1 molecular types of mrsa and mssa strains causing skin and
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Molecular Types of MRSA and MSSA Strains Causing Skin and Soft Tissue Infections and 1 Nasal Colonization – Identified in Community Health Centers in New York City 2 3 Running Title: MRSA in New York Community Health Centers 4 5 Maria Pardos de la Gandara1, Juan Antonio Raygoza Garay2, Michael Mwangi2, Jonathan N. 6 Tobin3,4, Amanda Tsang3*, Chamanara Khalida3, Brianna D’Orazio3, Rhonda G. Kost4, Andrea 7 Leinberger-Jabari4, Cameron Coffran4, Teresa H. Evering4, Barry S. Coller4, Shirish 8 Balachandra5, Tracie Urban5, Claude Parola5, Scott Salvato5, Nancy Jenks6, Daren Wu7, Rhonda 9 Burgess8, Marilyn Chung1, Herminia de Lencastre1,9, Alexander Tomasz1# 10 11 1Laboratory of Microbiology & Infectious Diseases, The Rockefeller University, New York 12 2Department of Biochemistry and Molecular Biology, Penn State University, University Park, 13 PA 14 3Clinical Directors Network (CDN), New York, NY 15 4The Rockefeller University Center for Clinical and Translational Science, New York, NY 16 5Urban Health Center, Bronx, NY 17 6Hudson River Health Care, Peekskill, NY 18 7Open Door Family Medical Center, Ossining, NY 19 8Manhattan Physicians Group – 125th Street Clinic, New York, NY 20
JCM Accepted Manuscript Posted Online 10 June 2015J. Clin. Microbiol. doi:10.1128/JCM.00591-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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9 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica (ITQB/UNL), 21 Oeiras, Portugal 22 23 # Corresponding author: Dr. Alexander Tomasz, Laboratory of Microbiology & Infectious 24 Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10065. Email: 25 [email protected] 26 27 * Present addresses: Amanda Tsang, Columbia University Medical Center, New York, New 28 York, United States. 29 30
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Abstract 31 In November 2011, The Rockefeller University Center for Clinical and Translational 32
Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors 33 Network (CDN) launched a research and learning collaborative project with six Community 34 Health Centers in the New York City metropolitan area to determine the nature (clonal type) of 35 community-acquired Staphylococcus aureus strains causing skin and soft tissue infections 36 (SSTIs). Between November 2011 to March 2013 wound and nasal samples from 129 patients 37 with active SSTIs suspicious of S. aureus were collected and characterized by molecular typing 38 techniques. In 63 of 129 patients the skin wounds were infected by S. aureus: MRSA was 39 recovered from 39 wounds and MSSA from 24. Most – 46 of the 63 – wound isolates belonged 40 to the CC8/PVL+ group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were 41 MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the 42 nares: 16 of the colonizing isolates were MRSA and 14 were MSSA and the majority of the 43 colonizing isolates belonged to the USA300 clonal group. In most cases (70%) the colonizing 44 isolate belonged to the same clonal type as the strain involved with the infection. In three of the 45 patients, the identity of invasive and colonizing MRSA isolates was further documented by 46 whole genome sequencing. (223 Words) 47 48
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Introduction 49 Staphylococcus aureus is the most common cause of bacterial infections in humans 50
worldwide (1) and methicillin-resistant Staphylococcus aureus (MRSA) is the main cause of skin 51 and soft tissue infections (SSTIs) in North America, with a single clone, USA300, accounting for 52 98% of these infections (2, 3). 53
The first human case of MRSA infection in the US was reported in Boston in 1968 (4). 54 MRSA was first detected in hospitals, and over the following decades, it became the main 55 nosocomial pathogen around the world (5). In 1998, the prevalence of MRSA in 12 hospitals 56 throughout the city of New York was assessed (6) and a single MRSA clone was found to be 57 responsible for an overwhelming majority of MRSA infections. The same MRSA clone was 58 subsequently identified as dominant in MRSA infections in 29 hospitals in the tri-state area (7) 59 and it was also identified in MRSA infections in Japan (8). This MRSA clone (MLST clonal 60 complex CC5, sequence type ST5, SCCmecII and a unique PFGE profile) – also known as the 61 “New York/Japan clone” or “MRSA clone USA100” – became the most prevalent MRSA clone 62 involved in MRSA infections in hospitals in the USA in the 1990s (9). 63
In 1993 a new MRSA clone emerged in Kimberley, Western Australia (10) in a 64 community of patients without previous health care contact [community acquired (CA)-MRSA]. 65 In the late 1990s CA-MRSA also appeared in the USA and was responsible for the death of four 66 otherwise healthy pediatric patients in Minnesota and North Dakota (11). These new CA-MRSA 67 strains belonged to a clone (USA400/CC1/SCCmecIV) unrelated to the major MRSA clones that 68 were frequent in hospitals in the USA during the same period. 69
In 2002-2003, another new clone of MRSA emerged in the USA among football players 70 in Pennsylvania, followed soon after by prison inmates in Mississippi and athletes in Colorado, 71
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Indiana and California (for a review see (12)). This clone, named USA300 72 (t008/ST8/SCCmecIVa/PVL+/ACME+), is currently the most common MRSA clone in the 73 community as well as in hospitals in the USA; it is frequently involved with infections of 74 younger patients and causes high rates of sepsis and high rates of spread and mortality when 75 infecting hospitalized patients (3). 76
In November 2011, The Rockefeller University Center for Clinical and Translational 77 Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors 78 Network (CDN) established a multidisciplinary collaboration, the Community-Acquired MRSA 79 (CA-MRSA) Project (CAMP) with six Community Health Centers strategically located in 80 different parts of New York City and surrounding areas with the objective of tracking the 81 presence of CA-MRSA among SSTIs, and with the aim of improving diagnosis, therapy and 82 community awareness of this bacterium (Tobin et al. in preparation). 83 84 Materials and Methods 85 Biological specimen collection and processing 86
Following CDC guidelines for managing SSTIs (13) wounds were incised and drained, 87 where possible, and in addition to wound specimens, additional surveillance specimens were 88 acquired via nasal swabs. Wound and nasal specimens were sent in liquid Amies transport 89 medium (Puritan Medical, Guilford, Maine, US) to BioReference Laboratories (Elmwood Park, 90 NJ, US) for identification and antibiogram by the MicroScan system (Siemens, Munich, 91 Germany). Cultures were tested against 12 antimicrobial agents, including: penicillin, 92 amoxicillin/clavulanic acid, cefazolin, oxacillin, clindamycin, erythromycin, gentamicin, 93
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levofloxacin, tetracycline, trimethoprim/sulfamethoxazole, vancomycin, and linezolid, following 94 the Clinical and Laboratory Standards Institute recommendations (14) and antibiograms were 95 provided to the CHC clinician who was responsible for the patient’s care. 96
S. aureus isolates and information on antibiotype were next sent to the Laboratory of 97 Microbiology and Infectious Diseases at The Rockefeller University, NY, for molecular 98 characterization. S. aureus species confirmation was performed by growth on Mannitol-Salt agar 99 (MSA, Difco, BBL, Becton Dickinson, Franklin Lakes, NJ, USA) and by testing coagulase 100 production by Staphaurex assay (Thermo Fisher Scientific, Lenexa, KS, USA). 101 102 Molecular identification: spa typing, MLST, PFGE, SCCmec typing 103
Characterization by spa typing was performed as previously described (15) and spa types 104 were determined using the RIDOM web server (http://spaserver.ridom.de/). The spa server was 105 also used to predict sequence types (ST). When STs could not be determined from the spa server, 106 the genetic background of the isolates was determined by MLST (16). Assignment of STs was 107 done by submission of the DNA sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, 108 pta, tpi, yqiL) to the online MLST database (http://www.mlst.net/). Clonal Complexes were 109 determined for the STs (17). 110
PFGE was then performed to discern isolates belonging to the same clonal complex. 111 Bacterial DNA was restricted with SmaI enzyme and the resulting fragments were separated by 112 electrophoresis (18). Band patterns were compared manually following guidelines to confirm 113 classification (9, 19). 114
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The structure of the staphylococcal cassette chromosome mec (SCCmec) was determined 115 by multiplex PCR (20) and SCCmec type IV subtyping was done by multiplex PCR (21). 116 Ambiguous results were further tested by amplification of the ccrB gene (22) and comparing the 117 sequences obtained with the online database (http://www.ccrbtyping.net/). SCCmec was 118 considered non-typable (NT) when it was not possible to ascertain a class of mec complex and/or 119 a type of ccrB. 120 121 Molecular characterization: detection of mecA, Panton-Valentine Leukocidin (PVL), and ACME 122 genes 123
Appropriate DNA probes were used to test for mecA gene, responsible for resistance to 124 oxacillin and all beta-lactam antibiotics, and for the lukS and lukF genes (which encode PVL, the 125 Panton-Valentine leukocidin) (23, 24). 126
The two main loci that make up ACME-I in USA300 strain FPR3757 (arcA and opp3) 127 were amplified by PCR (25). This element was typed according to its structure: type I (arc and 128 opp3 operons), type II (arc operon only), and type III (opp3 operon only) (26). 129
130 Antibiotic resistance phenotype of wound and nasal isolates 131 Invasive and colonizing isolates from three patients that were characterized by whole 132 genome sequencing were also characterized for their oxacillin resistant phenotypes by population 133 analysis profiles (PAPs) (27, 28). 134 135 Whole genome sequencing 136
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Bacterial isolates from three patients were selected for whole genome sequencing. Two 137 patients (UHP/CAMP-016 and UHP/CAMP-022) carried USA300 isolates in both the wounds 138 and the nares, while the third patient (UHP/CAMP-028) was infected and colonized by 139 USA1100 strains. From each isolate, genomic DNA was extracted using a Wizard® Genomic 140 DNA Purification kit as per the manufacturer’s instructions (Promega, Madison, WI, USA). 141
Sequencing was carried out at the Genomics Core Facility of the Huck Institutes of the 142 Life Sciences at Penn State University. DNA libraries were constructed with an Illumina TruSeq 143 DNA-seq Library Preparation Kit and had an average fragment size of 400 bp. The sequencing 144 involved a 500-cycle run on an Illumina MiSeq to yield 2 × 250 bp paired-end reads. 145 Complete (i.e. ungapped) genomic sequences for MRSA strains USA300-FPR3757, and MRSA 146 252 are in RefSeq and can be downloaded from the NCBI’s website 147 (http://www.ncbi.nlm.nih.gov/). Accession numbers are : NC_002952 and BX571856 148 respectively (25, 29). The BioProject numbers are as follows: for USA300 strain FPR3757 it is 149 PRJNA58555; and for strain MRSA252 it is PRJNA57839. 150 151 Read sequences for the CAMP isolates generated by this study 152 The sequences are in the Sequence Read Archive (SRA) and can be downloaded from the 153 NCBI’s website (http://www.ncbi.nlm.nih.gov/). The BioProject number is PRJNA277213. 154 155 Selection of USA300-FPR3757 and MRSA 252 – as reference strains 156
The software DNASTAR’s SeqMan NGen version 12 (http://www.dnastar.com/) was 157 used to generate de novo assemblies of the read sequences. For each CAMP isolate, the number 158 of contigs and the N50 statistic were in the ranges 7-11 and 481,266-1,121,951 bp respectively. 159
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Due to the small read size of 250 bp and the lack of mate pair sequencing, it is likely that the 160 contigs contain numerous assembly errors. Nevertheless, the contigs were useful because they 161 could be blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the most closely related 162 MRSA strain with a complete genomic sequence available in the NCBI nucleotide database. For 163 CAMP-18, -19, -28, and -29 (wound and nasal isolates from patients UHP/CAMP-016 and 164 UHP/CAMP-022 respectively) the most closely related strain was USA300-FPR3757, and for 165 CAMP-36 and -37 (nasal and wound isolates from patient UHP/CAMP-028), it was strain 166 MRSA252. 167 168 Reference-guided assemblies of the reads 169
The software NGen version 12 (http://www.dnastar.com/) was used to generate 170 reference-guided assemblies of the read sequences. For the isolates CAMP-18, -19, -28, and -29, 171 a whole genome multi-alignment was generated by mapping the read sequences of all four 172 isolates to the complete genomic sequence for USA300-FPR3757. For CAMP-36 and -37, 173 another whole genome multi-alignment was generated by mapping the read sequences of both 174 isolates to the complete genomic sequence for MRSA252. In each of the two multi-alignments, 175 the mean read coverage on the reference was ≥50X for every isolate. 176 177 DNA molecules in the reference strains and in the related CAMP isolates 178
From a whole genome multi-alignment of the reads, it was clear which DNA molecules, 179 besides the chromosome, were in both the reference and the CAMP isolates. Of the three 180 plasmids present in USA300-FPR3757, pUSA01 was identified in each of the isolates CAMP-181
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18, -19, -28, and -29, and pUSA02 and pUSA03 are in none of the four isolates. There are no 182 plasmids in MRSA252. 183 184 Polymorphism discovery 185
In each of the two whole genome multi-alignments of the reads, the software 186 DNASTAR’s SeqMan Pro version 12 (http://www.dnastar.com/) was used to call 187 polymorphisms in the DNA molecules of the reference strains and the CAMP isolates. In each 188 multi-alignment, polymorphisms of all types and sizes could be called relative to the reference 189 with expected false positive and false negative error rates of effectively zero in 98% of the 190 columns. Polymorphisms could not be called in the remaining 2% of the columns because they 191 consist of highly repetitive sequences for which the read sequences mapped to multiple positions. 192 Error rates were estimated by computer simulation as follows. For each of the reference strains - 193 USA300-FPR3757 and MRSA252 - a PERL script was used to introduce polymorphisms of all 194 types and sizes into the complete genomic sequence - in order to generate 100 new synthetic 195 mutated complete genomic sequences each with 1000 polymorphisms. Next the program WgSim 196 (https://github.com/lh3/wgsim) was applied to the new mutated sequences in order to generate 197 synthetic MiSeq read sequences with realistic sequencing error rates; then SeqMan NGen was 198 used to generate reference-guided assemblies of the reads as before; and then SeqMan Pro was 199 used to call polymorphisms like before. In the case of USA300-FPR3757 and MRSA252, this 200 meant that a reference-guided assembly always consisted of the complete genomic sequence of 201 the reference and also the reads from the clinical isolates (four for FPR3757 and two for 202 MRSA252). 203 204
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Phylogenetic tree construction 205 The phylogenetic trees were manually constructed based on the discovered numbers of 206
polymorphisms between the isolates. The more complex tree was constructed with the software 207 REALPHY using its default settings (http://realphy.unibas.ch/fcgi/realphy) (30). Using the 208 vernacular in the documentation for REALPHY: the reference genome was USA300- FPR3757; 209 the query genomes were the reads for the CAMP isolates (this study) and the northern Manhattan 210 isolates (31); the two reference genome alignments were merged in order to generate a single 211 tree; and the tree was inferred by PhyML. 212
213 214 Results 215
One hundred and twenty nine patients with active skin and soft tissue infections (SSTIs) 216 were enrolled from five Community Health Care Centers and samples were obtained from each 217 of the active SSTI lesions as well as the nares, for strain identification and characterization. The 218 morphology of the wounds was documented by digital camera (Figure 1) and a measuring tape 219 was applied adjacent to the wound. 220
The study yielded a total of 104 S. aureus isolates: 63 recovered from wounds and 41 221 from nasal carriage. Of the 63 (49%) patients infected by S. aureus, 39 patients (35%) were 222 infected by MRSA strains and twenty-four patients (19%) by MSSA strains. 223
224 Characterization of the S. aureus isolates by molecular typing techniques 225
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Most of the S. aureus isolates – 34 MRSA (54%) and 12 MSSA (19%) – belonged to the 226 USA300 clone (t008/ST8/SCCmecIVa/PVL+/ACME+) or to closely related clones (USA300 227 PFGE profile and ST8, but presenting differences in spa type, SCCmecIV subtype, and absence 228 of PVL or ACME). Forty-one patients (65%) carried S. aureus in their nares: 19 (46%) carried 229 MRSA and 22 (53%) carried MSSA: twenty-three of the nasal isolates belonged to the clonal 230 group of USA300 (56% of S. aureus nasal isolates) (Figures 2A and B). 231
MRSA was involved in 61% of the abscesses (27/44), and 92% (24/27) of these belonged 232 to the USA300 clone. Three patients presented with MRSA in the wounds and with MSSA in 233 the nares; two cases had MSSA infections but MRSA carriage. In all these cases the MRSA 234 isolates belonged to the clonal group of USA300 while the MSSA strains belonged to very 235 different clones (CC15; CC5; CC1; CC30, respectively). Table 1A and B show the clonal 236 distribution of nasal and wound isolates, and whether they were MRSA or MSSA. 237
Of the 19 patients with MRSA in the nares, 14 were colonized by the same strain that 238 caused the SSTI and all except one of these strains belonged to the USA300 clone or closely 239 related clones. Three patients carried MRSA in the nares but no S. aureus was isolated in the 240 wounds. 241
ACME was only found among isolates belonging to the CC8/USA300 clonal complex 242 and was most prevalent among MRSA strains (29/39 MRSA wound infections and 10/19 MRSA 243 nasal isolates). Four MSSA wound isolates that also carried the ACME virulence factor 244 belonged to the same CC8/USA300 clonal complex. None of the 22 nasal MSSA isolates carried 245 the ACME determinant. 246
The Panton-Valentine Leukocidin (pvl) genes lukSF were detected in half (52.8%) of the 247 wound isolates: in 94% of all MRSA and 75% of MSSA strains. Among all nasal isolates (84% 248
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MRSA and 31% MSSA) 46% carried the lukSF gene. All PVL+ strains belonged to the 249 CC8/USA300 complex except for three patients whose isolates belonged to CC30 and CC88 (all 250 of them also sharing with USA300 the SCCmecIVa cassette). And all strains belonging to the 251 USA300 clonal group carried these genes except two nasal MRSA isolates that belonged to the 252 “New York clone V” (ST8, SCCmecIVg/ACME—/PVL—) (24). 253
Twenty-two of the sixty-three patients (35%) that were identified with an SSTI carrying 254 S. aureus reported previous episodes with similar lesions and described the present lesion as a 255 recurrence. Of those 22 cases, 17 (77%) were identified as infections with MRSA, and all of 256 them by the USA300 clone. One patient had a previous diagnosis of MRSA and had a USA300 257 wound infection in the present study, but did not carry the strain in the nares. 258
259 Antibiotic resistance of MRSA and MSSA isolates 260 One hundred and four S. aureus isolates, representing all nasal isolates and all but five 261
wound cultures were analyzed with antibiograms. None of the isolates were resistant to 262 gentamicin, vancomycin, or linezolid. Forty-one isolates (28 MRSA and 13 MSSA strains) 263 showed multi-resistant profiles, i.e., were resistant to three or more families of antibiotics (Table 264 2). Four MRSA isolates were resistant to four families of antibiotics: ß-lactams (penicillin, 265 amoxicillin/clavulanic, cefazolin and oxacillin), lincosamides (clindamycin), macrolides 266 (erythromycin), and quinolones (levofloxacin), all belonging to USA300 or USA100 (‘New 267 York/Japan’) clones. The remaining twenty-four MRSA isolates that were resistant to three 268 families of antibiotics showed varying patterns of resistance: twenty-one isolates were resistant 269 to ß-lactams, macrolides, and quinolones (USA300 and USA100); one USA300 MRSA nasal 270 isolate was resistant to ß-lactams, lincosamides, and macrolides; one CC88 MRSA wound isolate 271
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was resistant to ß-lactams, macrolides, and tetracyclines, and one CC8 MRSA nasal isolate was 272 resistant to ß-lactams, quinolones, and trimethoprim/sulfamethoxazole. 273
One MSSA nasal isolate belonging to CC15 was resistant to antibiotics of five different 274 families: ß-lactams (penicillin, amoxicillin/clavulanic, and cefazolin, but not oxacillin), 275 lincosamides, macrolides, quinolones, and tetracyclines. Two wound MSSA isolates were 276 resistant to four families of antibiotics: some ß-lactams, lincosamides, macrolides, and 277 quinolones (CC15 and CC8). Nine isolates were resistant to three families of antibiotics: two 278 isolates resistant to ß-lactams, macrolides, and tetracyclines (nasal and wound of one single 279 patient with CC8/PVL+); and seven isolates resistant to ß-lactams, lincosamides, and macrolides 280 (CC5, CC8, CC15, CC30, CC121). 281
As mentioned above, 14 patients infected with MRSA also carried the same strain in the 282 nares and in 13 of these cases the MRSA strains belonged to the USA300 group; in one patient 283 the CA-MRSA recovered from the nares and the wound belonged to the “South-West Pacific 284 clone” (USA1100/t665/CC30/SCCmecIVa/PVL+/ACME—). 285 286
Antibiotic resistance phenotypes of invasive and colonizing isolates 287 Invasive and colonizing isolates from the three patients (UHP-CAMP-016, -CAMP-022 288
and -CAMP-028) that were also analyzed by whole genome sequencing – showed identical 289 oxacillin resistant phenotypes as indicated by the superimposable population analysis profiles 290 (Figure 3). 291
292 . 293
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Comparison of wound and nares isolates in three patients by whole genome sequencing 294 Two of the three patients (UHP/CAMP-016 and UHP/CAMP-022) were infected and 295
colonized by MRSA isolates belonging to the USA300 group (t008 and 296 t052/ST8/SCCmecIVa/PVL+/ACME+). The third patient (UHP/CAMP-028) was infected and 297 colonized by CA-MRSA USA1100. 298
For each of the three pairs of nares and wound isolates CAMP-18 and -19, CAMP-28 and 299 -29, and CAMP-36 and -37, we did whole genome shotgun sequencing to elucidate phylogenetic 300 relationships and identify polymorphisms of all types and sizes. For each of these six isolates, 301 we identified the most closely related MRSA strain available in the NCBI nucleotide database 302 (http://www.ncbi.nlm.nih.gov/) –with a complete (i.e. ungapped) genomic sequence. For 303 CAMP-18, -19, -28, and -29, that strain is USA300 FPR3757, and for CAMP-36 and -37, it is 304 MRSA252. 305
The tree in Figure 4 depicts the phylogenetic relationships and numbers of 306 polymorphisms. It can be seen that for each pair of nares and wound isolates, they differ by only 307 a few point mutations. Isolates CAMP-18 and -19 differed from one another by 5 point 308 mutations; isolates CAMP-28 and -29 differed by 7 point mutations; and CAMP-36 and -37 by 4 309 point mutations. These mutations are described in Table 3 and – in more detail - in Table S1. It 310 is unclear if any of the listed mutations are important for colonization of the nares or infection of 311 the wound. Curiously, one of the mutations in the wound isolate CAMP-19 is in a gene, 312 SAUSA300_1059, with homology to exotoxins. 313
We compared the CAMP isolates to isolates of the same MRSA clone characterized in a 314 recent outbreak by MRSA clone USA300 in Northern Manhattan (31). Uhlemann and 315 colleagues al. shotgun sequenced the whole genomes of about 400 MRSA isolates recovered 316
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during the outbreak (31). The tree in Figure 5 depicts the phylogenetic relationships between the 317 CAMP and the Northern Manhattan isolates. Figure 5 shows that the genotypes of the four 318 CAMP USA300 isolates are within the genetic variation seen in the Northern Manhattan isolates. 319 Therefore, the CAMP and the Northern Manhattan isolates of USA300 may all be part of the 320 same outbreak. 321
322 Discussion 323
S. aureus was identified in 63 of the 129 cases of infections in this study and 30% of 324 wounds were infected by MRSA. Of the six skin lesions shown in Figure 1, MRSA was 325 recovered from only two of the cases. Thus, S. aureus and more specifically MRSA SSTI, cannot 326 be diagnosed by observation alone – additional laboratory analysis is necessary for a correct 327 diagnosis and treatment. 328
Both ACME and PVL seem to play important roles in the prevalence of USA300. Many 329 of the isolates in our study (40/69 CC8 isolates) did not present with the complete characteristic 330 pattern t008/ST8/SCCmecIVa/PVL+/ACME+. Both the cassette chromosome and the mobile 331 element encoding enzymes involved with arginine catabolism are known to be susceptible to 332 genetic modifications, including a total or partial loss from the bacteria (32). Over the years, 333 evolution may have caused changes in the spa gene generating variants that still belong to the 334 same clonal complex (33). Many wound isolates characterized in this study were closely related 335 but not identical to CA-MRSA USA300 (t008/ST8/SCCmecIVa/ PVL+/ACME+). This may 336 indicate that the isolates were derivatives of the original clone, and while lacking some of the 337 virulence determinants, they still shared a genetic background that facilitates rapid clonal spread. 338
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CA-MRSA strains may not be more virulent than many MSSA clones. The clinical 339 importance of CA-MRSA may reside in a combination of factors: a better fitness related to the 340 smaller SCCmec cassettes; higher growth rates; ACME-mediated resistance to host defenses; 341 increased virulence due to the PVL toxin (34); and resistance to multiple antibiotics, which 342 complicates treatment and increases the risk of recurrence (35) 343
We recovered 39 MRSA strains from the wounds: thirty-eight of these were CA-MRSA 344 and one strain was a representative of the healthcare acquired (HA-) MRSA clone CC5/USA100 345 (Table 1). Most of the infections were due to a single MRSA clone, USA300, which is currently 346 the most prevalent clone of CA-MRSA in the USA and specifically in New York City (31). 347 ACME was restricted to strains belonging to USA300 CA-MRSA clone or clones closely related 348 to it. Interestingly, ACME was also found in four MSSA isolates recovered from wounds, 349 grouping all CC8/USA300 strains in what Tenover et al. described as a family of isolates 350 showing >80% similarity by PFGE typing (12). In all patients carrying an ACME+ strain in the 351 nares (all of them MRSA), the same strain was also recovered from the wounds. ACME was 352 absent from all MSSA nasal cultures. 353
The Panton-Valentine leukocidin is a virulence factor frequently associated with CA-354 MRSA (94% of all MRSA wound isolates in our study). However, in our study, the prevalence 355 of the genes lukS-lukF was also very high among MSSA (75%) wound isolates. Most (66%) of 356 these MSSA wound isolates belonged to the same CC8 clonal complex as the USA300 clone 357 which would suggest that these strains were derivatives of the original USA300 clone that have 358 lost the SCCmec cassette while preserving the genetic background that would still provide for 359 superior survival and virulence (36, 37). Most nasal MRSA isolates (84%) and some MSSA 360
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nasal isolates (30%) presented as PVL+, but in these cases the same strain was also detected in 361 the wound culture of the same patient. 362
363 Whole Genome Sequencing and PAPs 364 The identity of wound and carriage (nasal) isolates was documented by standard typing 365
methods. In three patients, pairs of wound and carriage (nasal isolates) were also compared by 366 whole genome sequencing and by detailed characterization of the resistance phenotypes using 367 population analysis profiles (28) (Figure 3). The three patients included: UHP/CAMP-016 and 368 UHP/CAMP-022, each infected and colonized by MRSA strains of the USA300 group (t008 and 369 t052/ST8/SCCmecIVa/PVL+/ACME+); and a third patient, UHP/CAMP-028, which was infected 370 and colonized by CA-MRSA USA1100 (t665/ST1472/SCCmecIVa/PVL+/ACME—). 371
Whole genome sequencing showed that the pairs of isolates recovered from the infection 372 and colonization sites of the same patient were very similar in terms of DNA sequence (see 373 Figure 4). Population analysis profiles (PAPs) showed that the bacterial isolates from the 374 wound and nares of the same patient also exhibited identical – heterogeneous – resistance to 375 oxacillin. The PAPs of bacteria recovered from the two patients infected by USA300 were 376 virtually identical and were different from the PAP of strains recovered from patient 377 UHP/CAMP-028 which was infected by an MRSA strain belonging to the USA1100 clone 378 (Figure 3). 379
In conclusion, the CA-MRSA clone USA300 was identified as the most prevalent S. 380 aureus causing SSTIs in metropolitan New York City community health centers. Methicillin-381 susceptible variants of the same clone that share most of the genetic background of USA300 382 were also prevalent among isolates causing SSTIs. 383
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Clinical presentation alone is not sufficient to predict the nature of the pathogen 384 associated with SSTIs; bacterial cultures and molecular identification is needed to determine the 385 true prevalence and spread of MRSA clones. 386
Nasal carriage cannot be used as a predictor of S. aureus SSTI infection. Many patients 387 that were found to have active infections by MRSA did not carry the pathogen in the nares (22 388 patients presenting with MRSA only in the wound versus 14 patients with MRSA both in the 389 wound and in the nares). Nevertheless, MRSA nasal carriage often accompanied MRSA wound 390 infections by the same strain. In the present study we performed an extensive screening on some 391 patients presenting with recurrent skin and soft tissue infections by MRSA in which carriage was 392 checked both in the nares and at nine additional body sites (pharynx, axillae, cubital folds, 393 inguinal folds, patellar folds). In some patients the nasal carriage was negative (data not shown), 394 and in one USA300 patient the infecting MRSA strain was only found in the inguinal and 395 patellar folds (38). 396
A recent study described an outbreak and extensive spread of MRSA belonging to the 397 clonal type of USA300 in Northern Manhattan (31) during the same time period that our CAMP 398 Study was performed. Comparison of the sequencing data presented in this communication with 399 the sequencing data available from the northern Manhattan outbreak clearly indicates the 400 involvement of strains with an identical clonal type. 401
Given the capacity of the USA300 MRSA clone to adhere and survive on skin and on a 402 variety of inert surfaces, control steps should include screening for carriage by close contacts and 403 by fomites in homes and public transportation (39) that may represent potential reservoirs of 404 these dangerous MRSA clones. 405
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Acknowledgements 407 This work was supported by NIH-NCATS Grant #8 UL1 TR000043 to B. Coller (PI) and 408
by a 2011 CTSA Administrative Supplement Award to J. Tobin (PI). Additional support was 409 provided by a US Public Health Service award 2 RO1 AI457838-15 to A. Tomasz (PI), AHRQ 410 Grant 1 P30-HS-021667 to J. Tobin (PI), and PCORI Grant # CER-1402-10800 to J. Tobin (PI). 411 We thank Dr. Vikas Koundal (Department of Biochemsitry and Molecular Biology, Penn State 412 University) for help in the additional purification and concentration of some DNA samples – 413 prior to genome sequencing. 414 We greatly appreciate the efforts of the participating Community Health Centers, including 415 Brookdale Family Center (Brooklyn NY), Hudson River Healthcare (Peekskill NY), Manhattan 416 Physicians Group (New York NY), Open Door Family Health Center (Ossining NY), Urban 417 Health Plan (Bronx NY); the BioReference Laboratories, Inc. and the collaboration of the 418 clinicians, office staff and patients. 419 420 on M
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35. Otto, M. 2013. Staphylococcal infections: mechanisms of biofilm maturation and 554 detachment as critical determinants of pathogenicity. Annu Rev Med 64:175-88. 555 36. Highlander, S. K., K. G. Hulten, X. Qin, H. Jiang, S. Yerrapragada, E. O. Mason, Jr., 556 Y. Shang, T. M. Williams, R. M. Fortunov, Y. Liu, O. Igboeli, J. Petrosino, M. 557 Tirumalai, A. Uzman, G. E. Fox, A. M. Cardenas, D. M. Muzny, L. Hemphill, Y. 558 Ding, S. Dugan, P. R. Blyth, C. J. Buhay, H. H. Dinh, A. C. Hawes, M. Holder, C. L. 559 Kovar, S. L. Lee, W. Liu, L. V. Nazareth, Q. Wang, J. Zhou, S. L. Kaplan, and G. M. 560 Weinstock. 2007. Subtle genetic changes enhance virulence of methicillin resistant 561 and sensitive Staphylococcus aureus. BMC Microbiol 7:99. 562 37. Miller, L. G., F. Perdreau-Remington, A. S. Bayer, B. Diep, N. Tan, K. Bharadwa, J. 563 Tsui, J. Perlroth, A. Shay, G. Tagudar, U. Ibebuogu, and B. Spellberg. 2007. 564 Clinical and epidemiologic characteristics cannot distinguish community-associated 565 methicillin-resistant Staphylococcus aureus infection from methicillin-susceptible S. 566 aureus infection: a prospective investigation. Clin Infect Dis 44:471-82. 567 38. Balachandra, S., M. Pardos de la Gandara, S. Salvato, T. Urban, C. Parola, C. 568 Khalida, R. G. Kost, T. H. Evering, M. Pastagia, B. M. D'Orazio, A. Tomasz, H. de 569 Lencastre, and J. N. Tobin. 2015. Recurrent furunculosis caused by a community-570 acquired Staphylococcus aureus strain belonging to the USA300 clone. Microb Drug 571 Resist 21:237-43. 572 39. Conceicao, T., F. Diamantino, C. Coelho, H. de Lencastre, and M. Aires-de-Sousa. 573 2013. Contamination of public buses with MRSA in Lisbon, Portugal: a possible 574 transmission route of major MRSA clones within the community. PLoS One 575 8:e77812. 576 577
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Figure Legends 580 Figure 1. Photographs of skin lesions in six patients enrolled in the study. Skin wounds of 581 all patients were photographed with a digital camera to document the morphology/appearance of 582 the Skin and Soft Tissue Infections. Samples taken from the wounds were subsequently used for 583 the identification of the causative agents. 584 585 Figure 2. (A). Molecular types of S. aureus isolates recovered from wounds (B). Molecular 586 types of S. aureus isolates recovered from nares. 587 588 Figure 3. Population Analysis Profiles of the nasal and wound isolates from three selected 589 patients. UHP/CAMP-016 is a patient that carries in both the wound and the nares the same 590 strain USA300 (t008/ST8/SCCmecIVa/ PVL+/ACME+); UHP/CAMP-022 is another patient, 591 which carries in wound and nares a variant of USA300 (t052/ST8/SCCmecIVa/ PVL+/ACME+); 592 UHP/CAMP-028 is a patient that carries a USA1100 strain 593 (t665/ST1472/SCCmecIVa/PVL+/ACME—) in both the wound and the nares. 594 595 Figure 4: Phylogeny of MRSA isolates recovered from wounds and nasal colonization sites 596 of three patients: comparison by whole genome sequencing. CAMP-18 and CAMP-19 are the 597 wound and nasal isolates, respectively, from patient UHP/CAMP-016; CAMP-28 and CAMP-29 598 are the nasal and wound isolates, from patient UHP/CAMP-022. CAMP-36 and CAMP-37 are 599 the nasal and wound isolates, respectively, of patient UHP/CAMP-028. CAMP-18 and CAMP-600 19 were both characterized as USA300 (t008/ST8/SCCmecIVa/ PVL+/ACME+); CAMP-28 and 601 CAMP-29 belonged to a variant of USA300 (t052/ST8/SCCmecIVa/ PVL+/ACME+); CAMP-36 602
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and CAMP-37 belonged to the clone USA1100 (t665/CC30/SCCmecIVa/PVL+/ACME—). 603 Besides each tree branch, listed are the number of point mutations (bold) and larger structural 604 variants (in parentheses) between the chromosomes of the ancestor and descendant. 605 606 Figure 5: The relationship of the CAMP USA300 isolates to isolates of the same clone from 607 the outbreak in northern Manhattan. 608 Uhlemann et al. shotgun sequenced the whole genomes of about 400 MRSA isolates recovered 609 during a USA300 outbreak in northern Manhattan (29). The tree illustrates the phylogenetic 610 relationship between the CAMP and Uhlemann et al. isolates as inferred from whole genomic 611 analysis. Due to the sheer number of isolates, the branches are densely packed. Of the northern 612 Manhattan isolates, the vast majority are USA300 and so comprise the main body of the tree 613 (highlighted in blue), and a very small minority are non-USA300 isolates and represent distant 614 outgroups (highlighted in red). The USA300 isolates CAMP-18, -19, -28, and -29 fall well 615 within the main body of the tree and not on the periphery (see arrows). Thus, the genotypes of 616 the four CAMP USA300 isolates are encompassed by the genetic variation seen in the northern 617 Manhattan USA300 isolates. Thus, the CAMP and northern Manhattan USA300 isolates may all 618 be part of the same outbreak. Not surprisingly, the non-USA300 isolates CAMP-36 and -37 fall 619 among the distant outgroups (see arrows). 620
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TABLE 1. Variation in clonal complex (MLST type) of S. aureus isolates 621 1A. Distribution of isolates grouped by clonal complex 622
MLST type No. isolates No. MRSA wound No. MSSA wound No. MRSA nasal No. MSSA nasal
CC8 69 34 12 17 6
CC30 12 2 4 1 5
CC5 6 1 2 1 2
CC15 6 0 2 0 4
CC121 3 0 1 0 2
ST72 2 1 0 0 1
CC1 1 0 0 0 1
CC45 1 0 1 0 0
CC88 1 1 0 0 0
CC97 1 0 0 0 1
CC152 1 0 1 0 0
CC398 1 0 1 0 0
Total No. Isolates 104 39 24 19 22
623
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1B. Distribution of wound isolates belonging to clonal complex CC8 624 USA300 clonal group in MRSA wounds USA300 clonal group in MSSA wounds
USA300 (t008/ST8/SCCmecIVa/PVL+/ACME+) 21 USA300-like (t008/ST8/PVL+/ACME+) 3
— other spa types 7 — other spa types 1
— PVL (-) 0 — PVL (-) 0
— ACME (-) 2 — ACME (-) 4
Other 4 Other 4
Total No. 34 Total No. 12
625 626 627 628 629 630
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TABLE 2. Antibiotic resistance profiles of S. aureus isolates 631 Antibiotic Families MRSA MSSA Clonal Complexes (CCs) Origin No. Patients with both wound + nasal USA300
BLAs* + CLI + ERY + LEV + TET 0 1 CC15 nasal —
BLAs* + CLI + ERY + LEV 4 2 CC5, CC8, CC15 wound, nasal 1 patient USA300
BLAs* + ERY + TET 1 2 CC8, CC88 wound, nasal 1 patient USA300
BLAs* + ERY + LEV 21 0 CC5, CC8 wound, nasal 2 patients USA300, 1 patient USA300-like
BLAs* + CLI + ERY 1 7 CC5, CC8, CC15, CC30 wound, nasal 1 patient USA300
BLAs* + LEV + TMP-SMX 1 0 CC8 nasal —
BLAs* + ERY 7 5 CC8, CC30, CC121 wound, nasal 1 patient USA300
BLAs* + TET 3 1 CC5, CC8 wound, nasal 1 patient USA300
BLAs* + TMP-SMX 0 1 CC8 nasal —
BLAs stand for ß-lactam antibiotics: penicillin, amoxicillin/clavulanic, cefazolin and oxacillin in the case of MRSA isolates, and 632 penicillin only for MSSA isolates. CLI: clindamycin; ERY: erythromycin; LEV: levofloxacin; TET: tetracycline; TMP-SMX: 633 trimethoprim-sulfamethoxazole. 634 CCs: Clonal complex as defined by MLST. CC5: Clonal complex of the HA-MRSA strain USA100 (‘New York/Japan’ clone); CC8: 635 Clonal complex of the CA-MRSA strain USA300 clone 636
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637 Table 3: Mutations in CAMP isolates* 638 639
Position in the reference Changec Type
Proximal gened Referencea Positionb Referencee N315f Name Function
Mutations in the nares isolate CAMP-18 that are not in the wound isolate CAMP-19
FPR3757 357,216 ct intergenic SAUSA300_0307 SA0295 acid phosphatase? 1,181,875 tc synonymous SAUSA300_1080 SA1029 ftsZ cell division
Mutations in the wound isolate CAMP-19 that are not in the nares isolate CAMP-18
FPR3757 1,159,271 ct nonsynonymous SAUSA300_1059 SA1009 exotoxin? 1,777,369 t‒ frameshift SAUSA300_1622 SA1499 tig molecular chaperone 1,858,349 ag synonymous SAUSA300_1686 SA1561 murC cell wall synthesis
Mutations in the nares isolate CAMP-28 that are not in the wound isolate CAMP-29: none
Mutations in the wound isolate CAMP-29 that are not in the nares isolate CAMP-28
FPR3757
157,471 ca nonsynonymous SAUSA300_0138 SA0131 deoD purine metabolism 1,043,552 -a intergenic SAUSA300_0954 SA0904 antibiotic resistance? 1,387,815 ga nonsynonymous SAUSA300_1260 SA1197 tyrA shikimate pathway 1,768,026 tc synonymous SAUSA300_1614 SA1491 hemL porphyrin biosynthesis 2,031,280 ta intergenic SAUSA300_1871 SA1706 2,335,992 ga intergenic SAUSA300_2158 2,348,652 a‒ intergenic SAUSA300_2167
Mutations in the nares isolate CAMP-36 that are not in the wound isolate CAMP-37
252 142,269 ca intergenic SAR0129 SA0122 butA acetoin formation 1,042,026 t‒ frameshift SAR0994 SA0881 2,105,113 ct nonsynonymous SAR2012 SA1735
Mutations in the wound isolate CAMP-37 that are not in the nares isolate CAMP-36 252 2,707,338 ga nonsynonymous SAR2622 SA2330 transcriptional regulator? aFor CAMP-18, -19, -28, and -29, the reference is MRSA strain USA300 FPR3757. For CAMP-36 and -37, the reference strain is MRSA 252. bNucleotide position on the chromosome of the reference. No mutations were observed on plasmids. cNucleotide change on the Watson strand where sense (Watson or Crick) is defined by the reference. The convention used is [original][new]. dIf the mutation is in a gene, that gene; if the mutation is in intergenic sequence between divergently or tandemly transcribed genes, the nearest downstream gene; and if the mutation is in intergenic sequence between convergently transcribed genes, the nearest gene. eName of the gene in the reference. fThe name of the orthologous gene in strain N315.
640 *For more detailed descriptions, see Table S1 in the supporting materials 641 642
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