Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak

ABSTRACT The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epid...

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Autores principales:	Santosh K. Bikkarolla, Viveka Nordberg, Fredrika Rajer, Vilhelm Müller, Muhammad Humaun Kabir, Sriram KK, Albertas Dvirnas, Tobias Ambjörnsson, Christian G. Giske, Lars Navér, Linus Sandegren, Fredrik Westerlund
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR/Cas9 optical DNA mapping antibiotic resistance intensive care unit plasmids Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/730b4c91ff0a497f8391279efab4bee3
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spelling	oai:doaj.org-article:730b4c91ff0a497f8391279efab4bee32021-11-15T16:22:10ZOptical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak10.1128/mBio.00347-192150-7511https://doaj.org/article/730b4c91ff0a497f8391279efab4bee32019-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00347-19https://doaj.org/toc/2150-7511ABSTRACT The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings. IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.Santosh K. BikkarollaViveka NordbergFredrika RajerVilhelm MüllerMuhammad Humaun KabirSriram KKAlbertas DvirnasTobias AmbjörnssonChristian G. GiskeLars NavérLinus SandegrenFredrik WesterlundAmerican Society for MicrobiologyarticleCRISPR/Cas9optical DNA mappingantibiotic resistanceintensive care unitplasmidsMicrobiologyQR1-502ENmBio, Vol 10, Iss 4 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 optical DNA mapping antibiotic resistance intensive care unit plasmids Microbiology QR1-502
spellingShingle	CRISPR/Cas9 optical DNA mapping antibiotic resistance intensive care unit plasmids Microbiology QR1-502 Santosh K. Bikkarolla Viveka Nordberg Fredrika Rajer Vilhelm Müller Muhammad Humaun Kabir Sriram KK Albertas Dvirnas Tobias Ambjörnsson Christian G. Giske Lars Navér Linus Sandegren Fredrik Westerlund Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
description	ABSTRACT The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings. IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.
format	article
author	Santosh K. Bikkarolla Viveka Nordberg Fredrika Rajer Vilhelm Müller Muhammad Humaun Kabir Sriram KK Albertas Dvirnas Tobias Ambjörnsson Christian G. Giske Lars Navér Linus Sandegren Fredrik Westerlund
author_facet	Santosh K. Bikkarolla Viveka Nordberg Fredrika Rajer Vilhelm Müller Muhammad Humaun Kabir Sriram KK Albertas Dvirnas Tobias Ambjörnsson Christian G. Giske Lars Navér Linus Sandegren Fredrik Westerlund
author_sort	Santosh K. Bikkarolla
title	Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
title_short	Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
title_full	Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
title_fullStr	Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
title_full_unstemmed	Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
title_sort	optical dna mapping combined with cas9-targeted resistance gene identification for rapid tracking of resistance plasmids in a neonatal intensive care unit outbreak
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/730b4c91ff0a497f8391279efab4bee3
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Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak

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