Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

Abstract

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

Keywords: PMA; STEC; Stx phages; VBNC; bacteriophages; qPCR.

Figure 1

Methods used to investigate the cause of PCR-positive/culture-negative samples for STEC. The methods were applied to the examination of sheep recto-anal junction swab samples. The methods were as follows: (a) real-time PCR (qPCR) and culture method; (b) propidium monoazide pre-treatment and real-time PCR (qPCR-PMA); (c) bacteriophage detection and plaque assay; (d) phage induction. qPCR = real-time PCR; CFS = cell free supernatant; PMA = propidium monoazide.

Figure 2

Results of the approaches used in this study. (a) Results of the STEC screening (method a) of the o-RAJ samples (n = 155). (b) Results of the combination of the methods used to investigate the qPCR-positive/culture-negative phenomenon (n = 53).