Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.

Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigat...

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Autores principales:	Vinicius Silva Castro, Rodrigo Ortega Polo, Eduardo Eustáquio de Souza Figueiredo, Emmanuel Wihkochombom Bumunange, Tim McAllister, Robin King, Carlos Adam Conte-Junior, Kim Stanford
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/43a9cb56079c419c8c2f9d1f2baf2468
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spelling	oai:doaj.org-article:43a9cb56079c419c8c2f9d1f2baf24682021-12-02T20:08:31ZInconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.1932-620310.1371/journal.pone.0257168https://doaj.org/article/43a9cb56079c419c8c2f9d1f2baf24682021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0257168https://doaj.org/toc/1932-6203Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.Vinicius Silva CastroRodrigo Ortega PoloEduardo Eustáquio de Souza FigueiredoEmmanuel Wihkochombom BumunangeTim McAllisterRobin KingCarlos Adam Conte-JuniorKim StanfordPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 9, p e0257168 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Vinicius Silva Castro Rodrigo Ortega Polo Eduardo Eustáquio de Souza Figueiredo Emmanuel Wihkochombom Bumunange Tim McAllister Robin King Carlos Adam Conte-Junior Kim Stanford Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
description	Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.
format	article
author	Vinicius Silva Castro Rodrigo Ortega Polo Eduardo Eustáquio de Souza Figueiredo Emmanuel Wihkochombom Bumunange Tim McAllister Robin King Carlos Adam Conte-Junior Kim Stanford
author_facet	Vinicius Silva Castro Rodrigo Ortega Polo Eduardo Eustáquio de Souza Figueiredo Emmanuel Wihkochombom Bumunange Tim McAllister Robin King Carlos Adam Conte-Junior Kim Stanford
author_sort	Vinicius Silva Castro
title	Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
title_short	Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
title_full	Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
title_fullStr	Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
title_full_unstemmed	Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.
title_sort	inconsistent pcr detection of shiga toxin-producing escherichia coli: insights from whole genome sequence analyses.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/43a9cb56079c419c8c2f9d1f2baf2468
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Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses.

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