AbstractWhole-genome sequencing of 24 Proteus mirabilis isolates revealed the clonal expansion of two cefoxitin-resistant strains among patients with community-onset infection. These strains harboured blaCMY-2 within a chromosomally located integrative and conjugative element and exhibited multidrug resistance phenotypes. A predominant strain, identified in 18 patients, also harboured the PGI-1 genomic island and associated resistance genes, accounting for its broader antibiotic resistance profile. The identification of these novel multidrug-resistant strains among community-onset infections suggests that they are endemic to this region and represent emergent P. mirabilis lineages of clinical significance

Crowley, B.

Mac Aogáin, M.

Rogers, T.R.

new microbes and new infections

PubMed

Elsevier - Publisher Connector 

NEW RESISTANT MICROBES IN HUMANSIdentiﬁcation of emergent blaCMY-2-
carrying Proteus mirabilis lineages
by whole-genome sequencingM. Mac Aogáin1, T. R. Rogers1,2 and B. Crowley1,2
1) Department of Clinical Microbiology, Sir Patrick Dun Translational
Research Laboratory, School of Medicine, Trinity College
Dublin and 2) Department of Microbiology, St James’s Hospital, Dublin,
IrelandAbstractWhole-genome sequencing of 24 Proteus mirabilis isolates revealed
the clonal expansion of two cefoxitin-resistant strains among
patients with community-onset infection. These strains harboured
blaCMY-2 within a chromosomally located integrative and
conjugative element and exhibited multidrug resistance
phenotypes. A predominant strain, identiﬁed in 18 patients, also
harboured the PGI-1 genomic island and associated resistance
genes, accounting for its broader antibiotic resistance proﬁle. The
identiﬁcation of these novel multidrug-resistant strains among
community-onset infections suggests that they are endemic to
this region and represent emergent P. mirabilis lineages of clinical
signiﬁcance.
New Microbes and New Infections © 2015 The Authors. Published
by Elsevier Ltd on behalf of European Society of Clinical
Microbiology and Infectious Diseases.
Keywords: blaCMY-2, genomics, ICE, PGI-1, Proteus mirabilis
Original Submission: 8 October 2015; Revised Submission:
18 November 2015; Accepted: 19 November 2015
Article published online: 27 November 2015Ne
Ne
Th
httCorresponding author: M. Mac Aogáin, Department of Clinical
Microbiology, Trinity College Dublin, Room 107, Central Pathology
Lab (CPL), St James’s Hospital, Dublin 8, Ireland
E-mail: m.macaogain@tcd.ieIntroductionProteus mirabilis is a signiﬁcant cause of urinary tract infections
and is a leading cause of catheter-associated urinary tractw Microbe and New Infect 2016; 9: 58–62
w Microbes and New Infections © 2015 The Authors. Published by Elsevier Ltd on behalf of
is is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4
p://dx.doi.org/10.1016/j.nmni.2015.11.012infections [1]. Treatment is complicated by the acquisition of
antibiotic resistance genes affording P. mirabilis a selective
advantage during therapy. Horizontal acquisition of AmpC-type
β-lactamase genes has been an important driver of resistance in
Europe and has been associated with the clonal expansion of
resistant strains [2,3]. Since the publication of the P. mirabilis
HI4320 reference genome, additional genome sequences have
become available providing a framework for genomic epide-
miology in this species [4–9]. Here, we applied whole-genome
sequencing to investigate the genomic epidemiology of emer-
gent cefoxitin-resistant P. mirabilis isolates causing community-
onset infections in Ireland.MethodsClinical P. mirabilis isolates, recovered by the Microbiology
Department of St James’s Hospital (Dublin, Ireland), were sub-
jected to antimicrobial sensitivity testing on a Vitek 2 system
(bioMérieux, Marcy l’Étoile, France). Infections with onset in the
community were categorized as either community acquired,
when onset of illness occurred outside a healthcare facility with
no reported discharge from a healthcare facility within the pre-
vious 12 weeks, or healthcare associated, when onset of illness
occurred within 4 weeks of discharge from a healthcare facility.
Whole-genome sequencing of P. mirabilis isolates was performed
on an Illumina MiSeq platform at the TrinSeq sequencing facility
(Trinity College Dublin, Ireland). Sequencing reads were aligned
to the P. mirabilis HI4320 genome (AM942759) using the
Burrows-Wheeler short-read aligner, while de novo assembly
was performed using the NSilico Simplicity pipeline (Simplicity
v1.2) [2,10]. Sequence data for the P. mirabilis integrative and
conjugative element ICEPmiJpn1 (AB525688.1) and Proteus
Genomic Island-1 (KJ411925) were also used as reference se-
quences for read mapping and contig alignment. Single-
nucleotide variants (SNVs) were resolved using SAMtools [11].
Phylogenetic trees were generated by neighbour-joining (BIONJ)
using PhyML and visualized with iTOL [12,13]. Raw short-read
data have been deposited at the European Nucleotide Archive
under study accession number PRJEB7631. Draft assemblies of
representative cefoxitin-resistant strains PM655 and PM593
have been deposited at DDBJ/EMBL/GenBank under accession
numbers JSUO00000000 and JSUP00000000, respectively.ResultsBetween December 2012 and November 2013, 33% of P.
mirabilis isolates (n = 79) recovered by the MicrobiologyEuropean Society of Clinical Microbiology and Infectious Diseases
.0/)
FIG. 1. Phylogenetic comparison of Irish Proteus mirabilis isolates with previously sequenced P. mirabilis strains. (A) A neighbour-joining tree was
generated on the basis of concatenated sequences of seven conserved genes present in all strains (adk; PMI0375, fumC; PMI0891, gyrB; PMI1296, icd;
PMI2184, mdh; PMI1732, purA; PMI3370 and recA; PMI3400). Sequences from 24 P. mirabilis isolates from this study and eight representative P. mirabilis
whole-genome data sets; C05028 (ANBT00000000), WGLW4 (AMGU00000000), HI4320 (AM942759), ATCC29906 (ACLE01000000), ATCC7002
(JOVJ00000000), PR03 (AORN00000000), WGLW6 (AMGT00000000) and BB2000 (CP004022) were compared. Black bar indicates average
nucleotide substitutions per site across the seven genes analysed (9 kb). To the right of the tree, under label CA, plus and minus symbols denote either
community-acquired or hospital-associated infections, respectively, while subsequent columns ‘AC,’ ‘GN,’ ‘FX,’ ‘CZ,’ ‘CP’ and ‘TP’ indicate the
resistance proﬁle of each isolate to amoxicillin–clavulanic acid, gentamicin, cefoxitin, ceftazidime, ciproﬂoxacin and piperacillin–tazobactam,
respectively. Colour indicates resistance level: black, full resistance; dark grey, intermediate resistance; light grey, susceptible. (B) Unrooted neighbour-
joining tree of the observed 18-strain clonal cluster based on whole-genome comparisons across 3,207,626 called sites relative to the P. mirabilis
HI4320 genome. Circles indicate genetically indistinguishable strains. Bar below the tree indicates length corresponding to one single-nucleotide
variant.
NMNI Mac Aogáin et al. Emergent blaCMY-2-carrying P. mirabilis lineages 59Department of St James’s Hospital were found to exhibit
resistance to cefoxitin (minimum inhibitory concentration
16 mg/L). Twenty-one cefoxitin-resistant isolates, conﬁrmed
as being from community-onset infections, were further
investigated by whole-genome sequencing. Cefoxitin-resistant
isolates had multidrug-resistance (MDR) phenotypes, exhibit-
ing coresistance to other antibiotics including amoxicillin–
clavulanic acid (100%), gentamicin (91%), ceftazidime (76%) and
ciproﬂoxacin (52%) but remained sensitive to piperacillin–
tazobactam, with the three control isolates exhibiting suscep-
tibility to all antibiotics tested (Fig. 1(A)). No epidemiologic
links between cases could be established on analysis of patient
records, suggesting that potential strain bias due to patient-to-
patient transmission was minimal.
Whole-genome sequence data indicated that our resistant
isolates exhibited signiﬁcant genetic divergence from available
P. mirabilis genome sequences present in the Refseq database
and included a large cluster of 18 genetically related isolates
(Fig. 1(A)). Genome-wide analysis of the 18-isolate cluster
(cluster I) revealed isolates to be highly clonal, exhibiting aNew Microbes and New Infections © 2015 The Authors. Published by Elsevier Ltd on beha
This is an ogenetic divergence of between 0–30 SNVs (Fig. 1(B)). Similarly,
within the smaller isolate cluster (cluster II, n = 3), isolates
PM100 and PM593 were genetically indistinguishable, whereas
PM063 diverged by 20 SNVs (isolates in cluster I diverged from
those of cluster II by more than 19,000 SNVs).
An R391/SXT-family integrative and conjugative element
(ICE), harbouring the blaCMY-2 AmpC-family β-lactamase gene,
was identiﬁed in all cefoxitin-resistant strains (both in cluster I
and cluster II). This ICE shared high sequence identity to ICE-
PmiJpn1 (AB525688.1) and similarly contained the blaCMY-2 gene
within a composite transposon insertion [14]. The identiﬁed
ICE region was integrated at the same chromosomal location in
both strains (Fig. 2(A)). Cluster I isolates also harboured addi-
tional chromosomally integrated resistance genes; a Tn7-asso-
ciated class 1 integron was present upstream of PMI3067 (glmS)
carrying the aadA1 and dfrA1 resistance genes (Fig. 2(B)), while
the Proteus Genomic Island 1 (PGI-1) was also identiﬁed [15].
PG1-1 harboured the resistance genes aadB, aad2, aphA1b,
blaTEM-1 and sul1 associated with diverse mobile elements
within its MDR region while lacking the “right-end” of the PGI-1lf of European Society of Clinical Microbiology and Infectious Diseases, NMNI, 9, 58–62
pen access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
FIG. 2.Observedmobile genetic elements associatedwith chromosomally integrated resistance genes among Proteus mirabilis strains identiﬁed in this study.
Genetic environs (black) of detected resistance genes (white) among resistant P. mirabilis isolates are illustrated. Identiﬁed insertion sequences are high-
lighted in grey boxes. (A) Both strains harboured the described integrative and conjugative element (ICE) element ICEPmiJpn1, which was integrated
between chromosomal genes PMI2422 (prfC) and PMI2949. The blaCMY-2 gene was present within an ICE-embedded composite transposon ﬂanked by IS10
elements, as observed in ICEPmiJpn1 (AB525688.1). The ICE observed in cluster I isolates also included three additional genes encoding hypothetical
proteins (underlined), which are absent from previously described ICE elements in P. mirabilis. (B) Cluster I isolates harboured the aadA1 and dfrA1 genes
associated within a Tn7-associated class 2 integron, chromosomally located between PMI3067 (glmS) and PMI3068. (C) PGI-1 genomic island was identiﬁed
among cluster I isolates, chromosomally integrated between PMI3127 (hipB) and PMI3127 (trmE). Resistance genes identiﬁed within the PGI-1 region
included aphA1b, blaTEM-1, sul1, aadA2 and aadB, which were embedded among a mosaic of mobile genetic elements in the PGI-1 MDR region.
60 New Microbes and New Infections, Volume 9 Number C, January 2016 NMNI
New Microbes and New Infections © 2015 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases, NMNI, 9, 58–62
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
NMNI Mac Aogáin et al. Emergent blaCMY-2-carrying P. mirabilis lineages 61MDR region as well as the IS26-mediated recombination event
originally described among French P. mirabilis isolates (Fig. 2(C))
[15]. The three antibiotic-susceptible isolates investigated
lacked the R391/SXT-family ICE, class 2 integron-borne aadA1
and dfrA1, and PGI-1. Although we failed to identify mutations
within the quinolone-resistance-determining regions of gyrA,
gyrB, parC or parE among ciproﬂoxacin-resistant isolates, loss-
of-function mutations in transcriptional regulators of efﬂux
acrR (frameshift: c.90_93dup) and soxR (stop gain;
p.Gln147Stop) were identiﬁed in four of ﬁve resistant isolates
exhibiting ciproﬂoxacin minimum inhibitory concentrations of
>4 mg/L. These mutations were absent from susceptible
(n = 13) or intermediately resistant (n = 6) isolates.DiscussionProteus mirabilis exhibits a clonal population structure whereby
the emergence of pathogenic strains is often observed in as-
sociation with the acquisition of exogenous antibiotic resistance
genes [3,14,16]. Here the application of whole-genome
sequencing allowed ﬁne-structure comparative genomic anal-
ysis of locally emergent P. mirabilis strains identifying two novel
clonal lineages among community-onset infections. While both
strains harboured the ICE-located blaCMY-2, accounting for
observed cephamycin resistance, cluster I strains were distin-
guished by the presence of a chromosomally located Tn7-
associated class 1 integron and the PGI-1 genomic island. These
gene mobilization platforms harboured several additional anti-
biotic resistance genes accounting for the broader resistance
proﬁle of the cluster I strain such as invariable gentamicin
resistance, which was accounted for by the presence of class 1
integron-associated aadB in the PGI-1 MDR region (Fig. 2(C)).
Thus, the acquisition of multiple resistance genes via distinct
gene mobilization platforms has likely been a key contributor to
the clonal expansion of this novel strain among community-
onset infections. Worryingly, a number of cluster I isolates
were also resistant to ciproﬂoxacin, further limiting therapeutic
scope for treatment of community-acquired infection. While
the mechanism of ﬂuoroquinolone resistance in these strains
awaits formal validation, mutational disruptions in soxR and acrR
were noted. Similar mutations in acrR and soxR are known to
contribute to ﬂuoroquinolone resistance in other pathogenic
members of the Enterobacteriaceae by altering expression of the
AcrAB efﬂux system, while increased expression of acrB has
been conﬁrmed among ﬂuoroquinolone-resistant P. mirabilis
isolates [17–19]. The predominance of the MDR strains
described here among apparently epidemiologically unrelated
patients suggests that they represent P. mirabilis lineages
endemic to this region, warranting further local surveillance. ItNew Microbes and New Infections © 2015 The Authors. Published by Elsevier Ltd on beha
This is an owill be interesting to assess whether these strains are localized
to this region or have a broader global distribution. In the
absence of a widely accepted and portable typing scheme in P.
mirabilis, whole-genome sequencing provides an important tool
in addressing such questions.AcknowledgementsSupported by the Department of Clinical Microbiology, Trinity
College Dublin. The authors gratefully acknowledge C. Roche
and L. Rose for their excellent technical assistance and NSilico
Life Science Ltd for access to bioinformatic software.Conﬂict of InterestNone declared.References[1] Armbruster CE, Mobley HL. Merging mythology and morphology: the
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.0/)


Identification of emergent blaCMY-2-carrying Proteus mirabilis lineages by whole-genome sequencing 

Whole-genome sequencing of 24 Proteus mirabilis isolates revealed the clonal expansion of two cefoxitin-resistant strains among patients with community-onset infection. These strains harboured blaCMY-2 within a chromosomally located integrative and conjugative element and exhibited multidrug resistance phenotypes. A predominant strain, identified in 18 patients, also harboured the PGI-1 genomic island and associated resistance genes, accounting for its broader antibiotic resistance profile. The identification of these novel multidrug-resistant strains among community-onset infections suggests that they are endemic to this region and represent emergent P. mirabilis lineages of clinical significance

M. Mac Aogáin

T.R. Rogers

B. Crowley

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Identification of emergent blaCMY-2-carrying Proteus mirabilis lineages by whole-genome sequencing

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Identification of emergent blaCMY-2-carrying Proteus mirabilis lineages by whole-genome sequencing

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