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Tracking antibiotic resistance Sequencing plasmids can reveal the transmission of resistance among bacteria from patients in a clinical setting
A
ccording to the World Health Organization (1), “Antimicrobial resistance…threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi.…A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.” Often, antibiotic-resistance genes acquired by bacteria are associated with mobile genetic elements that mediate their exchange between pathogens, or between commensal and pathogenic bacterial populations. Thus, the genetic context in which an antibiotic-resistance gene is placed can reflect its mobility within the bacterial population. Although the presence of resistance genes in clinical isolates can be detected with either polymerase chain reaction technology or high-throughput short–read length DNA sequencing, their precise genetic context may be overlooked because of the limitations of these methodologies. As Conlan et al. (2) demonstrate, the use of single-molecule long-read DNA sequencing can address this limitation and opens the way to track the transmission of antibiotic-resistant bacteria in a health care environment. The transfer of genetic elements between bacteria, known as horizontal transfer (3), can occur through plasmids, small DNA molecules that are separate from bacterial chromosomal DNA. Conlan et al. applied long-read genome sequencing to identify plasmids harboring the gene blaKPC, which encodes a carbapenemase that hydrolyzes carbapenem antibiotics. This analysis was carried out after an outbreak in 2011 at the U.S. National Institutes of Health Clinical Center in which six infected patients died from carbapenem-resistant Klebsiella pneumoniae (4). Conlan et al. surveyed more than 1000 patients for carbapenemresistant bacteria. The bacterial genomes from such infected patients were analyzed
1
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia. 2 Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, QLD 4072, Australia. E-mail: [email protected]
1454
with long-read sequencing (single-molecule real-time sequencing). Sixty-three plasmid sequences were completed from patient or hospital environmental isolates, providing a unique snapshot of plasmid complexity from a single geographical site. The sequences allowed elucidation of the fine detail of plasmid diversity that would not have been obvious otherwise. Two patients infected with near-identical
Long-read sequence Bacterial plasmids Track transmission by following plasmid diversity (resistance genes in context)
Hospital environment Patients with antibiotic-resistant bacteria
Antibioticresistant bacteria
Short-read sequence Plasmid fragments
Lacks detail to track plasmid diversity
Context. Long-read sequencing can resolve complete plasmids from bacterial genome assemblies. Plasmid comparisons can help to track the dissemination of antimicrobial-resistance genes among pathogenic bacteria in a clinical setting. By contrast, short-read sequencing will generally produce collapsed repeats at repeated locations such as insertion sequences (black) that are dispersed throughout the plasmid backbone (white), making the context of antibiotic-resistance genes (pink, blue, yellow) difficult to discern.
carbapenemase-resistant Enterobacter cloacae strains harbored blaKPC on the same plasmid (pKPC-47e), triggering increased surveillance measures. Shared carbapenemase-encoding plasmids were found in three different bacterial genera epidemiologically linked with hospital environments (sinks). Of five patients who presented with carbapenemase-resistant Klebsiella sp., long-read genome sequencing associated one of these patients to the original 2011 outbreak, indicating nosocomial transmission. Long-read genome sequencing unequivocally ruled out a link with the 2011 outbreak in the remaining cases, despite epidemiological evidence suggesting otherwise. Carriage of shared plasmids was observed between a patient isolate and isolates of other bacterial species present in the ward environment. For several cases,
Plasmids play a key role in disseminating antimicrobial-resistance genes among pathogenic bacteria. Resistance genes often aggregate in modular clusters (5) that enable resistance to persist even in the absence of direct selection. Some insertion sequences (IS) (6), such as IS26, sandwich resistance genes into a composite transposon that can mobilize elsewhere. Other elements, such as ISEcp1, mobilize adjacent genes, leading to multiple copies of resistance genes within the genome. The multiplicity of insertion sequences and other repetitive elements is a major confounding factor in standard short-read genome assembly, which can lead to the fragmentation of chromosome and plasmid sequence assemblies. In this sense, plasmids may be viewed as the “dark matter” of short-read bacterial genome assemblies, with many sciencemag.org SCIENCE
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
ILLUSTRATION: V. ALTOUNIAN/SCIENCE
By Scott A. Beatson1,2 and Mark J. Walker1,2
compelling evidence to exclude nosocomial transmission may have been gathered from short-read sequencing, but long-read sequencing not only produced the required diagnostic result, but additionally provided reference sequences for all chromosomes and plasmids to aid future surveillance. Plasmid comparisons also disproved the hypothesis that transfer of a blaKPC plasmid had occurred between K. pneumoniae and E. cloacae within a patient from the original outbreak (4); long-read genome sequencing showed discrete genetic contexts for the two blaKPC alleles, indicating independent acquisition. Long-read genome sequencing also proved critical for determining the precise genomic context of blaKPC alleles in cases where more than one blaKPC gene was present in a single isolate.
Downloaded from www.sciencemag.org on December 10, 2014
MICROBIOLOGY
large-scale genomic studies conspicuously avoiding the complexities of plasmid structure. Genomic comparisons such as that described by Conlan et al. reveal how the dynamism in the structure and arrangement of resistance elements can only be realized by “closing” plasmid genomes with long-read sequencing (see the figure). Traditional Sanger sequencing is the gold standard for the analysis and assembly of complete plasmid sequences from antibiotic-resistant strains of bacteria. This approach may suffer from the need to isolate or subclone individual high molecular weight plasmids before sequencing (7), which is often technically difficult, timeconsuming, and costly, and may be intractable for multiple plasmids. Short-read sequencing technologies can affordably produce an assembly of a bacterial genome that contains nonrepetitive sequences typically in hundreds of “contigs” separated by “collapsed repeats” indicative of multiple copies of the same sequence located in several different locations within the genome. These repeats are often mobile elements such as insertion sequences that may be found in multiple copies on plasmids, thus making it difficult to assemble plasmid sequences. Cataloging the collection of antibioticresistance genes in any particular bacterium is relatively straightforward, but determining how these genes fit together within plasmids, which is critical for understanding how these elements spread in clinical settings, can be more difficult. By contrast, the genome sequences produced through long-read sequencing offer a complete picture of the plasmid content of a bacterium, including the number, position, and context within mobile elements of every acquired antibiotic-resistance gene. Long-read genome assembly offers clear advantages for the resolution of complete plasmid sequences that can discriminate plasmid diversity, antimicrobial-resistance gene context, and multiplicity. Such information will enhance our understanding of plasmid carriage, transfer, epidemiology, and evolution. REFERENCES
1. WHO, Antimicrobial resistance: Global report on surveillance 2014 (2014). 2. S. Conlan et al., Sci. Transl. Med. 254, 254ra126 (2014). 3. H. Ochman, J. G. Lawrence, E. A. Groisman, Nature 405, 299 (2000). 4. E. S. Snitkin et al. Sci. Transl. Med. 4, 148ra116 (2012). 5. R. M. Hall, C. M. Collis, Mol. Microbiol. 15, 593 (1995). 6. J. Mahillon, M. Chandler, Microbiol. Mol. Biol. Rev. 62, 725 (1998). 7. C. Venturini, S. A. Beatson, S. P. Djordjevic, M. J. Walker, FASEB J. 24, 1160 (2010). 10.1126/science.1260471
NANOMATERIALS
Making strong nanomaterials ductile with gradients Microstructures that increase metal crystallite size from nanoscale with surface depth are both strong and ductile nisms of the extremely fine grains that induces cracking. By applying surface plastic deformation onto a bulk coarse-grained metal, a distinctive microstructure is generated from the strain gradient: a nanograined layer (several tens of micrometers thick) covers the coarse-grained substrate with a graded variation of grain size in between (see the first figure). Tensile tests of the heterogeneously structured Cu cylinder (pulling the sample along the long axis) showed that the top
By K. Lu
S
teels can be made stronger, tougher, or more resistant to corrosion either by changing composition (adding in more carbon or other elements) or by modifying their microstructures. An extreme microstructural route for strengthening materials is to reduce the crystallite size from the micrometer scale (“coarse-grained”) to the nanoscale. Nanograined aluminum or copper (Cu) may become even harder than highstrength steels, but these materials 0 (m) can be very brittle and crack when pulled (deformed in tension), apparently because strain becomes localized and resists deformation. However, nanograined metals can be plastically deformed under 50 compression or rolling at ambient temperature, implying that moderate deformation can occur if the cracking process is suppressed. Tremendous efforts have been made to explore how to suppress strain lo100 calization in tensioned nanomaterials and make them ductile. Gradient microstructures, in which the grain size increases from nanoscale at the surface to coarse-grained in the core, were recently discovered to be an effective approach to improving 150 ductility (1–4). One advantage of metals in structural applications is that they “sigGradient nanograined structure. After a surface mechanical nal” their impending failure—they grinding treatment to copper, grain sizes are about 20 nm in the can deform and crack to some extopmost treated surface (outlined by dashed line) and increase tent before they completely fail. gradually to the microscale with depth. However, when a piece of fully nanograined copper is pulled, catastrophic nanograined layer and the coarse-grained failure occurs immediately when the load core can be elongated coherently by as much exceeds its yield strength (the point at as ~60% before failure—comparable to that which permanent deformation begins), just in conventional Cu, but the sample’s yield like most ceramics and other normal fragstrength is doubled (1). Almost no tensile ile materials. Such tensile brittleness is an elongation was observed in the nanograined Achilles’ heel of nanomaterials that hinders layer as it was removed from the substrate. their technological applications; for examEvidently, the observed extraordinary tensile ple, they cannot be strengthened by work Shenyang National Laboratory for Materials Science, Institute hardening. The microscopic origin appears of Metal Research, Chinese Academy of Sciences, Shenyang to be early necking (decrease in cross sec110016, China, and Herbert Gleiter Institute of Nanoscience, tion) induced by strain localization prior Nanjing University of Science & Technology, Nanjing 210094, to activation of plastic deformation mechaChina. E-mail: [email protected]
SCIENCE sciencemag.org
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
1455
Tracking antibiotic resistance Scott A. Beatson and Mark J. Walker Science 345, 1454 (2014); DOI: 10.1126/science.1260471
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of December 10, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/345/6203/1454.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/345/6203/1454.full.html#related This article cites 6 articles, 3 of which can be accessed free: http://www.sciencemag.org/content/345/6203/1454.full.html#ref-list-1 This article has been cited by 1 articles hosted by HighWire Press; see: http://www.sciencemag.org/content/345/6203/1454.full.html#related-urls This article appears in the following subject collections: Microbiology http://www.sciencemag.org/cgi/collection/microbio
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on December 10, 2014
This copy is for your personal, non-commercial use only.
Tracking antibiotic resistance Sequencing plasmids can reveal the transmission of resistance among bacteria from patients in a clinical setting
A
ccording to the World Health Organization (1), “Antimicrobial resistance…threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi.…A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.” Often, antibiotic-resistance genes acquired by bacteria are associated with mobile genetic elements that mediate their exchange between pathogens, or between commensal and pathogenic bacterial populations. Thus, the genetic context in which an antibiotic-resistance gene is placed can reflect its mobility within the bacterial population. Although the presence of resistance genes in clinical isolates can be detected with either polymerase chain reaction technology or high-throughput short–read length DNA sequencing, their precise genetic context may be overlooked because of the limitations of these methodologies. As Conlan et al. (2) demonstrate, the use of single-molecule long-read DNA sequencing can address this limitation and opens the way to track the transmission of antibiotic-resistant bacteria in a health care environment. The transfer of genetic elements between bacteria, known as horizontal transfer (3), can occur through plasmids, small DNA molecules that are separate from bacterial chromosomal DNA. Conlan et al. applied long-read genome sequencing to identify plasmids harboring the gene blaKPC, which encodes a carbapenemase that hydrolyzes carbapenem antibiotics. This analysis was carried out after an outbreak in 2011 at the U.S. National Institutes of Health Clinical Center in which six infected patients died from carbapenem-resistant Klebsiella pneumoniae (4). Conlan et al. surveyed more than 1000 patients for carbapenemresistant bacteria. The bacterial genomes from such infected patients were analyzed
1
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia. 2 Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, QLD 4072, Australia. E-mail: [email protected]
1454
with long-read sequencing (single-molecule real-time sequencing). Sixty-three plasmid sequences were completed from patient or hospital environmental isolates, providing a unique snapshot of plasmid complexity from a single geographical site. The sequences allowed elucidation of the fine detail of plasmid diversity that would not have been obvious otherwise. Two patients infected with near-identical
Long-read sequence Bacterial plasmids Track transmission by following plasmid diversity (resistance genes in context)
Hospital environment Patients with antibiotic-resistant bacteria
Antibioticresistant bacteria
Short-read sequence Plasmid fragments
Lacks detail to track plasmid diversity
Context. Long-read sequencing can resolve complete plasmids from bacterial genome assemblies. Plasmid comparisons can help to track the dissemination of antimicrobial-resistance genes among pathogenic bacteria in a clinical setting. By contrast, short-read sequencing will generally produce collapsed repeats at repeated locations such as insertion sequences (black) that are dispersed throughout the plasmid backbone (white), making the context of antibiotic-resistance genes (pink, blue, yellow) difficult to discern.
carbapenemase-resistant Enterobacter cloacae strains harbored blaKPC on the same plasmid (pKPC-47e), triggering increased surveillance measures. Shared carbapenemase-encoding plasmids were found in three different bacterial genera epidemiologically linked with hospital environments (sinks). Of five patients who presented with carbapenemase-resistant Klebsiella sp., long-read genome sequencing associated one of these patients to the original 2011 outbreak, indicating nosocomial transmission. Long-read genome sequencing unequivocally ruled out a link with the 2011 outbreak in the remaining cases, despite epidemiological evidence suggesting otherwise. Carriage of shared plasmids was observed between a patient isolate and isolates of other bacterial species present in the ward environment. For several cases,
Plasmids play a key role in disseminating antimicrobial-resistance genes among pathogenic bacteria. Resistance genes often aggregate in modular clusters (5) that enable resistance to persist even in the absence of direct selection. Some insertion sequences (IS) (6), such as IS26, sandwich resistance genes into a composite transposon that can mobilize elsewhere. Other elements, such as ISEcp1, mobilize adjacent genes, leading to multiple copies of resistance genes within the genome. The multiplicity of insertion sequences and other repetitive elements is a major confounding factor in standard short-read genome assembly, which can lead to the fragmentation of chromosome and plasmid sequence assemblies. In this sense, plasmids may be viewed as the “dark matter” of short-read bacterial genome assemblies, with many sciencemag.org SCIENCE
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
ILLUSTRATION: V. ALTOUNIAN/SCIENCE
By Scott A. Beatson1,2 and Mark J. Walker1,2
compelling evidence to exclude nosocomial transmission may have been gathered from short-read sequencing, but long-read sequencing not only produced the required diagnostic result, but additionally provided reference sequences for all chromosomes and plasmids to aid future surveillance. Plasmid comparisons also disproved the hypothesis that transfer of a blaKPC plasmid had occurred between K. pneumoniae and E. cloacae within a patient from the original outbreak (4); long-read genome sequencing showed discrete genetic contexts for the two blaKPC alleles, indicating independent acquisition. Long-read genome sequencing also proved critical for determining the precise genomic context of blaKPC alleles in cases where more than one blaKPC gene was present in a single isolate.
Downloaded from www.sciencemag.org on December 10, 2014
MICROBIOLOGY
large-scale genomic studies conspicuously avoiding the complexities of plasmid structure. Genomic comparisons such as that described by Conlan et al. reveal how the dynamism in the structure and arrangement of resistance elements can only be realized by “closing” plasmid genomes with long-read sequencing (see the figure). Traditional Sanger sequencing is the gold standard for the analysis and assembly of complete plasmid sequences from antibiotic-resistant strains of bacteria. This approach may suffer from the need to isolate or subclone individual high molecular weight plasmids before sequencing (7), which is often technically difficult, timeconsuming, and costly, and may be intractable for multiple plasmids. Short-read sequencing technologies can affordably produce an assembly of a bacterial genome that contains nonrepetitive sequences typically in hundreds of “contigs” separated by “collapsed repeats” indicative of multiple copies of the same sequence located in several different locations within the genome. These repeats are often mobile elements such as insertion sequences that may be found in multiple copies on plasmids, thus making it difficult to assemble plasmid sequences. Cataloging the collection of antibioticresistance genes in any particular bacterium is relatively straightforward, but determining how these genes fit together within plasmids, which is critical for understanding how these elements spread in clinical settings, can be more difficult. By contrast, the genome sequences produced through long-read sequencing offer a complete picture of the plasmid content of a bacterium, including the number, position, and context within mobile elements of every acquired antibiotic-resistance gene. Long-read genome assembly offers clear advantages for the resolution of complete plasmid sequences that can discriminate plasmid diversity, antimicrobial-resistance gene context, and multiplicity. Such information will enhance our understanding of plasmid carriage, transfer, epidemiology, and evolution. REFERENCES
1. WHO, Antimicrobial resistance: Global report on surveillance 2014 (2014). 2. S. Conlan et al., Sci. Transl. Med. 254, 254ra126 (2014). 3. H. Ochman, J. G. Lawrence, E. A. Groisman, Nature 405, 299 (2000). 4. E. S. Snitkin et al. Sci. Transl. Med. 4, 148ra116 (2012). 5. R. M. Hall, C. M. Collis, Mol. Microbiol. 15, 593 (1995). 6. J. Mahillon, M. Chandler, Microbiol. Mol. Biol. Rev. 62, 725 (1998). 7. C. Venturini, S. A. Beatson, S. P. Djordjevic, M. J. Walker, FASEB J. 24, 1160 (2010). 10.1126/science.1260471
NANOMATERIALS
Making strong nanomaterials ductile with gradients Microstructures that increase metal crystallite size from nanoscale with surface depth are both strong and ductile nisms of the extremely fine grains that induces cracking. By applying surface plastic deformation onto a bulk coarse-grained metal, a distinctive microstructure is generated from the strain gradient: a nanograined layer (several tens of micrometers thick) covers the coarse-grained substrate with a graded variation of grain size in between (see the first figure). Tensile tests of the heterogeneously structured Cu cylinder (pulling the sample along the long axis) showed that the top
By K. Lu
S
teels can be made stronger, tougher, or more resistant to corrosion either by changing composition (adding in more carbon or other elements) or by modifying their microstructures. An extreme microstructural route for strengthening materials is to reduce the crystallite size from the micrometer scale (“coarse-grained”) to the nanoscale. Nanograined aluminum or copper (Cu) may become even harder than highstrength steels, but these materials 0 (m) can be very brittle and crack when pulled (deformed in tension), apparently because strain becomes localized and resists deformation. However, nanograined metals can be plastically deformed under 50 compression or rolling at ambient temperature, implying that moderate deformation can occur if the cracking process is suppressed. Tremendous efforts have been made to explore how to suppress strain lo100 calization in tensioned nanomaterials and make them ductile. Gradient microstructures, in which the grain size increases from nanoscale at the surface to coarse-grained in the core, were recently discovered to be an effective approach to improving 150 ductility (1–4). One advantage of metals in structural applications is that they “sigGradient nanograined structure. After a surface mechanical nal” their impending failure—they grinding treatment to copper, grain sizes are about 20 nm in the can deform and crack to some extopmost treated surface (outlined by dashed line) and increase tent before they completely fail. gradually to the microscale with depth. However, when a piece of fully nanograined copper is pulled, catastrophic nanograined layer and the coarse-grained failure occurs immediately when the load core can be elongated coherently by as much exceeds its yield strength (the point at as ~60% before failure—comparable to that which permanent deformation begins), just in conventional Cu, but the sample’s yield like most ceramics and other normal fragstrength is doubled (1). Almost no tensile ile materials. Such tensile brittleness is an elongation was observed in the nanograined Achilles’ heel of nanomaterials that hinders layer as it was removed from the substrate. their technological applications; for examEvidently, the observed extraordinary tensile ple, they cannot be strengthened by work Shenyang National Laboratory for Materials Science, Institute hardening. The microscopic origin appears of Metal Research, Chinese Academy of Sciences, Shenyang to be early necking (decrease in cross sec110016, China, and Herbert Gleiter Institute of Nanoscience, tion) induced by strain localization prior Nanjing University of Science & Technology, Nanjing 210094, to activation of plastic deformation mechaChina. E-mail: [email protected]
SCIENCE sciencemag.org
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203
Published by AAAS
1455
Tracking antibiotic resistance Scott A. Beatson and Mark J. Walker Science 345, 1454 (2014); DOI: 10.1126/science.1260471
If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this information is current as of December 10, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/345/6203/1454.full.html A list of selected additional articles on the Science Web sites related to this article can be found at: http://www.sciencemag.org/content/345/6203/1454.full.html#related This article cites 6 articles, 3 of which can be accessed free: http://www.sciencemag.org/content/345/6203/1454.full.html#ref-list-1 This article has been cited by 1 articles hosted by HighWire Press; see: http://www.sciencemag.org/content/345/6203/1454.full.html#related-urls This article appears in the following subject collections: Microbiology http://www.sciencemag.org/cgi/collection/microbio
Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS.
Downloaded from www.sciencemag.org on December 10, 2014
This copy is for your personal, non-commercial use only.
