Understanding the occurrence of polymerase chain reactions-positive and culture-negative for Shiga toxin producing Escherichia coli in samples from beef production chain

Abstract

Molecular epidemiological methods have been employed to detect pathogens and transmission pathways, for disease surveillance, outbreak investigation, outbreak monitoring and control. Molecular methods such as polymerase chain reactions (PCR) is used to assess the positivity rate of virulent gene(s) with pathogens, but in many cases, cultural isolation of the pathogen may not be possible in PCR positive cases. This dichotomy between the outcome of results may be associated with low number of cells compared with the large population of background microflora, presence of viable but non-culturable cells, loss of virulence gene (s) after subculture, and the high sensitivity of the PCR assay. Shiga-toxigenic Escherichia coli (STEC) was used as a model for investigating this phenomenon. In this study, duplex PCR was used to screen 335 abattoir and 303 beef retail outlets selective broth enriched samples for the presence of stx1 and stx2 genes. Subsequent culture isolation of stx-positive broth samples was carried out. The overall STEC positivity determined by PCR in 335 and 303 abattoir and beef retail outlets in selective enrichment broth samples, respectively was 35.2% (118/335; 95% CI: 30.1 - 40.6) and 12.5% (38/303; 95% CI: 9 - 16.8). Only 24 (20%; 24/118) abattoir and 8 (21%; 8/38) retail outlet stx-positive samples were culturable. Both yielded only 51 isolates; 30 isolates for abattoir samples and 21 isolates for beef retail outlets, respectively. Our results confirm the dichotomy of PCR positive/culture negative samples, and from an epidemiological perspective, it is recommended that the use of only PCR to detect virulence genes in broth cultures should be acceptable where isolation is not achievable. This may be the best method for generating relevant epidemiologic data for disease control.