Tracking antibiotic resistance

Scott A. Beatson and Mark J. Walker

Science 345, 1454 (2014);
DOI: 10.1126/science.1260471

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INSIGHTS | P E R S P E C T I V E S

MICROBIOLOGY compelling evidence to exclude nosocomial

transmission may have been gathered from

Tracking antibiotic resistance short-read sequencing, but long-read se-

quencing not only produced the required
diagnostic result, but additionally provided
Sequencing plasmids can reveal the transmission of reference sequences for all chromosomes
and plasmids to aid future surveillance.
resistance among bacteria from patients in a clinical setting Plasmid comparisons also disproved the
hypothesis that transfer of a blaKPC plas-
By Scott A. Beatson1,2 and Mark J. Walker1,2 with long-read sequencing (single-mol- mid had occurred between K. pneumoniae
ecule real-time sequencing). Sixty-three and E. cloacae within a patient from the

A
ccording to the World Health Or-
ganization (1), “Antimicrobial re-
sistance…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 popu-
lations. Thus, the genetic context in which
an antibiotic-resistance gene is placed
can reflect its mobility within the bacte-
rial 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 method-
ologies. 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 Clini-
cal Center in which six infected patients
died from carbapenem-resistant Klebsiella
pneumoniae (4). Conlan et al. surveyed
plasmid sequences were completed from original outbreak (4); long-read genome
patient or hospital environmental isolates, sequencing showed discrete genetic con-
providing a unique snapshot of plasmid texts for the two blaKPC alleles, indicating
complexity from a single geographical independent acquisition. Long-read ge-
site. The sequences allowed elucidation nome sequencing also proved critical for
of the fine detail of plasmid diversity that determining the precise genomic context of
would not have been obvious otherwise. blaKPC alleles in cases where more than one
Two patients infected with near-identical blaKPC gene was present in a single isolate.
Long-read sequence
Bacterial plasmids
Track transmission
by following
plasmid diversity
(resistance genes in context)
Antibiotic-
resistant
Hospital environment bacteria
Patients with Lacks detail to
antibiotic-resistant track plasmid
bacteria diversity
Short-read sequence
Plasmid fragments
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 clo- Plasmids play a key role in disseminat-
acae strains harbored blaKPC on the same ing antimicrobial-resistance genes among
plasmid (pKPC-47e), triggering increased pathogenic bacteria. Resistance genes of-
surveillance measures. Shared carbapen- ten aggregate in modular clusters (5) that
emase-encoding plasmids were found in enable resistance to persist even in the ab-
three different bacterial genera epidemio- sence of direct selection. Some insertion
logically linked with hospital environments sequences (IS) (6), such as IS26, sandwich
(sinks). Of five patients who presented with resistance genes into a composite transpo-
carbapenemase-resistant Klebsiella sp., son that can mobilize elsewhere. Other el-
long-read genome sequencing associated ements, such as ISEcp1, mobilize adjacent
one of these patients to the original 2011 genes, leading to multiple copies of resis-
outbreak, indicating nosocomial transmis- tance genes within the genome. The mul-
ILLUSTRATION: V. ALTOUNIAN/SCIENCE

more than 1000 patients for carbapenem-
resistant 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: mark.walker@uq.edu.au
sion. Long-read genome sequencing un-
equivocally ruled out a link with the 2011
outbreak in the remaining cases, despite
epidemiological evidence suggesting oth-
erwise. Carriage of shared plasmids was
observed between a patient isolate and iso-
lates of other bacterial species present in
the ward environment. For several cases,
tiplicity of insertion sequences and other
repetitive elements is a major confounding
factor in standard short-read genome as-
sembly, which can lead to the fragmenta-
tion 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

1454 19 SEP TEMBER 2014 • VOL 345 ISSUE 6203 sciencemag.org SCIENCE

Published by AAAS
large-scale genomic studies conspicuously
avoiding the complexities of plasmid struc-
ture. Genomic comparisons such as that
described by Conlan et al. reveal how the
dynamism in the structure and arrange-
ment 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 assem-
bly of complete plasmid sequences from
antibiotic-resistant strains of bacteria. This
approach may suffer from the need to iso-
late or subclone individual high molecular
weight plasmids before sequencing (7),
which is often technically difficult, time-
consuming, and costly, and may be intrac-
table for multiple plasmids. Short-read
sequencing technologies can affordably
produce an assembly of a bacterial genome
that contains nonrepetitive sequences typi-
cally in hundreds of “contigs” separated by
“collapsed repeats” indicative of multiple
copies of the same sequence located in sev-
eral 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 antibiotic-
resistance genes in any particular bac-
terium is relatively straightforward, but
determining how these genes fit together
within plasmids, which is critical for un-
derstanding how these elements spread
in clinical settings, can be more difficult.
By contrast, the genome sequences pro-
duced through long-read sequencing offer
a complete picture of the plasmid content
of a bacterium, including the number, posi-
tion, 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 infor-
mation will enhance our understanding of
plasmid carriage, transfer, epidemiology,
and evolution.
REFERENCES
1. WHO, Antimicrobial resistance: Global report on surveil-
lance 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
SCIENCE sciencemag.org
NANOMATERIALS
Making strong nanomaterials
ductile with gradients
Microstructures that increase metal crystallite size from
nanoscale with surface depth are both strong and ductile
By K. Lu nisms of the extremely fine grains that in-
duces cracking. By applying surface plastic
S
teels can be made stronger, tougher, deformation onto a bulk coarse-grained
or more resistant to corrosion either metal, a distinctive microstructure is gener-
by changing composition (adding in ated from the strain gradient: a nanograined
more carbon or other elements) or layer (several tens of micrometers thick)
by modifying their microstructures. covers the coarse-grained substrate with a
An extreme microstructural route graded variation of grain size in between
for strengthening materials is to reduce (see the first figure).
the crystallite size from the micrometer Tensile tests of the heterogeneously
scale (“coarse-grained”) to the nanoscale. structured Cu cylinder (pulling the sample
Nanograined aluminum or copper (Cu) along the long axis) showed that the top
may become even harder than high-
strength steels, but these materials 0 (␮m)
can be very brittle and crack when
pulled (deformed in tension), ap-
parently because strain becomes
localized and resists deformation.
However, nanograined metals can
be plastically deformed under
50
compression or rolling at ambient
temperature, implying that moder-
ate deformation can occur if the
cracking process is suppressed. Tre-
mendous efforts have been made to
explore how to suppress strain lo-
calization in tensioned nanomateri- 100
als 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 struc-
tural applications is that they “sig- Gradient 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 ex- topmost 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 frag- strength 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 exam- Evidently, the observed extraordinary tensile
ple, they cannot be strengthened by work
hardening. The microscopic origin appears Shenyang National Laboratory for Materials Science, Institute
to be early necking (decrease in cross sec- of Metal Research, Chinese Academy of Sciences, Shenyang
110016, 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 mecha- China. E-mail: lu@imr.ac.cn
19 SEP TEMBER 2014 • VOL 345 ISSUE 6203 1455
Published by AAAS