Molecular Types of Methicillin-Resistant Staphylococcus aureus andMethicillin-Sensitive S. aureus Strains Causing Skin and Soft TissueInfections and Nasal Colonization, Identified in Community HealthCenters in New York City

Maria Pardos de la Gandara,a Juan Antonio Raygoza Garay,b Michael Mwangi,b Jonathan N. Tobin,c,d Amanda Tsang,c*Chamanara Khalida,c Brianna D’Orazio,c Rhonda G. Kost,d Andrea Leinberger-Jabari,d Cameron Coffran,d Teresa H. Evering,d

Barry S. Coller,d Shirish Balachandra,e Tracie Urban,e Claude Parola,e Scott Salvato,e Nancy Jenks,f Daren Wu,g Rhonda Burgess,h

Marilyn Chung,a Herminia de Lencastre,a,i Alexander Tomasza

Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, New York, USAa; Department of Biochemistry and Molecular Biology, Penn StateUniversity, University Park, Pennsylvania, USAb; Clinical Directors Network (CDN), New York, New York, USAc; The Rockefeller University Center for Clinical and TranslationalScience, New York, New York, USAd; Urban Health Center, Bronx, New York, USAe; Hudson River Health Care, Peekskill, New York, USAf; Open Door Family Medical Center,Ossining, New York, USAg; Manhattan Physicians Group—125th Street Clinic, New York, New York, USAh; Laboratory of Molecular Genetics, Instituto de TecnologiaQuímica e Biológica (ITQB/UNL), Oeiras, Portugali

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory ofMicrobiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collabora-tive project with six community health centers in the New York City metropolitan area to determine the nature (clonaltype) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected andcharacterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24.Most— 46 of the 63–wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL�) group of S. aureus cloneUSA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonizedby S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolatesbelonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the straininvolved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further docu-mented by whole-genome sequencing.

Staphylococcus aureus is the most common cause of bacterialinfections in humans worldwide (1), and methicillin-resistant

Staphylococcus aureus (MRSA) is the main cause of skin and softtissue infections (SSTIs) in North America, with a single clone,USA300, accounting for 98% of these infections (2, 3).

The first human case of MRSA infection in the United Stateswas reported in Boston, MA, in 1968 (4). MRSA was first detectedin hospitals, and over the following decades, it became the mainnosocomial pathogen around the world (5). In 1998, the preva-lence of MRSA in 12 hospitals throughout the city of New Yorkwas assessed (6), and a single MRSA clone was found to be respon-sible for an overwhelming majority of MRSA infections. The sameMRSA clone was subsequently identified as dominant in MRSAinfections in 29 hospitals in the tristate area (7), and it was alsoidentified in MRSA infections in Japan (8). This MRSA clone(multilocus sequence typing [MLST] clonal complex CC5, se-quence type ST5, SCCmecII, and unique pulsed-field gel electro-phoresis [PFGE] profile)—also known as the “New York/Japanclone” or “MRSA clone USA100”— became the most prevalentMRSA clone involved in MRSA infections in hospitals in theUnited States in the 1990s (9).

In 1993, a new MRSA clone emerged in Kimberley, WesternAustralia (10), in a community of patients without previoushealth care contact (community-acquired MRSA [CA-MRSA]).In the late 1990s, CA-MRSA also appeared in the United States

and was responsible for the death of four otherwise healthy pedi-atric patients in Minnesota and North Dakota (11). These newCA-MRSA strains belonged to a clone (USA400/CC1/SCCmecIV)

Received 4 March 2015 Returned for modification 14 April 2015Accepted 3 June 2015

Accepted manuscript posted online 10 June 2015

Citation Pardos de la Gandara M, Raygoza Garay JA, Mwangi M, Tobin JN, Tsang A,Khalida C, D’Orazio B, Kost RG, Leinberger-Jabari A, Coffran C, Evering TH, Coller BS,Balachandra S, Urban T, Parola C, Salvato S, Jenks N, Wu D, Burgess R, Chung M, deLencastre H, Tomasz A. 2015. Molecular types of methicillin-resistantStaphylococcus aureus and methicillin-sensitive S. aureus strains causing skin andsoft tissue infections and nasal colonization, identified in community healthcenters in New York City. J Clin Microbiol 53:2648 –2658.doi:10.1128/JCM.00591-15.

Editor: N. A. Ledeboer

Address correspondence to Alexander Tomasz, tomasz@rockefeller.edu.

* Present address: Amanda Tsang, Columbia University Medical Center, New York,New York, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00591-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00591-15

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unrelated to the major MRSA clones that were frequent in hospi-tals in the United States during the same period.

In 2002 to 2003, another new clone of MRSA emerged in theUnited States among football players in Pennsylvania, followedsoon after by prison inmates in Mississippi and athletes in Colo-rado, Indiana, and California (for a review, see reference 12). Thisclone, named USA300 (t008/ST8/SCCmecIVa/Panton-Valentineleukocidin positive [PVL�]/ACME�), is currently the most com-mon MRSA clone in the community as well as in hospitals in theUnited States; it is frequently involved with infections of youngerpatients and causes high rates of sepsis and high rates of spreadand mortality when infecting hospitalized patients (3).

In November 2011, The Rockefeller University Center forClinical and Translational Science (CCTS), the Laboratory of Mi-crobiology and Infectious Diseases, and Clinical Directors Net-work (CDN) established a multidisciplinary collaboration, theCommunity-Acquired MRSA (CA-MRSA) Project (CAMP) withsix Community Health Centers (CHCs) strategically located indifferent parts of New York City and surrounding areas with theobjective of tracking the presence of CA-MRSA among SSTIs andwith the aim of improving diagnosis, therapy, and communityawareness of this bacterium (40).

MATERIALS AND METHODSThe study was reviewed and approved by the Institutional Review Boards(IRBs) at Clinical Directors Network and The Rockefeller University.

Biological specimen collection and processing. Following the Infec-tious Diseases Society of America guidelines for managing SSTIs (13),wounds were incised and drained, where possible, and in addition towound specimens, additional surveillance specimens were acquired vianasal swabs. Wound and nasal specimens were sent in liquid Amies trans-port medium (Puritan Medical, Guilford, Maine, USA) to BioReferenceLaboratories (Elmwood Park, NJ, USA) for identification and antibi-ogram by the MicroScan system (Siemens, Munich, Germany). Cultureswere tested against 12 antimicrobial agents, including penicillin, amoxi-cillin-clavulanic acid, cefazolin, oxacillin, clindamycin, erythromycin,gentamicin, levofloxacin, tetracycline, trimethoprim-sulfamethoxazole,vancomycin, and linezolid, according to the Clinical and Laboratory Stan-dards Institute recommendations (14), and antibiograms were providedto the CHC clinician who was responsible for the patient’s care.

S. aureus isolates and information on antibiotype were next sent to theLaboratory of Microbiology and Infectious Diseases at The RockefellerUniversity, New York, NY, for molecular characterization. S. aureus spe-cies confirmation was performed by growth on mannitol-salt agar (MSA;Difco, BBL, Becton Dickinson, Franklin Lakes, NJ, USA) and by testingcoagulase production by the Staphaurex assay (Thermo Fisher Scientific,Lenexa, KS, USA).

Molecular identification: spa typing, MLST, PFGE, and staphylo-coccal cassette chromosome mec (SCCmec) typing. Characterization byspa typing was performed as previously described (15), and spa types weredetermined using the RIDOM web server (http://spaserver.ridom.de/).The spa server was also used to predict sequence types (STs). When STscould not be determined from the spa server, the genetic background ofthe isolates was determined by MLST (16). Assignment of STs was done bysubmission of the DNA sequences of seven housekeeping genes (arcC,aroE, glpF, gmk, pta, tpi, and yqiL) to the online MLST database (http://www.mlst.net/). Clonal complexes were determined for the STs (17).

PFGE was then performed to identify isolates belonging to the sameclonal complex. Bacterial DNA was restricted with SmaI enzyme, and theresulting fragments were separated by electrophoresis (18). Band patternswere compared manually according to guidelines to confirm classification(9, 19).

The structure of the staphylococcal cassette chromosome mec(SCCmec) was determined by multiplex PCR (20), and SCCmec type IV

subtyping was done by multiplex PCR (21). Ambiguous results were fur-ther tested by amplification of the ccrB gene (22) and comparing thesequences obtained with the ccrB typing tool available at the Instituto deTecnologia Quimica e Biologica (ITQB) in Portugal. SCCmec was consid-ered nontypeable (NT) when it was not possible to ascertain a class of meccomplex and/or a type of ccrB.

Molecular characterization: detection of mecA, Panton-Valentineleukocidin (PVL), and ACME genes. Appropriate DNA probes were usedto test for the mecA gene, responsible for resistance to oxacillin and allbeta-lactam antibiotics, and for the lukS and lukF genes (which encodePVL [Panton-Valentine leukocidin]) (23, 24).

The two main loci that make up ACME-I in USA300 strain FPR3757(arcA and opp3) were amplified by PCR (25). This element was typedaccording to its structure: type I (arc and opp3 operons), type II (arcoperon only), and type III (opp3 operon only) (26).

Antibiotic resistance phenotype of wound and nasal isolates. Inva-sive and colonizing isolates from three patients that were characterized bywhole-genome sequencing were also characterized for their oxacillin re-sistance phenotypes by population analysis profiles (PAPs) (27, 28).

Whole-genome sequencing. Bacterial isolates from three patientswere selected for whole-genome sequencing. Two patients (UHP/CAMP-016 and UHP/CAMP-022) carried USA300 isolates in both the woundsand the nares, while the third patient (UHP/CAMP-028) was infected andcolonized by USA1100 strains. From each isolate, genomic DNA was ex-tracted using a Wizard genomic DNA purification kit per the manufac-turer’s instructions (Promega, Madison, WI, USA).

Sequencing was carried out at the Genomics Core Facility of the HuckInstitutes of the Life Sciences at Penn State University. DNA libraries wereconstructed with an Illumina TruSeq DNA-seq library preparation kit andhad an average fragment size of 400 bp. The sequencing involved a 500-cycle run on an Illumina MiSeq to yield 2 by 250-bp paired-end reads.

Complete (i.e., ungapped) genomic sequences for MRSA strainUSA300-FPR3757 and MRSA252 are in RefSeq and can be downloadedfrom the NCBI’s website (http://www.ncbi.nlm.nih.gov/).

Read sequences for the CAMP isolates generated by this study. Thesequences for the CAMP isolates are in the Sequence Read Archive (SRA)and can be downloaded from the NCBI’s website (http://www.ncbi.nlm.nih.gov/).

Selection of USA300-FPR3757 and MRSA252 as reference strains.The DNAStar software SeqMan NGen version 12 was used to generate denovo assemblies of the read sequences. For each CAMP isolate, the num-ber of contigs and the N50 statistic were in the ranges 7 to 11 and 481,266to 1,121,951 bp, respectively. Due to the small read size of 250 bp and thelack of mate pair sequencing, it is likely that the contigs contain numerousassembly errors. Nevertheless, the contigs were useful because they couldbe blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the mostclosely related MRSA strain with a complete genomic sequence availablein the NCBI nucleotide database. For CAMP-18, -19, -28, and -29 (woundand nasal isolates from patients UHP/CAMP-016 and UHP/CAMP-022,respectively), the most closely related strain was USA300-FPR3757, andfor CAMP-36 and -37 (nasal and wound isolates from patient UHP/CAMP-028), it was strain MRSA252.

Reference-guided assemblies of the reads. The DNAStar softwareNGen version 12 was used to generate reference-guided assemblies of theread sequences. For the isolates CAMP-18, -19, -28, and -29, a whole-genome multialignment was generated by mapping the read sequences ofall four isolates to the complete genomic sequence for USA300-FPR3757.For CAMP-36 and -37, another whole-genome multialignment was gen-erated by mapping the read sequences of both isolates to the completegenomic sequence for MRSA252. In each of the two multialignments, themean read coverage on the reference was �50� for every isolate.

DNA molecules in the reference strains and in the related CAMPisolates. From a whole-genome multialignment of the reads, it was clearwhich DNA molecules, besides the chromosome, were in both the refer-ence and the CAMP isolates. Of the three plasmids present in USA300-

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FPR3757, pUSA01 was identified in each of the isolates CAMP-18, -19,-28, and -29, and pUSA02 and pUSA03 were in none of the four isolates.There are no plasmids in MRSA252.

Polymorphism discovery. In each of the two whole-genome multi-alignments of the reads, the DNAStar software SeqMan Pro version 12 wasused to call polymorphisms in the DNA molecules of the reference strainsand the CAMP isolates. In each multialignment, polymorphisms of alltypes and sizes could be called relative to the reference with expectedfalse-positive and false-negative error rates of effectively zero in 98% ofthe columns. Polymorphisms could not be called in the remaining 2%of the columns because they consist of highly repetitive sequences forwhich the read sequences mapped to multiple positions. Error rates wereestimated by computer simulation as follows. For each of the referencestrains—USA300-FPR3757 and MRSA252—a PERL script was used tointroduce polymorphisms of all types and sizes into the complete genomicsequence—in order to generate 100 new synthetic mutated completegenomic sequences each with 1,000 polymorphisms. Next, the programWgSim (https://github.com/lh3/wgsim) was applied to the new mutatedsequences in order to generate synthetic MiSeq read sequences with real-istic sequencing error rates; then, SeqMan NGen was used to generatereference-guided assemblies of the reads as before; and then, SeqMan Prowas used to call polymorphisms as before. In the case of USA300-FPR3757and MRSA252, this meant that a reference-guided assembly always con-sisted of the complete genomic sequence of the reference and also thereads from the clinical isolates (four for FPR3757 and two for MRSA252).

Phylogenetic tree construction. The phylogenetic trees were manu-ally constructed based on the discovered numbers of polymorphisms be-tween the isolates. The more complex tree was constructed with the soft-ware REALPHY using its default settings (http://realphy.unibas.ch/fcgi/realphy) (29). Using the vernacular in the documentation for REALPHY,the reference genome was USA300-FPR3757, the query genomes were thereads for the CAMP isolates (this study) and the northern Manhattanisolates (30), the two reference genome alignments were merged in orderto generate a single tree, and the tree was inferred by PhyML.

Accession numbers. Nucleotide sequence accession numbers areCP000255 and BX571856, respectively (25, 31). The BioProject numbersfor microarray data are as follows: USA300 strain FPR3757, PRJNA58555;strain MRSA252, PRJNA57839; CAMP isolates, PRJNA277213.

RESULTS

One hundred twenty-nine patients with active skin and soft tissueinfections (SSTIs) were enrolled from five Community HealthCenters, and samples were obtained from each of the active SSTIlesions as well as the nares, for strain identification and character-ization. The morphology of the wounds was documented by dig-ital camera (Fig. 1), and a measuring tape was applied adjacent tothe wound.

The study yielded a total of 104 S. aureus isolates: 63 recoveredfrom wounds and 41 from nasal carriage. Of the 63 (49%) patientsinfected by S. aureus, 39 patients (35%) were infected by MRSAstrains and 24 patients (19%) were infected by MSSA strains.

Characterization of the S. aureus isolates by molecular typ-ing techniques. Most of the S. aureus isolates—34 MRSA (54%)and 12 MSSA (19%) isolates— belonged to the USA300 clone(t008/ST8/SCCmecIVa/PVL�/ACME�) or to closely relatedclones (USA300 PFGE profile and ST8 but presenting differencesin spa type, SCCmecIV subtype, and absence of PVL or ACME).Forty-one patients (65%) carried S. aureus in their nares: 19(46%) carried MRSA and 22 (53%) carried MSSA; 23 of the nasalisolates belonged to the clonal group of USA300 (56% of S. aureusnasal isolates) (Fig. 2A and B).

MRSA was involved in 61% of the abscesses (27/44), and 92%(24/27) of these belonged to the USA300 clone. Three patientspresented with MRSA in the wounds and with MSSA in the nares;two cases had MSSA infections but MRSA carriage. In all these

FIG 1 Photographs of skin lesions in six patients enrolled in the study. Skin wounds of all patients were photographed with a digital camera to document themorphology/appearance of the skin and soft tissue infections. Samples taken from the wounds were subsequently used for the identification of the causativeagents.

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cases, the MRSA isolates belonged to the clonal group of USA300while the MSSA strains belonged to very different clones (CC15,CC5, CC1, and CC30, respectively). Tables 1 and 2 show the clonaldistribution of nasal and wound isolates and whether they wereMRSA or MSSA.

Of the 19 patients with MRSA in the nares, 14 were colonizedby the same strain that caused the SSTI, and all except one of thesestrains belonged to the USA300 clone or closely related clones.Three patients carried MRSA in the nares, but no S. aureus wasisolated in the wounds.

ACME was found only among isolates belonging to the CC8/USA300 clonal complex and was most prevalent among MRSA

strains (29/39 MRSA wound infections and 10/19 MRSA nasalisolates). Four MSSA wound isolates that also carried the ACMEvirulence factor belonged to the same CC8/USA300 clonal com-plex. None of the 22 nasal MSSA isolates carried the ACME deter-minant.

The Panton-Valentine leukocidin (pvl) genes lukSF were detectedin half (52.8%) of the wound isolates: in 94% of all MRSA strains and75% of MSSA strains. Among all nasal isolates (84% MRSA and 31%MSSA) 46% carried the lukSF gene. All PVL� strains belonged to theCC8/USA300 complex except for three patients whose isolates be-longed to CC30 and CC88 (all of them also sharing with USA300 theSCCmecIVa cassette). Also, all strains belonging to the USA300 clonal

FIG 2 (A) Molecular types of S. aureus isolates recovered from wounds. (B) Molecular types of S. aureus isolates recovered from nares.

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group carried these genes except two nasal MRSA isolates that be-longed to the “New York clone V” (ST8, SCCmecIVg/ACME�/PVL�) (24).

Twenty-two of the 63 patients (35%) who were identified withan SSTI carrying S. aureus reported previous episodes with similarlesions and described the present lesion as a recurrence. Of those22 cases, 17 (77%) were identified as infections with MRSA, andall of them were infections by the USA300 clone. One patient hada previous diagnosis of MRSA and had a USA300 wound infectionin the present study but did not carry the strain in the nares.

Antibiotic resistance of MRSA and MSSA isolates. One hun-dred four S. aureus isolates, representing all nasal isolates and allbut five wound cultures, were analyzed with antibiograms. Noneof the isolates were resistant to gentamicin, vancomycin, or lin-ezolid. Forty-one isolates (28 MRSA and 13 MSSA strains) showedmultiresistant profiles, i.e., were resistant to three or more familiesof antibiotics (Table 3). Four MRSA isolates were resistant to fourfamilies of antibiotics: �-lactams (penicillin, amoxicillin-clavu-lanic acid, cefazolin, and oxacillin), lincosamides (clindamycin),macrolides (erythromycin), and quinolones (levofloxacin), all be-longing to USA300 or USA100 (“New York/Japan”) clones. Theremaining 24 MRSA isolates that were resistant to three families ofantibiotics showed various patterns of resistance: 21 isolates wereresistant to �-lactams, macrolides, and quinolones (USA300 andUSA100); one USA300 MRSA nasal isolate was resistant to �-lac-tams, lincosamides, and macrolides; one CC88 MRSA wound iso-late was resistant to �-lactams, macrolides, and tetracyclines; andone CC8 MRSA nasal isolate was resistant to �-lactams, quino-lones, and trimethoprim-sulfamethoxazole.

One MSSA nasal isolate belonging to CC15 was resistant toantibiotics of five different families: �-lactams (penicillin, amoxi-cillin-clavulanic acid, and cefazolin, but not oxacillin), lincos-amides, macrolides, quinolones, and tetracyclines. Two woundMSSA isolates were resistant to four families of antibiotics: some�-lactams, lincosamides, macrolides, and quinolones (CC15 andCC8). Nine isolates were resistant to three families of antibiotics:two isolates resistant to �-lactams, macrolides, and tetracyclines(nasal and wound isolates of a single patient with a CC8/PVL�

profile) and seven isolates resistant to �-lactams, lincosamides,and macrolides (CC5, CC8, CC15, CC30, and CC121).

As mentioned above, 14 patients infected with MRSA also car-ried the same strain in the nares, and in 13 of these cases, theMRSA strains belonged to the USA300 group; in one patientthe CA-MRSA recovered from the nares and the wound be-longed to the “South-West Pacific clone” (USA1100/t665/CC30/SCCmecIVa/PVL�/ACME�).

Antibiotic resistance phenotypes of invasive and colonizingisolates. Invasive and colonizing isolates from three patients(UHP-CAMP-016, -CAMP-022, and -CAMP-028) whose isolateswere also analyzed by whole-genome sequencing showed identicaloxacillin-resistant phenotypes as indicated by the superimposablepopulation analysis profiles (Fig. 3).

Comparison of wound and nares isolates in three patients bywhole-genome sequencing. Two of the three patients (UHP/CAMP-016 and UHP/CAMP-022) were infected and colonized byMRSA isolates belonging to the USA300 group (t008 and t052/ST8/SCCmecIVa/PVL�/ACME�). The third patient (UHP/CAMP-028)was infected and colonized by CA-MRSA USA1100.

For each of the three pairs of nares and wound isolatesCAMP-18 and -19, CAMP-28 and -29, and CAMP-36 and -37,we did whole-genome shotgun sequencing to elucidate phylo-genetic relationships and identify polymorphisms of alltypes and sizes. For each of these six isolates, we identified themost closely related MRSA strain available in the NCBI nucle-otide database (http://www.ncbi.nlm.nih.gov/) with a complete(i.e., ungapped) genomic sequence. For CAMP-18, -19, -28, and -29,that strain is USA300 FPR3757, and for CAMP-36 and -37, it isMRSA252.

The tree in Fig. 4 depicts the phylogenetic relationships andnumbers of polymorphisms. It can be seen that for each pair ofnares and wound isolates, they differ by only a few point muta-tions. Isolates CAMP-18 and -19 differed from one another by 5point mutations, isolates CAMP-28 and -29 differed by 7 pointmutations, and CAMP-36 and -37 differed by 4 point mutations.These mutations are described in Table 4 and—in more detail—inTable S1 in the supplemental material. It is unclear if any of thelisted mutations are important for colonization of the nares orinfection of the wound. Curiously, one of the mutations in the

TABLE 2 Distribution of wound isolates belonging to clonal complexCC8

S. aureus type and clone typeNo. of woundisolates

MRSAUSA300 (t008/ST8/SCCmecIVa/PVL�/ACME�) 21Other spa types 7PVL� 0ACME� 2Other 4

Total 34

MSSAUSA300-like (t008/ST8/PVL�/ACME�) 3Other spa types 1PVL� 0ACME� 4Other 4

Total 12

TABLE 1 Distribution of S. aureus isolates grouped by clonal complex

MLST

No. of isolates

Total

Wound Nasal

MRSA MSSA MRSA MSSA

CC8 69 34 12 17 6CC30 12 2 4 1 5CC5 6 1 2 1 2CC15 6 0 2 0 4CC121 3 0 1 0 2ST72 2 1 0 0 1CC1 1 0 0 0 1CC45 1 0 1 0 0CC88 1 1 0 0 0CC97 1 0 0 0 1CC152 1 0 1 0 0CC398 1 0 1 0 0

Total 104 39 24 19 22

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wound isolate CAMP-19 is in a gene, SAUSA300_1059, with ho-mology to exotoxins.

We compared the CAMP isolates to isolates of the same MRSAclone characterized in a recent outbreak by MRSA clone USA300in northern Manhattan (30). Uhlemann et al. shotgun sequencedthe whole genomes of about 400 MRSA isolates recovered duringthe outbreak (30). The tree in Fig. 5 depicts the phylogenetic rela-tionships between the CAMP and the northern Manhattan iso-lates. Figure 5 shows that the genotypes of the four CAMP USA300isolates are within the genetic variation seen in the northern Man-hattan isolates. Therefore, the CAMP and the northern Manhat-tan isolates of USA300 may all be part of the same outbreak.

DISCUSSION

S. aureus was identified in 63 of the 129 cases of infections in thisstudy, and 30% of wounds were infected by MRSA. Of the six skinlesions shown in Fig. 1, MRSA was recovered from only two of thecases. Thus, S. aureus, and more specifically MRSA-caused SSTI,cannot be diagnosed by observation alone—additional laboratoryanalysis is necessary for a correct diagnosis and treatment.

Both ACME and PVL seem to play important roles in the prev-alence of USA300. Many of the isolates in our study (40/69 CC8isolates) did not present with the complete characteristic patternt008/ST8/SCCmecIVa/PVL�/ACME�. Both the cassette chromo-some and the mobile element encoding enzymes involved witharginine catabolism are known to be susceptible to genetic modi-fications, including a total or partial loss from the bacteria (32).Over the years, evolution may have caused changes in the spa gene,generating variants that still belong to the same clonal complex(33). Many wound isolates characterized in this study were closelyrelated but not identical to CA-MRSA USA300 (t008/ST8/SCCmecIVa/PVL�/ACME�). This may indicate that the isolateswere derivatives of the original clone, and while lacking some ofthe virulence determinants, they still shared a genetic backgroundthat facilitates rapid clonal spread.

CA-MRSA strains may not be more virulent than many MSSAclones. The clinical importance of CA-MRSA may reside in a com-bination of factors: a better fitness related to the smaller SCCmeccassettes; higher growth rates; ACME-mediated resistance to hostdefenses; increased virulence due to the PVL toxin (34); and resis-tance to multiple antibiotics, which complicates treatment andincreases the risk of recurrence (35).

We recovered 39 MRSA strains from the wounds: 38 of thesewere CA-MRSA and one strain was a representative of the healthcare-acquired MRSA (HA-MRSA) clone CC5/USA100 (Table 1).Most of the infections were due to a single MRSA clone, USA300,which is currently the most prevalent clone of CA-MRSA in theUnited States and specifically in New York City (30). ACME wasrestricted to strains belonging to the USA300 CA-MRSA clone orclones closely related to it. Interestingly, ACME was also found infour methicillin-sensitive S. aureus (MSSA) isolates recoveredfrom wounds, grouping all CC8/USA300 strains in what Tenoverand Goering described as a family of isolates showing �80% sim-ilarity by PFGE typing (12). In all patients carrying an ACME�

strain in the nares (all of them MRSA), the same strain was alsorecovered from the wounds. ACME was absent from all MSSAnasal cultures.

Panton-Valentine leukocidin is a virulence factor frequentlyassociated with CA-MRSA (94% of all MRSA wound isolates inour study). However, in our study, the prevalence of the geneslukS-lukF was also very high among MSSA (75%) wound isolates.Most (66%) of these MSSA wound isolates belonged to the sameCC8 clonal complex as the USA300 clone, which would suggestthat these strains were derivatives of the original USA300 clonethat have lost the SCCmec cassette while preserving the geneticbackground that would still provide for superior survival and vir-ulence (36, 37). Most nasal MRSA isolates (84%) and some MSSAnasal isolates (30%) presented as PVL�, but in these cases, thesame strain was also detected in the wound culture of the samepatient.

Whole-genome sequencing and PAPs. The identity of woundand carriage (nasal) isolates was documented by standard typingmethods. In three patients, pairs of wound and carriage isolates(nasal isolates) were also compared by whole-genome sequencingand by detailed characterization of the resistance phenotypes us-ing population analysis profiles (28) (Fig. 3). The three patientsincluded UHP/CAMP-016 and UHP/CAMP-022, each infectedand colonized by MRSA strains of the USA300 group (t008 andt052/ST8/SCCmecIVa/PVL�/ACME�), and a third patient, UHP/CAMP-028, who was infected and colonized by CA-MRSAUSA1100 (t665/ST1472/SCCmecIVa/PVL�/ACME�).

Whole-genome sequencing showed that the pairs of isolatesrecovered from the infection and colonization sites of the samepatient were very similar in terms of DNA sequence (Fig. 4).

TABLE 3 Antibiotic resistance profiles of S. aureus isolates

Antibiotic family(-ies)a

No. of isolates

Clonal complex(es)b OriginNo. of patients with both wound and nasalUSA300 isolatesMRSA MSSA

BLAs � CLI � ERY � LEV � TET 0 1 CC15 NasalBLAs � CLI � ERY � LEV 4 2 CC5, CC8, CC15 Wound, nasal 1 patient with USA300BLAs � ERY � TET 1 2 CC8, CC88 Wound, nasal 1 patient with USA300BLAs � ERY � LEV 21 0 CC5, CC8 Wound, nasal 2 patients with USA300, 1 patient with USA300-likeBLAs � CLI � ERY 1 7 CC5, CC8, CC15, CC30 Wound, nasal 1 patient with USA300BLAs � LEV � TMP-SMX 1 0 CC8 NasalBLAs � ERY 7 5 CC8, CC30, CC121 Wound, nasal 1 patient with USA300BLAs � TET 3 1 CC5, CC8 Wound, nasal 1 patient with USA300BLAs � TMP-SMX 0 1 CC8 Nasala BLAs, �-lactam antibiotics; penicillin, amoxicillin-clavulanic acid, cefazolin, and oxacillin in the case of MRSA isolates and penicillin alone for MSSA isolates. CLI, clindamycin;ERY, erythromycin; LEV, levofloxacin; TET, tetracycline; TMP-SMX, trimethoprim-sulfamethoxazole.b Clonal complex as defined by MLST. CC5, clonal complex of the HA-MRSA strain USA100 (“New York/Japan” clone); CC8, clonal complex of the CA-MRSA strain USA300clone.

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Population analysis profiles (PAPs) showed that the bacterialisolates from the wound and nares of the same patient alsoexhibited identical— heterogeneous—resistance to oxacillin.The PAPs of bacteria recovered from the two patients infectedby USA300 were virtually identical and were different from thePAP of strains recovered from patient UHP/CAMP-028, who

was infected by a MRSA strain belonging to the USA1100 clone(Fig. 3).

In conclusion, the CA-MRSA clone USA300 was identified asthe most prevalent S. aureus clone causing SSTIs in metropolitanNew York City community health centers. Methicillin-susceptiblevariants of the same clone that share most of the genetic back-

FIG 3 Population analysis profiles of the nasal and wound isolates from three selected patients. UHP/CAMP-016 is a patient who carries in both the wound andthe nares the same USA300 strain (t008/ST8/SCCmecIVa/PVL�/ACME�); UHP/CAMP-022 is another patient who carries in wound and nares a variant ofUSA300 (t052/ST8/SCCmecIVa/PVL�/ACME�); UHP/CAMP-028 is a patient who carries a USA1100 strain (t665/ST1472/SCCmecIVa/PVL�/ACME�) in boththe wound and the nares.

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ground of USA300 were also prevalent among isolates causingSSTIs.

Clinical presentation alone is not sufficient to predict the na-ture of the pathogen associated with SSTIs; bacterial cultures and

molecular identification are needed to determine the true preva-lence and spread of MRSA clones.

Nasal carriage cannot be used as a predictor of S. aureus SSTIinfection. Many patients who were found to have active infections

FIG 4 Phylogeny of MRSA isolates recovered from wounds and nasal colonization sites of three patients: comparison by whole-genome sequencing. CAMP-18and CAMP-19 are the wound and nasal isolates, respectively, from patient UHP/CAMP-016. CAMP-28 and CAMP-29 are the nasal and wound isolates, frompatient UHP/CAMP-022. CAMP-36 and CAMP-37 are the nasal and wound isolates, respectively, of patient UHP/CAMP-028. CAMP-18 and CAMP-19 wereboth characterized as USA300 (t008/ST8/SCCmecIVa/PVL�/ACME�); CAMP-28 and CAMP-29 belonged to a variant of USA300 (t052/ST8/SCCmecIVa/PVL�/ACME�); CAMP-36 and CAMP-37 belonged to the clone USA1100 (t665/CC30/SCCmecIVa/PVL�/ACME�). Beside each tree branch are listed thenumbers of point mutations (boldface) and larger structural variants (in parentheses) between the chromosomes of the ancestor and descendant.

TABLE 4 Mutations in CAMP isolatesg

Mutation presencea

ReferenceMRSAstrain andposition Positionb Changec Type

Proximal gened

Referencee N315f Name Function

CAMP-18, not CAMP-19 FPR3757 357216 C¡T Intergenic SAUSA300_0307 SA0295 Acid phosphatase?1181875 T¡C Synonymous SAUSA300_1080 SA1029 ftsZ Cell division

CAMP-19, not CAMP-18 FPR3757 1159271 C¡T Nonsynonymous SAUSA300_1059 SA1009 Exotoxin?1777369 T¡— Frameshift SAUSA300_1622 SA1499 tig Molecular chaperone1858349 A¡G Synonymous SAUSA300_1686 SA1561 murC Cell wall synthesis

CAMP-28, not CAMP-29h

CAMP-29, not CAMP-28 FPR3757 157471 C¡A Nonsynonymous SAUSA300_0138 SA0131 deoD Purine metabolism1043552 —¡A Intergenic SAUSA300_0954 SA0904 Antibiotic resistance?1387815 G¡A Nonsynonymous SAUSA300_1260 SA1197 tyrA Shikimate pathway1768026 T¡C Synonymous SAUSA300_1614 SA1491 hemL Porphyrin biosynthesis2031280 T¡A Intergenic SAUSA300_1871 SA17062335992 G¡A Intergenic SAUSA300_21582348652 A¡— Intergenic SAUSA300_2167

CAMP-36, not CAMP-37 252 142269 C¡A Intergenic SAR0129 SA0122 butA Acetoin formation1042026 T¡— Frameshift SAR0994 SA08812105113 C¡T Nonsynonymous SAR2012 SA1735

CAMP-37, not CAMP-36 252 2707338 G¡A Nonsynonymous SAR2622 SA2330 Transcriptional regulator?a CAMP-18, CAMP-28, and CAMP-36 are nares isolates. CAMP-19, CAMP-29, and CAMP-37 are wound isolates.b Nucleotide position on the chromosome of the reference strain. No mutations were observed on plasmids.c Nucleotide change on the Watson strand where sense (Watson or Crick) is defined by the reference. The convention used is [original]¡[new], where “a to —” means there is adeletion and “— to a” means there is an insertion.d If mutation is in a gene, that gene; if mutation is in intergenic sequence between divergently or tandemly transcribed genes, the nearest downstream gene; and if mutation is inintergenic sequence between convergently transcribed genes, the nearest gene.e Name of gene in reference.f Name of orthologous gene in strain N315.g For more detailed descriptions, see Table S1 in the supplemental material.h No mutations were found in CAMP-28 that were not found in CAMP-29.

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by MRSA did not carry the pathogen in the nares (22 patientspresenting with MRSA only in the wound versus 14 patients withMRSA both in the wound and in the nares). Nevertheless, MRSAnasal carriage often accompanied MRSA wound infections by thesame strain. In the present study, we performed an extensivescreening on some patients presenting with recurrent skin and softtissue infections with MRSA in which carriage was checked bothin the nares and at nine additional body sites (pharynx, axillae,cubital folds, inguinal folds, and patellar folds). In some patients,the nasal carriage was negative (data not shown), and in oneUSA300-infected patient, the infecting MRSA strain was foundonly in the inguinal and patellar folds (38).

A recent study described an outbreak and extensive spread ofMRSA belonging to the clonal type USA300 in northern Manhat-tan (30) during the same time period in which our CAMP studywas performed. Comparison of the sequencing data presented inthis communication with the sequencing data available from thenorthern Manhattan outbreak clearly indicates the involvementof strains with an identical clonal type.

Given the capacity of the USA300 MRSA clone to adhere to andsurvive on skin and on a variety of inert surfaces, control stepsshould include screening for carriage by close contacts and byfomites in homes and public transportation (39), which may rep-resent potential reservoirs of these dangerous MRSA clones.

ACKNOWLEDGMENTS

This work was supported by NIH-NCATS grant 8 UL1 TR000043 to B. S.Coller (principal investigator [PI]) and by a 2011 CTSA AdministrativeSupplement Award to J. N. Tobin (PI). Additional support was providedby U.S. Public Health Service award 2 RO1 AI457838-15 to A. Tomasz(PI), AHRQ grant 1 P30-HS-021667 to J. N. Tobin (PI), and PCORI grantCER-1402-10800 to J. N. Tobin (PI).

We thank Vikas Koundal (Department of Biochemistry and Molecu-lar Biology, Penn State University) for help in the additional purificationand concentration of some DNA samples prior to genome sequencing.We greatly appreciate the efforts of the participating community healthcenters, including Brookdale Family Center (Brooklyn, NY), HudsonRiver Healthcare (Peekskill, NY), Manhattan Physicians Group (NewYork, NY), Open Door Family Health Center (Ossining, NY), and UrbanHealth Plan (Bronx, NY); the BioReference Laboratories, Inc.; and thecollaboration of the clinicians, office staff, and patients.

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