Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples
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...
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MDPI AG
2020-04-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/4/587 |
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| author | Guerrino Macori Siobhán C. McCarthy Catherine M. Burgess Séamus Fanning Geraldine Duffy |
| author_facet | Guerrino Macori Siobhán C. McCarthy Catherine M. Burgess Séamus Fanning Geraldine Duffy |
| author_sort | Guerrino Macori |
| collection | DOAJ |
| description | 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 <i>Escherichia coli</i> (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 <i>stx1</i> and <i>stx2</i> 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 <i>stx</i> phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free <i>stx</i> 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. |
| first_indexed | 2024-03-10T20:23:35Z |
| format | Article |
| id | doaj.art-c8ab0beb04794fccbc3eb7ff44c3330f |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T20:23:35Z |
| publishDate | 2020-04-01 |
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| series | Microorganisms |
| spelling | doaj.art-c8ab0beb04794fccbc3eb7ff44c3330f2023-11-19T22:01:32ZengMDPI AGMicroorganisms2076-26072020-04-018458710.3390/microorganisms8040587Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative SamplesGuerrino Macori0Siobhán C. McCarthy1Catherine M. Burgess2Séamus Fanning3Geraldine Duffy4Teagasc Food Research Centre, Ashtown, Dublin 15, IrelandTeagasc Food Research Centre, Ashtown, Dublin 15, IrelandTeagasc Food Research Centre, Ashtown, Dublin 15, IrelandUCD-Centre for Food Safety, School of Public Health, Physiotherapy & Sports Science, University College Dublin, Belfield, Dublin D04 N2E5, IrelandTeagasc Food Research Centre, Ashtown, Dublin 15, IrelandMolecular 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 <i>Escherichia coli</i> (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 <i>stx1</i> and <i>stx2</i> 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 <i>stx</i> phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free <i>stx</i> 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.https://www.mdpi.com/2076-2607/8/4/587STECqPCRbacteriophagesStx phagesPMAVBNC |
| spellingShingle | Guerrino Macori Siobhán C. McCarthy Catherine M. Burgess Séamus Fanning Geraldine Duffy Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples Microorganisms STEC qPCR bacteriophages Stx phages PMA VBNC |
| title | Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples |
| title_full | Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples |
| title_fullStr | Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples |
| title_full_unstemmed | Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples |
| title_short | Investigation of the Causes of Shigatoxigenic <i>Escherichia coli</i> PCR Positive and Culture Negative Samples |
| title_sort | investigation of the causes of shigatoxigenic i escherichia coli i pcr positive and culture negative samples |
| topic | STEC qPCR bacteriophages Stx phages PMA VBNC |
| url | https://www.mdpi.com/2076-2607/8/4/587 |
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