Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of r...

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Autores principales:	Claudia Stein, Oliwia Makarewicz, Yvonne Pfeifer, Christian Brandt, João Costa Ramos, Mareike Klinger, Mathias W Pletz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/20bb9b8792804a3cbda413a501efd23c
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spelling	oai:doaj.org-article:20bb9b8792804a3cbda413a501efd23c2021-11-18T08:48:13ZDirect RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).1932-620310.1371/journal.pone.0080079https://doaj.org/article/20bb9b8792804a3cbda413a501efd23c2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24224038/?tool=EBIhttps://doaj.org/toc/1932-6203The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.Claudia SteinOliwia MakarewiczYvonne PfeiferChristian BrandtJoão Costa RamosMareike KlingerMathias W PletzPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e80079 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Claudia Stein Oliwia Makarewicz Yvonne Pfeifer Christian Brandt João Costa Ramos Mareike Klinger Mathias W Pletz Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
description	The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.
format	article
author	Claudia Stein Oliwia Makarewicz Yvonne Pfeifer Christian Brandt João Costa Ramos Mareike Klinger Mathias W Pletz
author_facet	Claudia Stein Oliwia Makarewicz Yvonne Pfeifer Christian Brandt João Costa Ramos Mareike Klinger Mathias W Pletz
author_sort	Claudia Stein
title	Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
title_short	Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
title_full	Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
title_fullStr	Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
title_full_unstemmed	Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
title_sort	direct rna-based detection and differentiation of ctx-m-type extended-spectrum β-lactamases (esbl).
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/20bb9b8792804a3cbda413a501efd23c
work_keys_str_mv	AT claudiastein directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT oliwiamakarewicz directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT yvonnepfeifer directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT christianbrandt directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT joaocostaramos directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT mareikeklinger directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl AT mathiaswpletz directrnabaseddetectionanddifferentiationofctxmtypeextendedspectrumblactamasesesbl
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Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).

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