Cargando…

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates

BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study...

Descripción completa

Detalles Bibliográficos
Autores principales: Smiljanic, M., Kaase, M., Ahmad-Nejad, P., Ghebremedhin, B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504714/
https://www.ncbi.nlm.nih.gov/pubmed/28693493
http://dx.doi.org/10.1186/s12941-017-0223-z
_version_ 1783249329947934720
author Smiljanic, M.
Kaase, M.
Ahmad-Nejad, P.
Ghebremedhin, B.
author_facet Smiljanic, M.
Kaase, M.
Ahmad-Nejad, P.
Ghebremedhin, B.
author_sort Smiljanic, M.
collection PubMed
description BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla (KPC), bla (VIM), bla (NDM) or bla (OXA) were detected. Both RT-PCR assays detected all bla (KPC), bla (VIM) and bla (NDM) in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.
format Online
Article
Text
id pubmed-5504714
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-55047142017-07-12 Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates Smiljanic, M. Kaase, M. Ahmad-Nejad, P. Ghebremedhin, B. Ann Clin Microbiol Antimicrob Research BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla (KPC), bla (VIM), bla (NDM) or bla (OXA) were detected. Both RT-PCR assays detected all bla (KPC), bla (VIM) and bla (NDM) in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii. BioMed Central 2017-07-10 /pmc/articles/PMC5504714/ /pubmed/28693493 http://dx.doi.org/10.1186/s12941-017-0223-z Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Smiljanic, M.
Kaase, M.
Ahmad-Nejad, P.
Ghebremedhin, B.
Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title_full Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title_fullStr Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title_full_unstemmed Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title_short Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates
title_sort comparison of in-house and commercial real time-pcr based carbapenemase gene detection methods in enterobacteriaceae and non-fermenting gram-negative bacterial isolates
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504714/
https://www.ncbi.nlm.nih.gov/pubmed/28693493
http://dx.doi.org/10.1186/s12941-017-0223-z
work_keys_str_mv AT smiljanicm comparisonofinhouseandcommercialrealtimepcrbasedcarbapenemasegenedetectionmethodsinenterobacteriaceaeandnonfermentinggramnegativebacterialisolates
AT kaasem comparisonofinhouseandcommercialrealtimepcrbasedcarbapenemasegenedetectionmethodsinenterobacteriaceaeandnonfermentinggramnegativebacterialisolates
AT ahmadnejadp comparisonofinhouseandcommercialrealtimepcrbasedcarbapenemasegenedetectionmethodsinenterobacteriaceaeandnonfermentinggramnegativebacterialisolates
AT ghebremedhinb comparisonofinhouseandcommercialrealtimepcrbasedcarbapenemasegenedetectionmethodsinenterobacteriaceaeandnonfermentinggramnegativebacterialisolates