to selective agents. Two-dimensional gel electrophoresis was used to evaluate the
effect of selective agents on gene expression.
Selective agents reduce the recovery of viable-but non-culturable pathogens such as
E. coli O157, which has implications for the efficacy of cultural isolation techniques. A
sensitive and accurate identification technique is required and RT-PCR may facilitate the
detection of viable STEC in quality control, environmental and clinical samples.
Conditions that positively regulate virulence genes could be utilised to induce the
transcription of these genes in viable-but-non-culturable bacteria and the subsequent
detection of STEC specific virulence gene transcripts would provide evidence that
samples contain infective STEC. The work reported here was completed with an aim to
developing such an RT-PCR assay.
Although the stx genes are obvious targets, toxigenic strains could not be employed in the
present study due to the absence of a category three facility. Consequently, RT-PCR
assays were designed to target the pO157 hlyA and katP genes and studies were
completed with non-toxigenic E. coliOlSl: H7 NCTC 12900. E. coli O157: H7 NCTC
12900 lacks the stx genes but appears to survive as well as toxigenic E. coli O157 strains
despite its non-toxigenic phenotype (Bolton et al, 1999). Other non-toxigenic E. coli
0157 strains are available, but NCTC 12900 is sold commercially and is recommended
for use in quality control studies as the sole representative of STEC strains in a panel of
other intestinal pathogens (www.phls.co.uk/labservices/nctc/qcrefsets.htm).
Consequently, although the use of virulent clinical strains would have provided definitive evidence concerning the factors regulating hlyA and katP, E. coli O157: H7 NCTC 12900
was considered a suitable model for use in developing and assessing initial RT-PCR
based viability assays. The work reported in subsequent chapters aimed to identify
conditions that can be employed to initiate hlyA and katP transcription in the laboratory,
facilitating the development and optimisation of an RT-PCR assay to detect viable-butnon-
culturable E. coli 0157 in environmental samples. [Taken from section 1.9 in absence of an abstract]

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