Detection of mobile-genetic-element variation between colonizing and infecting hospital-associated methicillin-resistant Staphylococcus aureus isolates.

Abstract

Whole-genome analysis by 62-strain microarray showed variation in resistance and virulence genes on mobile genetic elements (MGEs) between 40 isolates of methicillin-resistant Staphylococcus aureus (MRSA) strain CC22-SCCmecIV but also showed (i) detection of two previously unrecognized MRSA transmission events and (ii) that 7/8 patients were infected with a variant of their own colonizing isolate.

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Published Ahead of Print 14 December 2011.
10.1128/JCM.05938-11.
2012, 50(3):1073. DOI:
J. Clin. Microbiol.
Lindsay
Alex J. McCarthy, Aodhan S. Breathnach and Jodi A.
Staphylococcus aureus Isolates
Hospital-Associated Methicillin-Resistant
Variation between Colonizing and Infecting
Detection of Mobile-Genetic-Element
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Detection of Mobile-Genetic-Element Variation between Colonizing
and Infecting Hospital-Associated Methicillin-Resistant
Staphylococcus aureus Isolates
Alex J. McCarthy, Aodhan S. Breathnach, and Jodi A. Lindsay
Centre for Infection, Division of Clinical Sciences, St George’s University of London, London, United Kingdom
Whole-genomeanalysisby62-strainmicroarrayshowedvariationinresistanceandvirulencegenesonmobilegeneticelements
(MGEs) between 40 isolates of methicillin-resistant Staphylococcus aureus (MRSA) strain CC22-SCCmecIV but also showed (i)
detectionoftwopreviouslyunrecognizedMRSAtransmissioneventsand(ii)that7/8patientswereinfectedwithavariantof
theirowncolonizingisolate.
H
tions. Control of MRSA incidence and spread requires precise
approaches for detecting epidemiological relationships between
bacteria.
Comparative genomics has revealed that the majority of hu-
man S. aureus isolates belong to 10 major lineages (6). Only five
lineages that have acquired methicillin resistance are responsible
for the majority of hospital-associated MRSA (HA-MRSA) infec-
tions (9). Lineages are defined by genes encoding surface-
expressedproteinsandtheirregulators,andthesegenesarehighly
stable(7).Incontrast,20%ofthegenomeisconstitutedofmobile
genetic elements (MGEs), including bacteriophages, plasmids, S.
aureus pathogenicity islands (SaPIs), transposons, and staphylo-
coccal chromosome cassettes (SCCs) (5). MGEs move between
bacteria by horizontal gene transfer (HGT) and carry clinically
relevantgenes,includingantibioticresistanceandvirulencegenes.
Current genotypic methods can successfully identify HA-
MRSA clones (1–3). Studies using such methods have concluded
that infections in MRSA-colonized individuals are caused by the
patient’s own colonizing strain (10, 12). However, these ap-
proachesdonotdistinguishvariationwithinHA-MRSAclonesor
have sufficient power to identify close epidemiological relation-
ships between isolates.
In this study, we used a newly developed 62-strain S. aureus
microarray(SAM-62)toassessMGEgenedistributions.Weasked
whether patients colonized with the major HA-MRSA strain in
our hospital, CC22-SCCmecIV, who developed subsequent infec-
tion were infected by their own strain variant. As a control, we
selected random MRSA isolates from multiple wards in our hos-
pital during the same time period. Using this approach, we show
that the patient’s own colonizing flora is the major reservoir of
infecting HA-MRSA, and we identified two previously unrecog-
nized MRSA transmission events.
We analyzed 40 MRSA CC22 isolates from patients of St
George’s Healthcare NHS Trust, London, United Kingdom, in-
cluding previously described paired colonization (at hospital ad-
mission),subsequentinfectingisolatesfrom8patients(4),and24
invasiveisolatestakenatrandomfromarangeofwardsandspec-
imentypesduringthesame2-monthperiodthatthecolonization
isolates were collected. A sequenced CC22 MRSA isolate (5096)
was also analyzed. Microarray experiments were performed using
ospitals are reservoirs of methicillin-resistant Staphylococcus
aureus (MRSA) isolates that commonly cause invasive infec-
SAM-62 as previously described (8). The array design is available
at B?G@Sbase (accession no. A-BUGS-38; http://bugs.sgul.ac.uk
/A-BUGS-38) and ArrayExpress (accession no. A-BUGS-38). We
performed hierarchical clustering analysis using a Euclidean dis-
tance metric based on 11,715 60-mer oligonucleotides repre-
senting MGE genes. Fully annotated microarray data have been
deposited in B?G@Sbase (accession no. E-BUGS-128; http:
//bugs.sgul.ac.uk/E-BUGS-128) and also ArrayExpress (acces-
sion no. E-BUGS-128).
Thecoregenomewashighlyconserved(datanotshown);how-
ever, the MGE content of isolates varied considerably (Fig. 1).
Some MGEs were highly frequent, such as ?2 (100%) and ?3
(98%)bacteriophagesandrep10(83%).Thebacteriophage?3car-
ries the immune evasion cluster (IEC) genes chp, scn, and sak that
are prevalent in human-associated S. aureus (11, 13). rep10plas-
midswereassociatedwithermC,encodingresistancetomacrolide
antibiotics. The Panton-Valentine leukocidin (PVL) toxin, typi-
cally carried on the bacteriophage ?2, was absent in our isolates.
The distribution of other MGEs was more varied, including ?1,
?6, SaPI2, SaPI3, SaPI4, SaPI5, rep5, rep7, rep15, rep20, rep22, rep29,
and rep30(Fig. 1). Many resistance genes had variable distribu-
tions,includingaacAandaphD(24%),cadA(39%),cadDX(5%),
dfrA (2%), ermC (80%), merAB (2%), mupA (7%), qacA (17%),
smr (7%), and tetK (2%). Therefore, a diverse range of MGEs and
virulenceandresistancegeneswerepresentinMRSAstrainCC22-
SCCmecIV in our hospital at the same time.
Isolates were clustered by the presence and absence of MGE
genes. A total of 7/8 pairs of colonization versus invasive isolates
werecloselyrelatedandcarriedthesameMGEs(Fig.1).Isolates3c
and 3i did not cluster together, but differed only by carriage of 3
SaPI genes, which could represent a single genetic event (data not
shown). Isolates 7c and 7i did not cluster together and differed by
carriage of SaPI2, SaPI3, rep7, and rep15, representing multiple
Received 27 September 2011 Returned for modification 3 November 2011
Accepted 5 December 2011
Published ahead of print 14 December 2011
Address correspondence to Jodi A. Lindsay, jlindsay@sgul.ac.uk.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.05938-11
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genetic events (5); therefore, this patient was probably colonized
and infected with different isolates. These data support previous
findings that invasive infections in MRSA-colonized individuals
arelikelytobecausedbythepatient’sowncolonizingstrainrather
than by circulating hospital strains (10, 12). This analysis demon-
strates that MGE profiling can accurately detect epidemiological
relationships between MRSA isolates.
Clustering analysis revealed two groups of closely related
MRSAisolates.Cluster1representsfourinvasiveisolates(7i,A,B,
and C) carrying the same MGEs, implying a close relationship
between isolates (Fig. 1). We analyzed patient notes to search for
evidence of interaction between patients (Fig. 2A). Patients 7 and
A were present on the same ward, X, and their isolates were pres-
ent in wound/peg site and wound site specimens on 14 May 2009
and on 4 June 2009, respectively. Interestingly, patient 7 did not
develop an infection with his or her own nasal colonizing isolate.
Patient B moved onto ward Y, a ward that shares medical teams
withwardX,on4June2009,andthispatienthadMRSAinawrist
siteinfectionspecimenonthesameday.PatientBmovedtoward
Z on 11 June 2009 and, subsequently, patient C on ward Z ac-
quired a MRSA infection (suprapubic catheter site swab sample
taken on 15 June 2009). These data show that previously unrec-
ognized MRSA transmission events can be identified using MGE
profiling.
Cluster2representsfourinvasiveisolates(D,E,F,andG)from
three patients carrying the same MGEs (Fig. 1 and 2B). Patient D
FIG1 Clustering of HA-MRSA CC22-SCCmecIV isolates and distribution of bacteriophage int, SaPI int, and plasmid rep genes. Each vertical line represents an
isolate.Carriage/invasiveisolatepairsaredenotedbyanumberandeithercforcarriageoriforinvasiveisolate.IsolatesA,B,andCareHA-invasiveisolatesthat
haveacloseevolutionaryrelationshipandformcluster1withisolate7i.IsolatesD,E,F,andGareHA-invasiveisolatesthathaveacloseevolutionaryrelationship
and form cluster 2. Note that isolates F and G originate from the same person. Isolates without labels are all HA-invasive isolates. Isolates have been clustered
using data from 60-mer oligonucleotides that represent all genes on mobile genetic elements (MGEs). Horizontal lines represent different 60-mer-
oligonucleotide probes specific to 8 bacteriophage int genes, 5 SaPI int genes, and 18 plasmid rep genes. The color in the main figure depicts whether the gene is
present in the respective isolate (red or yellow, present; black or blue, absent).
McCarthy et al.
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waspresentonwardU,andtheisolatewastakenfromsputumon
9June2009.Interestingly,patientEwaspresentonwardUfrom6
June 2009 and moved onto ward W on 17 June 2009, and this
patient had a wound site infection specimen on 23 June 2009.
IsolatesFandGwerefromthesamepatientonthesameward,W,
3dayslater,indicatingthatthesameMRSAstrainmayhavetrans-
ferredbetweentwopatients.IsolateFwasfromagraftsite,andwe
had the opportunity to include a second isolate, isolate G, from a
tissue swab. Interestingly, isolate F carried an additional plasmid
(rep25) and resistance gene (smr) that were not found in isolate G,
suggesting that MGE can transfer between isolates from the same
patient.
In conclusion, we found that a highly diverse range of MGEs
waspresentinaHA-MRSACC22cloneinahospitalsettingwithin
a short time frame. In at least 7/8 cases, the colonizing strain of a
patient at admission was highly similar to the strain causing sub-
sequent infection. Our study demonstrates that unrecognized
MRSAtransmissioneventscanbeidentifiedusingMGEprofiling.
ACKNOWLEDGMENTS
We acknowledge Jason Hinds, Kate Gould, Denise Waldron, and Adam
Witney from B?G@S (the Bacterial Microarray Group at St George’s,
UniversityofLondon)forsupplyofthemicroarrayandadvice.Wethank
Juliane Krebes, Emma Budd, and Alastair Thornley for technical and lo-
gistical assistance.
ThisworkwassupportedbythePILGRIMFP7grantfromtheEU.We
thank The Wellcome Trust for funding the multicollaborative microbial
pathogen microarray facility under its Functional Genomics Resources
Initiative.
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FIG 2 Detection of two unrecognized transmission events. Each row represents a patient, and each shade of gray represents a different ward. The first time a
patient was identified as MRSA positive is shown with a white spot. (A) Four patients on three different hospital wards are shown for each day by color (black,
wardX;lightgray,wardY;darkgray,wardZ).WardXandwardYaresisterwardsofgeneralmedicine,locatedonthesamefloor,andtheysharethesamemedical
teams. (B) Three patients on two different hospital wards are shown for each day by color (black, ward U; gray, ward W).
MGE Profiling of MRSA
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