Demography and Intercontinental Spread of the

Transcription

Demography and Intercontinental Spread of the USA300
Community-Acquired Methicillin-Resistant
Staphylococcus aureus Lineage.
Philippe Glaser, Patrı́cia Martins-Simões, Adrien Villain, Maxime Barbier,
Anne Tristan, Christiane Bouchier, Laurence Ma, Michele Bes, Frederic
Laurent, Didier Guillemot, et al.
To cite this version:
Philippe Glaser, Patrı́cia Martins-Simões, Adrien Villain, Maxime Barbier, Anne Tristan, et
al.. Demography and Intercontinental Spread of the USA300 Community-Acquired MethicillinResistant Staphylococcus aureus Lineage.. mBio, American Society for Microbiology, 2015, 7
(1), pp.e02183-15. <10.1128/mBio.02183-15>. <hal-01312831>
HAL Id: hal-01312831
http://hal.upmc.fr/hal-01312831
Submitted on 9 May 2016
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Demography and Intercontinental Spread of the USA300 CommunityAcquired Methicillin-Resistant Staphylococcus aureus Lineage
Philippe Glaser,a Patrícia Martins-Simões,b,c,d,e,f,g Adrien Villain,h Maxime Barbier,i,j Anne Tristan,b,c,d,e,f,g Christiane Bouchier,k
Laurence Ma,k Michele Bes,b,c,d,e,f,g Frederic Laurent,b,c,d,e,f,g Didier Guillemot,l Thierry Wirth,i,j François Vandeneschb,c,d,e,f,g
Institut Pasteur, Unité de Biologie des Bactéries Pathogènes à Gram-Positif, Paris, France; CNRS UMR3525, Paris, Francea; CIRI, International Center for Infectiology
Research, Lyon, Franceb; Inserm U1111, Lyon, Francec; Université Lyon 1, Lyon, Franced; École Normale Supérieure de Lyon, Lyon, Francee; CNRS UMR5308, Lyon, Francef;
CNR des Staphylocoques, Hospices Civils de Lyon, CBPE, Lyon, Franceg; Institut Pasteur, Bioinformatics Platform, Paris, Franceh; Laboratoire Biologie Intégrative des
Populations, Evolution Moléculaire, École Pratique des Hautes Etudes, Paris, Francei; Institut de Systématique, Evolution, Biodiversité, UMR-CNRS 7205, Muséum National
d’Histoire Naturelle, Université Pierre et Marie Curie, École Pratique des Hautes Etudes, Sorbonne Universités, Paris, Francej; Institut Pasteur, Genomics Platform, Paris,
Francek; Inserm UMR 1181 Biostatistics, Biomathematics, Pharmacoepidemiology and Infectious Diseases (B2PHI), Institut Pasteur, Versailles Saint-Quentin University, Paris,
Francel
P.G., P.M.-S., T.W., A.V., M.B., and F.V. contributed equally to this work.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized worldwide during
the 1990s; in less than a decade, several genetically distinct CA-MRSA lineages carrying Panton-Valentine leukocidin genes have
emerged on every continent. Most notably, in the United States, the sequence type 18-IV (ST8-IV) clone known as USA300 has
become highly prevalent, outcompeting methicillin-susceptible S. aureus (MSSA) and other MRSA strains in both community
and hospital settings. CA-MRSA bacteria are much less prevalent in Europe, where the European ST80-IV European CA-MRSA
clone, USA300 CA-MRSA strains, and other lineages, such as ST22-IV, coexist. The question that arises is whether the USA300
CA-MRSA present in Europe (i) was imported once or on very few occasions, followed by a broad geographic spread, anticipating an increased prevalence in the future, or (ii) derived from multiple importations with limited spreading success. In the present study, we applied whole-genome sequencing to a collection of French USA300 CA-MRSA strains responsible for sporadic
cases and micro-outbreaks over the past decade and United States ST8 MSSA and MRSA isolates. Genome-wide phylogenetic
analysis demonstrated that the population structure of the French isolates is the product of multiple introductions dating back
to the onset of the USA300 CA-MRSA clone in North America. Coalescent-based demography of the USA300 lineage shows that a
strong expansion occurred during the 1990s concomitant with the acquisition of the arginine catabolic mobile element and antibiotic resistance, followed by a sharp decline initiated around 2008, reminiscent of the rise-and-fall pattern previously observed
in the ST80 lineage. A future expansion of the USA300 lineage in Europe is therefore very unlikely.
ABSTRACT
To trace the origin, evolution, and dissemination pattern of the USA300 CA-MRSA clone in France, we sequenced
a collection of strains of this lineage from cases reported in France in the last decade and compared them with 431 ST8 strains
from the United States. We determined that the French CA-MRSA USA300 sporadic and micro-outbreak isolates resulted from
multiple independent introductions of the USA300 North American lineage. At a global level, in the transition from an MSSA
lineage to a successful CA-MRSA clone, it first became resistant to multiple antibiotics and acquired the arginine catabolic mobile element and subsequently acquired resistance to fluoroquinolones, and these two steps were associated with a dramatic demographic expansion. This expansion was followed by the current stabilization and expected decline of this lineage. These findings highlight the significance of horizontal gene acquisitions and point mutations in the success of such disseminated clones
and illustrate their cyclic and sporadic life cycle.
IMPORTANCE
Received 21 December 2015 Accepted 15 January 2016 Published 16 February 2016
Citation Glaser P, Martins-Simões P, Villain A, Barbier M, Tristan A, Bouchier C, Ma L, Bes M, Laurent F, Guillemot D, Wirth T, Vandenesch F. 2016. Demography and
intercontinental spread of the USA300 community-acquired methicillin-resistant Staphylococcus aureus lineage. mBio 7(1):e02183-15. doi:10.1128/mBio.02183-15.
Editor Alex van Belkum, bioMérieux
Copyright © 2016 Glaser et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported
license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to François Vandenesch, [email protected].
S
taphylococcus aureus remains one of the most challenging and
costly sources of bacterial infection worldwide. It asymptomatically colonizes about one-third of the human population and
may cause infections with outcomes that range from mild to severe and are occasionally life threatening (1). Notably, it is the
most common causative agent of nosocomial infections and a
leading cause of death in hospitalized patients (2). Until the mid-
January/February 2016 Volume 7 Issue 1 e02183-15
1990s, methicillin-resistant S. aureus (MRSA) infections were reported exclusively in hospital settings and most hospitalassociated MRSA (HA-MRSA) diseases resulted from a limited
number of successful clones (3). However, in the beginning of the
2000s, MRSA infections began to be reported in healthy individuals without risk factors or discernible connections to health care
institutions (4, 5). These community-acquired MRSA (CA®
mbio.asm.org 1
Downloaded from mbio.asm.org on May 9, 2016 - Published by mbio.asm.org
RESEARCH ARTICLE
MRSA) strains had genetic backgrounds distinct from those of
traditional HA-MRSA strains. Moreover, international strains of
CA-MRSA belonged to a series of different lineages and specific
clones were predominant on different continents (6). The dominant CA-MRSA clone in the United States, referred to as the
USA300 North American (USA300-NA) MRSA clone in this
study, belongs to pandemic multilocus sequence type 8 (ST8) constituted by methicillin-susceptible S. aureus (MSSA) and MRSA
strains with the same pulsed-field gel electrophoresis type,
USA300. This epidemiologically successful clone carries the IVa
subtype of the staphylococcal cassette chromosome mec (SCCmec)
element, agr allele 1, arginine catabolic mobile element (ACME)
type I, and a set of virulence genes including lukS-PV/lukF-PV, sek,
and seq (7–10).
It is generally recognized that this USA300-NA MRSA clone
descended from an ancestral USA500-like MSSA strain by the
acquisition of various mobile genetic elements (MGEs) and
shows very recent clonal expansion (2, 11). These MGEs,
namely, SCCmec type IV carrying the mecA gene, S. aureus pathogenicity island 5 containing sek and seq, phage phiSA2 carrying the
Panton-Valentine leukocidin genes, and ACME type I, are
thought to contribute to the success and high virulence of the
commonly known USA300-NA MRSA strains (12–16). ACME
type I is found exclusively in the USA300-NA MRSA lineage (16–
21). It has been shown that the functional modularity (arginine
deiminase system [arc] and the speG gene, which encodes a polyamine resistance enzyme) plays a major role in the enhanced success of this clone during colonization and skin infections (22, 23).
In recent years, multiple studies have shown that the USA300-NA
MRSA clone has spread worldwide, with reports of outbreaks in
South America, the Middle East, the western Pacific, and Europe
(for a detailed review, see reference 24). Interestingly, in South
America, the most prevalent CA-MRSA is a USA300 variant, the
so-called Latin American variant or USA300-LV (i.e., ST8, spa
type t008, SCCmec type IVc, lukSF-PV⫹, arcA) present in Colombia, Venezuela, and Ecuador (24–26). It has recently been proposed that the USA300 MRSA lineage has evolved since the 1980s
into two parallel epidemics, one in North America with the acquisition of the ACME by the ancestral USA300 lineage, forming the
USA300-NA MRSA epidemic, and one in South America, with the
acquisition of a novel copper and mercury resistance (COMER)
element by the ancestral lineage (27).
In Europe, CA-MRSA epidemiology is characterized by clonal
heterogeneity, although the most common European strain is the
European clone (ST80-IV, pvl⫹) (28). This clone originally
emerged from a West African ancestor (29) and expanded in the
Mediterranean area, the Middle East, and North Africa, as many
of the first patients infected with this clone in Europe had histories
of travel to these regions. Although the prevalence of this “European” clone is relatively high in some countries, like Algeria and
Tunisia, with a widespread distribution in both hospital and community settings (28–30), in Europe, the prevalence of this clone, as
well as that of CA-MRSA in general, remains low (31, 32) and even
seems to be declining (33, 34). This decay is in keeping with the
calculation of a recent slow decrease in its effective population size
based on a Bayesian skyline model (29). However, the question
arose as to whether the CA-MRSA clone (USA300-NA or
USA300-LV) could replace ST80 CA-MRSA and expand in a worrisome scenario similar to the one observed in the United States in
the past decade. Consistent with this hypothesis is the fact that the
2
®
mbio.asm.org
USA300-NA MRSA clone has been reported to be present in many
European countries for several years (31, 32, 35–45) and is occasionally associated with infection clusters (46–48). A recent study
showed that an increase in CA-MRSA USA300 prevalence in 2013
in Geneva, Switzerland, was due to distinct importations from the
North and South American continents (49).
This study addressed the question of the USA300 lineage’s capacity to expand successfully in Europe by applying wholegenome sequencing to a collection of French USA300 CA-MRSA
strains responsible for sporadic cases, as well as micro-outbreaks,
over the past decade, followed by a comparison with available
genomes of USA300 CA-MRSA, USA300 MSSA strains, and other
ST8 isolates from the United States (50, 51). Genome-wide phylogenetic relationships and coalescent-based analyses showed that
the population structure of the French isolates reflects multiple
introductions of the USA300-NA MRSA clone. Furthermore, the
coalescent-based demography of USA300-NA lineage confirmed
its success in the late 1990s but also suggested a sharp decline
initiated around 2008, reminiscent of the rise-and-fall pattern observed in the ST80 lineage.
RESULTS
USA300 phylogeography and resistance makeup. Prior to phylogenetic reconstruction, we checked if the data set provided some
evidence of homologous recombination. We first made a visual
inspection of the concatenated single-nucleotide polymorphisms
(SNPs), and no contiguous SNPs consisting of three or more
SNPs, mirroring homologous recombination, were detected. In a
second step, neighbor nets were inferred in order to detect putative recombination signatures that would result in networks
rather than trees. No major splits were found, and the pairwise
homoplasy index (PHI) test failed to detect recombination signatures (P ⫽ 0.475). We then performed whole-genome phylogenetic analysis of the 498 ST8 isolates (including MRSA, MSSA, and
USA300-LV and USA300-NA MRSA isolates) obtained from
France (n ⫽ 67) and the United States (n ⫽ 431) within a time
span of 15 years (see Table S1 in the supplemental material). A
total of 12,840 SNPs were used to generate a maximum-likelihood
(ML) tree that shows, as previously reported, that strains from San
Diego (51) and New York City (50) are interspersed (Fig. 1). The
closest relative sister group of the USA300-NA lineage corresponded to seven strains, all isolated from the New York City area.
These seven isolates carry the COMER element characteristic of
the USA300-LV South American clade (27) and belong to this
lineage. Adding French USA300 CA-MRSA strains to the phylogenetic analysis did not unravel any new branching pattern or
hidden sublineages and revealed that all of the French isolates
belong to the USA300-NA lineage. Among the U.S. isolates, a
fluoroquinolone-resistant lineage with the same mutations in
gyrA (Ser84Leu) and parC (Ser80Tyr) has emerged within the
USA300-NA MRSA clone and disseminated globally (Fig. 1). We
observed a similar distribution in France, with 18 fluoroquinolonesusceptible strains, including 10 from an outbreak in Le Puy-en-Velay
(a town in central France) (47) and 46 resistant isolates, including
the 28 isolates from an outbreak in a long-term care facility (Paris
16th District) showing the same two mutations in the gyrA and
parC loci. Plasmid analysis showed that all except three French
isolates carry plasmids related to previously described plasmid
p18805-p03 (52). This plasmid encodes multiple antibiotic and
heavy metal resistance genes [aminoglycosides, ant(6)-Ia and
Glaser et al.
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Third circle : Methicililin resistance
Susceptible
Susceptible
Resistant
Resistant
Second circle : ACME
Absence
Fourth circle : Year of isolation in blue gradients
Darkest blue (2014) Lightest blue (2000)
Putative intercontinental transmission event
*
FIG 1 Phylogenetic reconstruction of ST8 and its derived USA300 lineage. ML tree based on 498 genomes and a total of 12,840 concatenated SNPs, rooted by
Presence
using distantly related MSSA strain ERS092996. Branch colors indicate the geographic origins of the strains: red for New York State; yellow for California; green
for Minnesota, and blue for France. Outer circles represent ACME and antibiotic resistance profiles, whereas the inner circle represents the years of isolation of
the strains. The stars correspond to 12 predicted independent intercontinental USA300-NA CA-MRSA transfers from North America to France. The different
assignments in the ST8 “sublineages” are also indicated by colored areas within the circular tree. Fq, fluoroquinolone.
aph(3=)-III; beta-lactams, blaZ; macrolides mph(C); macrolideslincosamides-streptogramin B, msr(A); cadmium, cadD and
cadX]. These plasmids showed almost 100% nucleotide sequence identity indicative of a single origin but different regions of deletion. Similar plasmids are also present in the vast
majority of the American isolates (50, 51) but absent from the
strains of the USA300-LV lineage. It is therefore likely that an
ancestor of this multiresistance plasmid was acquired by the
common ancestor of the USA300-NA clone and contributed to
its expansion. The 28 isolates from the outbreak in a long-term
care facility (Paris 16th District) carried in their chromosome a
3.2-kb transposable element containing the dfrG gene, which
encodes a dihydrofolate reductase conferring trimethoprim resistance.
Temporal Bayesian analysis reveals the complexity of
USA300 demography. In a second step, we used the Bayesian co®
mbio.asm.org 3
Intercontinental Phylogeography of USA300
104
Effective population size
Acquisition of fluoroquinolone resistance
1998
103
102
1996
101
Acquisition of ACME
+ Methicillin resitance
1960
1970
1980
1990
2000
2010
Time
FIG 2 Bayesian skyline plot indicating population size changes in the USA300
lineage over time with a relaxed molecular clock. The shaded area represents
the 95% confidence interval, and the arrows point to major phenotypic events
driven by ACME and antibiotic resistance that might have contributed directly
to the success of the USA300-NA MRSA lineage.
alescent analysis implemented in BEAST (53) to generate a phylogenetic time tree and to derive estimated dates for the common
ancestors of the strains (see Fig. S1 in the supplemental material).
The tree was calibrated by using the sampling dates of the isolates
ranging from 2000 to 2014. We tested the performance of various
demographic models that favored a Bayesian skyline model with a
relaxed molecular clock (data not shown). The estimated shortterm mutation rate corresponded to 1.34 ⫻ 10⫺6 (95% confidence
interval of 1.11 ⫻ 10⫺6 to 1.56 ⫻ 10⫺6) substitutions per nucleotide site per year, similar to that reported previously in USA300
MRSA (50, 54) and other S. aureus evolutionary lineages (55, 56).
On the basis of this substitution rate, the estimated time of the
most recent common ancestor (TMRCA) of the USA300-NA
clone is 1996 (see Fig. S1 in the supplemental material), a result
that slightly shifts the ancestor of the CA-MRSA clone toward the
21st century, compared to the estimates obtained by Uhlemann
and colleagues (between 1970 and 1993) (50). In addition, our
analyses revealed that the acquisition of ACME, methicillin resistance, and a multiresistance plasmid dates back to 1996 and that
fluoroquinolone resistance was gained around 1998 (see Fig. S1 in
the supplemental material). It is noteworthy that, because of the
lack of intermediate strains harboring only one of those mobile
elements, we could not determine whether the acquisition of the
SCCmec element harboring the methicillin resistance gene mecA,
the multiresistance plasmid, and the ACME type I element occurred simultaneously or one event occurred very shortly after the
other. The divergence with the USA300-LV MRSA South American lineage dates back to 1983, an estimate slightly more recent
than that proposed by Planet et al. (around 1975) (27).
We then generated a Bayesian skyline plot that estimated the
pathogen’s demographic changes over time (Fig. 2) on the basis of
the 498 ST8 genomes. The ST8 USA300 effective population size
was relatively stable from the early 1960s to the mid-1990s. Then,
a bottleneck followed, giving rise to the so-called USA300-NA
clone. Interestingly, the USA300-NA clone went through a sharp
two-phase expansion event that perfectly matches the acquisitions
of the ACME and the antibiotic resistance elements, thus giving
4
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rise to the USA300-NA MRSA lineage, followed by the acquisition
of fluoroquinolone resistance, as mentioned above. Strikingly,
each of these major gains of function was accompanied by a
1-order-of-magnitude population size increase, resulting in a
global 100-fold effective population size increase (Fig. 2). However, after 2008, the coalescent-based analysis detects a sharp decline of the USA300-NA MRSA clone. These results need further
investigation to see if ongoing epidemiological surveys can confirm this model. We next searched for additional SNPs under positive selection that might explain the success of the USA300 lineage. To address this issue, we used the PCAdapt software (57, 58)
based on a Bayesian factor model to identify candidate mutations
(Fig. 3). In Table S2 in the supplemental material are shown the 20
SNPs with the highest scores (log10 Bayesian factors of ⬎6 ⫻ 1050).
Although such statistic-based predictions must be viewed cautiously and require experimental validation, the 20 mutations
were all located in protein coding sequences (see Table S2 in the
supplemental material). The six synonymous mutations might
affect the expression of the encoded protein. Strikingly, several
mutated genes might be involved in the interaction with the
human host or the environment, as they encode surface proteins, a lipoprotein, an autolysin, a putative transporter involved in teichoic acid transport, and a protein involved in
lipid biosynthesis.
French isolates are interspersed in United States isolate collections. Strikingly, the 67 strains in the French panel clustered in
12 lineages of 1 to 38 isolates. Furthermore, these clusters are
scattered within the North American MRSA isolates (USA300NA) (Fig. 1). This indicates a common origin of USA300-NA isolates and European USA300 CA-MRSA isolates, likely resulting
from several transfers from the United States to Europe. Interestingly, only one case could be related to travel to the United States,
in 2003 (strain HT20030124 from Annecy; see Table S1 in the
supplemental material).
We then calculated the genome-wide pairwise distances of the
most closely related French-American strains and compared them
with pairwise distances between strains collected from the same
patient at multiple sites, multiple colonies from the same nasal
sample, or strains collected from the same patient upon different
hospital admissions (59, 60) (Fig. 4). The median number of SNP
differences that separated the closest French-American epidemic
link was 74 (range, 59 to 120) and significantly differed from the
other two settings mentioned above (P ⬎ 0.0001). However, this
number of differences remains smaller than the median number
of SNP differences (n ⫽ 104) for USA300 CA-MRSA isolates collected from different households in a New York City community
(50). In addition, bootstrap support for clades encompassing the
French strains and their closest American homologs was ⬎70%
for ⬎50% of the different subsamples (see Fig. S2 in the supplemental material). Therefore, the epidemic links we detected between American and French strains are likely to reflect direct, or at
least very short, transmission paths.
The largest French cluster contains 28 isolates from an outbreak in a Parisian long-term care facility but also 3 strains isolated
in Aubervilliers nearby Paris, as well as 6 unlinked isolates from
different parts of France (Fig. 5A and B). This cluster is very likely
the product of a single introduction from the United States. Its
TMRCA is 1998 (Fig. 5C; see Fig. S1 in the supplemental material),
suggesting that this subclone had been circulating for at least a
decade in France before being noticed.
Glaser et al.
A
B
FIG 3 SNP-based Bayesian factor model analysis for detecting genes involved in positive selection in the USA300-NA lineage. (A) Latent factors of the 12,840
SNPs and 498 strains with the first two factors. (B) Manhattan plot representing the selection scan and the outliers that are related to the different latent factors.
The dotted line highlights the top 1% of the SNPs associated with the highest Bayes factor (BF) values.
DISCUSSION
Different admissions
Here we show that, in most instances, the USA300 CA-MRSA
cases identified in France in the last decade corresponded to sporadic and independent importations from the United States
(USA300-NA MRSA clone) without further indication of spreading in French territory. This is consistent with another observation
in Switzerland, where the local increased prevalence of USA300
CA-MRSA in 2013 in Geneva was shown to result from multiple
French-American
closest epidemic link
P < 0.0001
Intra Patients
P < 0.0001
0
200
400
600
800
1,000
Genomewise pairwise distances (number of SNPs)
FIG 4 Box plots representing pairwise SNP comparisons of strains from the
same patient (black), the closest strains from France and their American relative (blue), and strains from different hospital admissions of the same patient
(grey). These data were gathered from the present study and those of Golubchik et al. (59) and Price et al. (60).
importations from America (49). As the Institut de Veille Sanitaire (a disease surveillance agency of the Ministry of Health)
strongly recommend referring strains associated with multiple
cases of skin and soft tissue infections (SSTIs) (within a household
or other communities) to the French National Reference Laboratory, it is thus likely that the lack of observed spreading truly
is due to the absence or a very limited number of secondary
cases and not to underreporting. The reasons for the apparent
lack of success of these USA300-NA CA-MRSA isolates in diffusing within the French community are unknown. However, it
is in keeping with the low prevalence of CA-MRSA observed in
Europe and specifically in France (32), despite the isolation of
various CA-MRSA lineages on the European continent as early
as 1993 (38, 61). Moreover, these observations also argue
against the hypothesis that the USA300-NA MRSA clone is
intrinsically more successful than European CA-MRSA ST80
(3, 62, 63) and thus that the USA300-NA MRSA clone could be
a potential threat in Europe.
In one instance of the present study, however, one imported
lineage appeared to have a remarkable geographic spread, which
was subsequently responsible for two outbreaks, one in a longterm care facility (Paris 16th District) and another limited outbreak in a facility for the disabled also in the Paris area (Aubervilliers) in 2013 and 2014 (Fig. 5A). The same lineage was also
responsible for sporadic cases scattered in French territory, i.e., in
Paris (75019, June 2011), northeastern France (Strasbourg, January 2013), the French Alps (Grenoble, September 2011), central
France (Orleans, January 2011), and western France (St-Nazaire,
June 2012). Our Bayesian analysis predicts a TMRCA for this
clone of 1998 (Fig. 5C). This observation suggests that introduction of the USA300-NA MRSA clone can be successful but to a
limited extent. We did not detect genes specific to this lineage or
determine which genetic mechanisms might be behind the successful dissemination of certain isolates.
A recent report by Planet et al. (27) suggested that the history of
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Glaser et al.
A
LTCF Paris 75016
Aubervilliers
Orléans
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Paris
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Strasbourg
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63
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Most recent common ancestor ot the LTCF Paris 75016
(in years back to 2014)
FIG 5 Evolutionary relationships of the strains from an outbreak at a long-term care facility (LTCF) in the Paris area. (A) Minimum spanning tree based on SNP
data. Circle sizes are a function of the number of strains with the same genotype. Dashed lines correspond to links with ⬎25 SNPs; thin lines correspond to links
with ⬍25 SNPs, and thick lines correspond to links with ⱕ10 SNPs. Dates of isolation are indicated. Colors represent the geographical or epidemiological origins
according to the key. (B) ML tree based on the alignment of 488 SNPs of the dominant French lineage. Numbers of SNPs are mapped on the branches. Strains
from the outbreak group in three clusters colored green, blue, and red. (C) Posterior distribution of the Paris LTCF TMRCA generated with the BEAST algorithm.
The common ancestor (median) appeared in Europe in 1998 (95% highest posterior density, 1996 to 2003).
the USA300 CA-MRSA lineage followed a parallel evolution since
the mid-1970s on the American continents; one epidemic sublineage disseminated in South America (so-called USA300-LV) and
another disseminated in North America (referred to here as
USA300-NA MRSA). The separation of these two sublineages is
concomitant with the acquisition of the ACME by the North
American USA300 MRSA clone, while USA300-LV acquired an-
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other mobile element—the COMER element. They also acquired,
likely independently, two different subtypes of the SCCmec elements, type IVc in the LV clade and type IVa in the NA clade. The
demography of the USA300-NA lineage based on Bayesian analysis is in agreement with this model, showing a considerable demographic expansion around 1996, which coincides with the acquisition of the ACME and the SCCmec element by this USA300-NA
lineage, followed 2 years later by a second expansion peak that
likely corresponds to the acquisition of fluoroquinolone resistance. This period of expansion assessed by Bayesian analysis is in
line with the observed global spread of the USA300-NA CAMRSA clone in North America and worldwide, although with a
more limited impact in Europe (9, 32, 35, 37, 38, 44, 45, 64–67).
Strikingly, the effective population size of the lineage is predicted
to be declining during the first decade of this century, a prediction
that should be interpreted cautiously since there is no surveillance
of the prevalence of USA300-NA CA-MRSA at the worldwide
level. However, a recent meta-analysis retrieving 1,004 publications covering the three continents showed that USA300 CAMRSA is far from expanding on the various continents (68) and is
instead declining in many countries (33, 34). Observations on the
European continent suggest that the USA300-NA lineage, which
has been described for decades as having a rather low prevalence
and, according to our study, was imported into France on multiple instances many years ago, is apparently not expanding (32, 34,
44, 69, 70). Overall, it appears that the USA300-NA clone is behaving like many other MRSA clones, in particular, HA-MRSA,
for which a model of cyclic periods of expansion, equilibrium, and
decline has been observed (56, 71–76). It is worth mentioning that
in only very few cases could a plausible account be given to explain
the final population expansions, such as acquisition of resistance
to antibiotics or accumulation of mutations and increased fitness
(71–73). In the cases of USA300 CA-MRSA and the worldwide
emergence of CA-MRSA in general, there is no satisfactory explanation for the concomitant emergence and expansion of the various CA-MRSA clones in different countries or on different continents at the end of the 20th century (5).
The most plausible scenario explaining CA-MRSA clone dynamics seems to be a complex combination of acquisition and loss
of traits under selection (resistance to antibiotics, resistance to
human host defenses), stochastic random processes, and eventually, replacement of bacterial communities by others competing
for the same niche. The latter factor might explain the population
decline observed in our Bayesian analyses; however, we have to
keep in mind that the natural habitat of S. aureus is the nose and
the skin. Therefore, focusing on population genomics of only
pathogenic lineages might blur our global understanding of species communities and population dynamics.
MATERIALS AND METHODS
Bacterial strains. The bacterial strains used in this study were from 24
sporadic cases that were geographically and temporally dispersed and
from three limited outbreaks reported to the French National Reference
Center for Staphylococci (CNR Staphylococci). All strains were isolated
from cases (infection and/or carriage) that occurred in continental French
territory in the past 11 years (see Table S1 in the supplemental material).
The first outbreak occurred in Le Puy en Velay, a small town located in a
rural area in Auvergne, France, and involved 12 individuals with
community-acquired SSTIs, i.e., 6 children attending the same day care
facility and 6 adult relatives in six households, between June 2011 and May
2012 (47). Two outbreaks occurred in long-term health care units. One
was in a nursing home for mentally disabled patients in Aubervilliers in
the Paris area; four patients were detected as carriers or infected with
USA300 CA-MRSA between July 2013 and February 2014. The other outbreak occurred in a long-term care facility in the Paris 16th District between June 2012 and December 2013 and involved 10 infected patients
and 12 carriers.
Four strains from the United States corresponded to sporadic cases of
SSTIs that occurred before April 2003 and were provided by Barry Kre-
iswirth (77); three other strains originated from patients in Minnesota
before 2001 and were provided by Timothy Naimi (78). The genomic
backgrounds of these strains were assessed by diagnostic DNA microarray
analysis (Identibac S. aureus Genotyping; Alere, Jena, Germany) as previously described (79). This microarray covers 332 different target sequences corresponding to 185 distinct genes and their allelic variants. The
affiliation of isolates with clonal complexes was determined by a comparison of their hybridization profiles with those of reference strains previously characterized by multilocus sequence typing (79).
Published genomic data used for comparative analysis. Genomic
data from 36 isolates from the first large-scale genomic studies on USA300
CA-MRSA were retrieved (51). These strains had been isolated between
2003 and 2007 in California. In addition, the genomic data from 387 ST8
isolates from an extensive analysis of a New York City community performed between 2009 and 2011 were included. Metadata from these two
studies were graciously provided by the authors (50, 51).
DNA sequencing and SNP detection. S. aureus genomes were sequenced by using the Illumina HiSeq 2000 (101 nucleotide reads) or
MiSeq (150 nucleotide reads) sequencer, with a coverage of ⬎75⫻. Libraries were constructed with the Illumina TruSeq kit. Sequence reads
from previously described USA300 strains were downloaded from the
European Nucleotide Archive website (http://www.ebi.ac.uk/ena) or obtained directly from the authors. Sequence reads were aligned with the
first completely sequenced and annotated US300-NA genome, FPR3757
(GenBank accession no. CP000255.1) (10) by using the Burrows-Wheeler
Alignment tool (BWA mem 0.7.5a) (80). SNP calling was done with the
Genome Analysis Toolkit (GATK 2.7-2) (81) Unified Genotyper by following Broad Institute best practices. Candidate SNPs were further filtered by requiring coverage of greater than half of the genome mean coverage and 95% read agreement to validate the call. SNPs selected with this
stringent filter were regarded as true positives and were searched for in the
original set for every strain in which they had been filtered out. SNPs,
short indels, and coverage were visualized with SynTView (82). Specific
analysis of clones from the main French lineage was done by manually
checking read alignments around SNPs with Tablet (83). Four regions
corresponding to MGEs not present or showing a high density of SNPs in
other USA300 isolates and in closely related ST8 strains were removed
from the analysis, leaving a total of 12,840 polymorphic sites. These regions are the ACME and the SCCmec cassette (41 to 125 kb), pathogenicity island 3 (encoding enterotoxins K and Q; 881 to 896 kb) (84), and
integrated phages phiSA2usa (encoding the Panton-Valentine toxin;
1,546 to 1,644 kb) and phiSA3usa (2,084 to 2,127 kb). These regions
represent a total of 240 kb or 8.3% of the strain USA300_FPR3757 chromosome.
Accessory genome analysis. Plasmids and chromosomal regions specific to the French isolates compared to the FPR3757 reference genome
were analyzed following de novo assembly of unmapped reads. Assembly
was performed with Velvet by using an optimized k value (85). Plasmids
and phages were identified by BLASTn searches with plasmid and phage
sequences from S. aureus as the queries. Antibiotic resistance genes were
identified with the Center for Genomic Epidemiology web tool (http://
www.genomicepidemiology.org/).
Recombination detection. Most of the analyses developed in our analytical framework (phylogenetics and Bayesian inference) are based on
the assumptions that S. aureus evolution is mostly clonal and that recombination can be neglected. Therefore, in a preliminary step, we tested for
the presence of mosaic genomes with the algorithm SplitsTree v4.13.1
(86). Putative recombination signatures were inferred with Neighbor-Net
(87), and each data set was analyzed for the presence of recombinant
sequences with the PHI test in SplitsTree with an alpha value of 0.001.
Phylogenetic analyses. Phylogenic reconstructions were performed
by considering the 12,840 polymorphic sites retained in the core genome
of the 498 isolates. Phylogenetic relationships were reconstructed by the
ML approach implemented in PhyML 3.0 (88). The robustness of the ML
tree topology was assessed with bootstrapping analyses of 1,000 pseu-
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doreplicated data sets. A transversion substitution model was selected on
the basis of Akaike’s information criterion with jModelTest 2.1.3 (89).
Phylogenies were rooted with MSSA strain ERS092996.
Coalescent-based analyses. Evolutionary rates and tree topologies
were analyzed with the generalized time reversible (GTR) and HasegawaKishino-Yano (90) (HKY) substitution models with gamma distributed
among-site rate variation with four rate categories (⌫4). We tested both a
strict molecular clock (which assumes the same evolutionary rate for all of
the branches of the tree) and a relaxed clock that allows different rates
among the branches. Constant-size, logistic, exponentially growing coalescent models were used. We also considered the Bayesian skyline plot
model (91), based on a general, nonparametric prior that enforces no
particular demographic history. We used a piecewise linear skyline model
with 20 groups and then compared the marginal likelihood of each model
with Bayes factors estimated in Tracer 1.5. Bayes factors represent the
ratio of the marginal likelihood of the models being compared. Approximate marginal likelihoods for each coalescent model were calculated via
importance sampling (1,000 bootstrap replications) with the harmonic
mean of the sampled likelihoods. A ratio between 3 and 10 indicates moderate support of the idea that one model fits the data better than another,
whereas values of ⬎10 indicate strong support. For each analysis, two
independent runs of 100 million steps were performed and the chain was
sampled every 10,000th generation. Examination of the Markov chain
Monte Carlo (MCMC) samples with Tracer 1.5 indicated convergence
and adequate mixing of the Markov chains, with effective sample sizes for
each parameter in the hundreds or thousands. The first 10% of each chain
was discarded as burn-in. We found the maximum clade credibility topology with TreeAnnotator 1.7.5 (52), and we reconstructed the Bayesian
skyline plot with Tracer 1.5. The relaxed clock models provided a better fit
to the data (Bayes factor, ⬎12) and under the different models tested, the
Bayesian skyline model provided a (marginally) better fit, overall.
Analysis of genes under positive selection. To capture SNPs under
positive selection, we used the PCAdapt software to perform a genome
scan based on a Bayesian factor model (57). We chose K ⫽ 2 factors
because the third and the fourth factors did not correspond to population
structure and distinguished individuals within the same clades. The factor
analysis was performed on the centered genotype matrix that was not
scaled. The MCMC algorithm was initialized by singular value decomposition, and the total number of steps was 400 with a burn-in of 200 steps.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mbio.asm.org/
lookup/suppl/doi:10.1128/mBio.02183-15/-/DCSupplemental.
Figure S1, PDF file, 0.9 MB.
Figure S2, PDF file, 0.1 MB.
Table S1, XLS file, 1.7 MB.
Table S2, DOCX file, 0.1 MB.
ACKNOWLEDGMENTS
We are grateful to all of the clinicians and microbiologists from the different laboratories who provided samples and/or clinical data included in
this study. In particular, we gratefully acknowledge Tim Naimi and Barry
Kreiswirth in the United States for providing isolates, Beate Heym and
Isabelle Simon of Assistance Publique des Hôpitaux de Paris, and Olivier
Baud and Olivier Lesens of the Clermont-Ferrand Hospital for their contribution to the investigation of the two first major outbreaks that took
place in France in a long-term care facility in the Paris area and in a day
care center at Le Puy en Velay and for providing us with all of the isolates
involved in these outbreaks. We thank the technicians of the National
Reference Center for Staphylococci for their skillful technical contribution and Jerome Etienne and Gerard Lina for scientific input. We thank
Pierre Lechat of the bioinformatics platform of the Pasteur Institute for
SynTView integration. We also thank Anne-Catrin Uhlemann and Ryan
Tewhey, who graciously provided metadata concerning the USA300 genomes used in their studies (50, 51).
This work was supported by the Fondation pour la Recherche Médi-
8
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mbio.asm.org
cale (grant ING20111223510); Labex ECOFECT, Labex IBEID, and Infrastructure d’Avenir France Génomique (ANR10-IBNS-09-08); and the Institut de Veille Sanitaire (INVS).
FUNDING INFORMATION
Infrastructure d’Avenir France Genomique provided funding to Philippe
Glaser under grant number ANR10-IBNS-09-08. Institut de Veille Sanitaire provided funding to François Vandenesch. Fondation pour la Recherche Médicale (FRM) provided funding to Patricia Martins Simões
under grant number ING20111223510.
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