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Virulence Genes and Genotypic Associations in Nasal Carriage, Community-

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Virulence Genes and Genotypic Associations in Nasal Carriage, Community-

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 35823592 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00657-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Virulence Genes and Genotypic Associations in Nasal Carriage, Community-

    Associated Methicillin-Susceptible and Methicillin-Resistant USA400

    Staphylococcus aureus Isolates

    12111Sanjay K. Shukla,* Mary E. Karow,Jennifer M. Brady,Mary E. Stemper,Jennifer Kislow, 2213Natalie Moore,Katherine Wroblewski,Po-Huang Chyou,David M. Warshauer, 452Kurt D. Reed,Ruth Lyneld,and William R. Schwan

    12Marsheld Clinic Research Foundation, Marsheld, Wisconsin; University of WisconsinLa Crosse, La Crosse, Wisconsin; 3Wisconsin State Laboratory of Hygiene, Madison, Wisconsin; Feinberg School of Medicine, Northwestern University, 45Chicago, Illinois; and Minnesota Department of Public Health, St. Paul, Minnesota

    Received 29 March 2010/Returned for modication 18 May 2010/Accepted 20 July 2010

It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus

    (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the

    place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of

    differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically dened clinical CA-MRSA (n 77), clinical MSSA (n 103), and nasal carriage MSSA (n 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identied putative toxin genes from the USA400 lineage of CA-MRSA. Genotypi- cally, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene proles of the three groups showed that CA-MRSA

    strains harbored signicantly higher percentages (>95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional

    virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.

     Staphylococcus aureus both is a benign commensal and com- (PFGE), multilocus sequence typing (MLST), and spa typing mon pathogen in humans and is responsible for a variety of have helped in distinguishing the genotypes of CA-MRSA infections, ranging from supercial skin and soft tissue infec- strains from those of other S. aureus strains (2, 14, 26). In tions to bacteremia, endocarditis, and osteomyelitis (33). PFGE, SmaI-restricted S. aureus genomes are compared to Based on its susceptibility to beta-lactams, S. aureus is com- determine their genetic relatedness and also compared against monly described as methicillin-susceptible S. aureus (MSSA) the reference USA genotypes (USA100, USA200, etc., up to or methicillin-resistant S. aureus (MRSA) (15). Infections due USA1200) as described by the Centers for Disease Control and to health care-associated MRSA (HA-MRSA) have been a Prevention (CDC) (34, 53). Of these, CA-MRSA isolates problem since the 1970s; however, starting in the 1990s, new mostly belong to USA300 and USA400 clones and in some strains of MRSA, referred to as community-associated MRSA cases USA1000 and USA1100 clones as well. HA-MRSA iso- (CA-MRSA), appeared in community dwellers and were ge- lates generally belong to USA100, USA200, and USA500 (34, netically different from HA-MRSA strains (7). Until recently, 35). One of the two major clones of CA-MRSA, USA400, individuals who presented with infections due to CA-MRSA recognized in the early 1990s and initially referred to as the typically have had none of the established risk factors associ- MW2 clone, was the predominant CA-MRSA clone that ini- ated with HA-MRSA, such as recent hospitalization, surgery, tially circulated in the midwestern United States in the 1990s dialysis, long-term care residence, or indwelling medical de- (8, 17, 40, 52). The second and more recent CA-MRSA clone, vices. Lately however, CA-MRSA strains have been reported USA300, was rst recognized in 2000 and has since spread from both the community and the health care settings (25, 45). throughout the world (54). More than a thousand MLST allelic Genotyping tools such as pulsed-eld gel electrophoresis proles for S. aureus have been identied so far, of which CA-MRSA strains are primarily represented by sequence type 1 (ST1) (USA400) and ST8 (encompasses USA300 and USA500). Of the several thousand spa types in the Ridom * Corresponding author. Mailing address: Molecular Microbiology Laboratory, Marsheld Clinic Research Foundation, 1000 North Oak database (http://spaserver.ridom.de/), the most predominant Avenue-MLR, Marsheld, WI 54449. Phone: (715) 389-5363. Fax: CA-MRSA spa types are t008 and t128. (715) 389-4950. E-mail: shukla.sanjay@mcrf.mdclin.edu. CA-MRSA strains, besides their distinct PFGE, ST, and spa Supplemental material for this article may be found at http://jcm proles, almost ubiquitously possess the Panton-Valentine leu- .asm.org/. kocidin (PVL or lukSF-PV) genes, in contrast to only 1% to 5% Published ahead of print on 28 July 2010.

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    TABLE 1. Grouping of 293 S. aureus isolates studied and their of S. aureus strains overall having these genes (33, 50, 57). The years of collection PVL toxin has been implicated in many skin and soft tissue infections and lethal necrotizing pneumonia, but the exact role Collection No. of Group period of PVL during S. aureus infections remains controversial (10, isolates Collection site(s) 28, 31, 58). The genome sequence of the CA-MRSA strain Clinical MSSA 19872006 103 WI, MN MW2 of the USA400 lineage showed the presence of several Nasal carriage MSSA 20002005 113 Upper Midwest CA-MRSA 19901999 77 WI additional putative toxin genes (e.g., ear, sec4, sel2, seg2, ssl1

    [set16], and lpl10) compared with other S. aureus strains that

    had been sequenced (1). Some of these toxin genes share homology with classic staphylococcal enterotoxin genes that

    strain. They were mainly recovered from skin and soft tissue infections and in encode pyrogenic exotoxins (49) typically produced during the rare instances from invasive cases such as blood. The CA-MRSA strains were post-exponential phase of growth, and the genes encoding primarily ( 90%) isolated from a variety of skin and soft tissue infections from these exotoxins are most often carried on plasmids, bacterio- outpatient clinics and were genotypically dened. gCA-MRSA strains in this phages, or pathogenicity islands. The classic staphylococcal study were strains whose PFGE was related to the MW2 PFGE type and har- enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, bored staphylococcal chromosome cassette mec (SCCmec) IV. In addition they were recovered from patients visiting outpatient clinics. sem, sen, and seo) are commonly found in strains of S. aureus Genotypic methods. All isolates were typed by PFGE, MLST, and spa typing (12, 23, 39, 42, 43, 46, 62). Pyrogenic exotoxin genes are com- (2, 14, 26). SCCmec types were determined for MRSA isolates by the method of mon in S. aureus, and as many as 73% of S. aureus isolates carry Oliveira and de Lencastre (41). PFGE clonal groups were dened by the Tenover at least one of the genes encoding a classic pyrogenic exotoxin; et al. (53) criteria and 80% genetic similarity using the Dice coefcient (1.25% tolerance) and UWPGMA (unweighted-pair group method using average link- however, the distribution among various clonal types differs ages) method. An MLST-based clonal complex (CC) was dened as a group that (3). Strains of USA400 CA-MRSA typically have been shown shared six of the seven MLST alleles through the use of eBURST (http://eburst to possess the sea, sec, seh, and sek enterotoxin genes, whereas .mlst.net). HA-MRSA strains usually carry the sed, seg, sei, sej, sem, sen, Screening for virulence factor genes. All isolates were screened for 40 known and seo enterotoxin genes (40). Currently, the distribution of and putative S. aureus virulence genes located on bacteriophages and pathoge- nicity and genomic islands on the S. aureus genome. Of the 40 virulence genes, the newly identied putative toxin genes (seg2, sel2, sec4, ssl1, the following have been shown to have clinical relevance: classical staphylococcal and lpl10) (1) from the MW2 strain has not been reported from enterotoxin genes (sea to see), the toxic shock syndrome toxin gene (tst), entero- among CA-MRSA strains in general or from clinical and nasal toxin gene cluster or egc (seg, sei, sem, sen, and seo), the collagen binding protein carriage MSSA strains. Since CA-MRSA isolates are able to gene (cna), bronectin binding protein genes (fnbA and fnbB), clumping factor genes (clfA and clfB), genes for the serine-aspartate repeat family of proteins cause disease in humans without predisposing risk factors and (sdrC, sdrD, and sdrE), the intracellular adhesion protein gene (icaA), hemolysin have spread rapidly in communities, these strains may possess genes (hla, hlb, hld, and hlgB), leukocidin genes (lukSF-PV, lukD, and lukE), and a greater number of toxin genes than do the other strains of S. exfoliative toxin genes (eta and etb). Additionally seven putative virulence genes aureus. (ear, sec4, sel2, bsa, seg2, lpl10, and ssl1) were screened because of their specu- lated role in S. aureus virulence (1). The presence or absence of these genes was The aim of this study was to compare the genotypes of screened by a combination of singleplex or four multiplex PCRs. The list of clinical CA-MRSA USA400, clinical MSSA, and colonizing primers for the virulence genes, their locations on the genome, and expected nasal carriage MSSA isolates and determine the frequency and product sizes are given in Table S1 in the supplemental material. The PCR distribution of the classic enterotoxin genes as well as the new primers were commercially synthesized at Integrated DNA Technology (Cor- putative toxin genes in them. Our results showed that MSSA alville, IA). All the control strains were obtained from the Network on Antimi- crobial Resistance in Staphylococcus aureus (NARSA). The multiplex PCRs strains were much more diverse in their genotypes than were included multiplex 1 (sea to see), multiplex 2 (seg to sej), multiplex 3 (sek to seo), CA-MRSA USA400 strains. In addition, CA-MRSA USA400 and multiplex 4 (seg2, sec4, and ear). Singleplex PCRs were run for the remainder strains possessed a distinct array of toxin genes compared to of the genes. Using the genomic DNA as templates, PCR was performed using MSSA strains. These data may provide insight into the success the HotStarTaq Master Mix kit (Qiagen, Valencia, CA). The sea-to-see multiplex of CA-MRSA USA400 and its ability to cause severe disease in reaction mixtures were set up in a 50- l PCR mixture and consisted of 25 l of the HotStarTaq Master Mix; 40 pmol of each of the forward and reverse primers previously healthy people. for sea and seb; 10 pmol of each of the forward and reverse primers for sec, sed, 2and see; 3 l of 25 mM Mg; 7 l of water; and 4 l of the extracted DNA template. The seg-to-sej multiplex reaction mixture consisted of 25 l of the MATERIALS AND METHODS HotStarTaq Master Mix; 10 pmol of each of the forward and reverse primers for 2S. aureus strains. Two hundred ninety-three S. aureus isolates collected over a seg, seh, sej, and sei; 0.5 l of 25 mM Mg; 16.5 l of water; and 4 l of the extracted DNA template. For sek to seo, the reaction mixture consisted of 25 l 19-year period from 1987 to 2005 from outpatient and surveillance cultures from of the HotStarTaq Master Mix; 10 pmol of each of the forward and reverse Wisconsin and the Minnesota Department of Health were included in the study. 2primers for sek, sel, sem, sen, and seo; 1.0 l of 25 mM Mg; 15 l of water; and The collection periods for the three groups of the isolates differed but did 4.0 l of the template. The rst three multiplex reaction mixtures went through overlap. These isolates were classied into three groups, (i) nasal carriage MSSA an initial activation at 95?C for 15 min, followed by 35 cycles of denaturation at (n 113), (ii) clinical MSSA (n 103), and (iii) clinical CA-MRSA (n 77), 94?C for 2 min, annealing at 57?C for 2 min, and extension at 72?C for 1 min. A based on the collection and phenotypic criteria (Table 1). Sixty-six percent of nal extension was carried out at 72?C for 7 min followed by a nal hold of 4?C. nasal carriage strains were from Wisconsin and were collected from college- A PCR mix for multiplex PCR 4 included the HotStarTaq Master Mix kit, sterile going healthy individuals. Anterior nares of these individuals were screened for double-distilled water, 50 pmol of seg2, 40 pmol each of sec4 and ear primers, 1 S. aureus followed by determination of their resistance to methicillin by MIC g of DNA template to the master mix that contained 2.5 U of HotStar Taq testing using oxacillin. Putative MRSA strains were then conrmed using growth on cation-adjusted Mueller-Hinton II agar plates containing 6 g/ml of oxacillin. DNA polymerase, and 3.0 mM MgCl. The cycling conditions consisted of 35 2cycles of denaturation at 94?C for 1 min, annealing at 55?C for 1 min, and None of the subjects were recently hospitalized or had exposure to health care extension at 72?C for 1 min. environments. Thirty-four percent of the nasal colonization isolates were from At least two separate PCR amplications per gene were performed for each S. the Minnesota Department of Health and were from a collection of MSSA aureus isolate to ensure reproducibility. The PCR products were electrophoresed isolates obtained from outpatients in the upper Midwest. Clinical MSSA strains on 1.5% agarose gels and stained with ethidium bromide to visualize the DNA were strains recovered from patients with conrmed disease due to an MSSA

FIG. 1. PFGE-based dendrogram of S. aureus isolates consisting of at least one unique spa type from each of the three cohorts: clinical

CA-MRSA, clinical MSSA, and nasal carriage MSSA. The dendrogram also includes PFGE-based USA genotypes as reference strains. The dendrogram was created using 1.25% tolerance, the Dice similarity coefcient, and the unweighted-pair group method using arithmetic averages. Clonal relatedness was determined by 80% genetic similarity. PT, pulsotype.

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FIG. 1Continued.

     3586 SHUKLA ET AL. J. CLIN. MICROBIOL.

bands. A 100-bp DNA ladder (New England Biolabs) was used to determine the TABLE 2. Range of percentages of major CCs in three size of each PCR product. All PCRs were done in a PE9700 Thermal Cycler groups of isolates (Perkin-Elmer, Wellesley, MA). A positive control for each respective gene and % of CC in isolate group: a negative control represented by a strain of E. coli were included for each run. CC A no-template control was also run for each PCR run to rule out the possibility Clinical Nasal carriage CA-MRSA of contamination of the reagents. A positive PCR was determined by the pres- MSSA MSSA ence of an appropriately sized DNA band in the gel. CC1 3.5 5.8 100 Identity of the amplicons. At least two randomly chosen amplicons for each of aCC5 14.1 14.5 NF the 40 virulence genes tested were sequenced to ensure that primers amplied NF CC8 2.7 1.9 the expected genes. Gene identities were conrmed by comparison with the CC12 0.9 NF NF DNA sequence database at GenBank using the BLAST search. CC15 15.0 4.9 NF Statistical analyses. All data analyses were carried out using the Statistical CC20 NF 4.9 NF Analysis System (SAS, Cary, NC) and StatXact statistical software (Cytel Soft- CC22 0.9 NF NF ware Corp., Cambridge, MA). Fishers exact test was used to derive the P value. CC25 9.7 1.9 NF Any P value less than 0.05 indicated statistical signicance. Simpsons diversity CC30 29.2 39.8 NF index was determined as described previously (20). NF CC45 5.3 8.7 CC50 0.9 NF NF RESULTS NF CC59 9.7 2.9 CC97 2.7 1.9 NF Range of genotypic diversity in nasal carriage, clinical NF CC121 NF 7.8 MSSA, and clinical CA-MRSA isolates. The nasal isolates (n CC395 NF 2.9 NF CC398 0.9 NF NF 113) were represented by 41 MLST allelic proles or 15 clonal CC509 0.9 NF NF complexes (6 major and 9 minor). A major CC was dened as CC1021 2.7 NF NF a CC that was represented by at least 4% of the isolates in that Singletons 0.9 1 NF group. The six major CCs were CC30 (29.2%), CC15 (15%), a NF, not found. CC5 (14.1%), CC25 (9.7%), CC59 (9.7%), and CC45 (5.3%).

    The remaining CCs were CC1, CC8, CC97, CC1021, CC12,

    CC22, CC50, CC398, and CC509, and their frequency ranged

    from 1% to 3.5%. These isolates were also represented by 68 in this group, with ve major ones being t012 (13.6%), t159 unique PFGE types or pulsotypes that could be grouped into (5.8%), t018 (4.9%), t164 (3.8%), and t209 (3.8%). The per- 18 PFGE-based clonal groups (CGs) using the criteria of cent ranges of the remaining spa types were from 0.9% to

    Tenover et al. (53). The distribution of major CGs in order of 2.9%.

    their percentages was as follows: CG-B CC30 or USA200 The CA-MRSA isolates were the least diverse and were (23.9%), CG-C CC15 (15.9%), CG-D CC5 (12.4%), represented by nine PFGE types. However, they all belonged CG-F CC25 (9.7%), CG-G CC59 (8.8%), CG-A CC15 to CC1 (CG-A or USA400) but were still represented by six or USA400 (5.3%), CG-E CC45 or USA600 (5.3%), and spa types: t128 (80.5%), t559 (6.5%), t125 (5.2%), t558 (5.2%), CG-L CC30 (4.4%). The frequencies of the remaining nine t175 (2.6%), and t555 (2.6%). Using the representative strains minor CGs were below 3%, and they were as follows: CG-H from each of the three groups, their comparative genotypic CC8 and CC30, CG-K CC9, CG-P CC9, CG-Q CC1, characteristics and diversity are summarized in Fig. 1. Overall, CG-R CC1021, CG-S CC8, CG-WW CC398, CG- 18 CCs were identied in these three groups of isolates, of YY CC22, CG-Z CC50, and CC-ZZ CC509. By our which CC20, CC121, and CC395 were not present in the nasal analysis, the non-USA types were represented by 65.5% of the carriage group whereas CC12, CC22, CC50, C398, CC509, and isolates. Fifty-three spa types were identied, of which the CC1021 were absent in the clinical MSSA group (Table 2). seven major ones were t084 (10.6%), t012 (9.7%), t078 (8%), Nasal carriage and clinical MSSA strains were genetically t216 (8%), t021 (4.4%), t548 (4%), and t2917 (3.5%). more diverse by all three typing methods, unlike CA-MRSA Clinical MSSA isolates (n 103) were represented by 38 isolates, which showed very little diversity by PFGE and MLST allelic proles, which were grouped into 12 CCs. Seven MLST. Interestingly, there was no signicant difference in the

    major CCs were CC30 (39.8%), CC5 (14.6%), CC45 (8.7%), percentages of unique PFGE types, clonal groups, or MLST CC121 (7.8%), CC1 (5.8%), CC15 (4.9%), and CC20 (4.9%). allelic proles between the clinical MSSA strains and nasal The remaining CCs were CC59, CC395, CC8, CC25, and carriage isolates. However, clinical MSSA strains were repre- CC97, and their frequency ranged from 1% to 2.9%. This sented by signicantly higher percentages of spa types than

    group was also represented by 59 pulsotypes which were were the nasal carriage isolates. Overall, S. aureus isolates that

    grouped into 16 CGs. Three clones matched with the USA were susceptible to methicillin were more diverse than were types: CG-B CC30 or USA200 (38.8%), CG-A CC-1 or the MRSA isolates by PFGE. In terms of distinguishing abil- USA400 (7.8%), and CG-E CC45 or USA600 (5.8%). The ities of the three typing methods, spa typing and PFGE were

    non-USA types were represented by 47.6% of the isolates. comparable but more discriminatory than MLST for clinical Their breakdowns were as follows: CG-J CC121 (6.8%), MSSA strains (Table 3). However, once PFGE types were CG-D CC5 (5.8%), CG-K CC9 (5.8%), CG-C CC15 grouped into clonal groups according to the Tenover et al. (53) (4.8%), and CG-M CC20 (4.8%); the frequencies of the criteria, spa typing was found to be more discriminatory. Clin- remaining 9 CGs (CG-F CC25, CG-G CC59, CG-H ical MSSA strains were found to have a higher percentage of CC8 and CC30, CG-L CC30, CG-P CC9, CG Q CC1, spa types than were nasal carriage isolates. In addition to the CG-U CC395, CG-V CC395 and CC1148, and CG-Y simple descriptive analysis of diversity of genotypes described CC121) were below 3%. Seventy-two spa types were identied above, the diversity indices (DI) of nasal carriage and clinical

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    TABLE 3. Number of pulsotypes, PFGE-based clonal groups, STs, TABLE 4. Simpsons index of diversity of nasal carriage MSSA, and spa types in study isolates collected during 1987 to 2005 clinical MSSA, and CA-MRSA isolates by PFGE, MLST, and spa typing No. (% diversity) of: Group (n) Index of diversity for isolate group (n): Pulsotypes Clonal groups STs spa types Genotypic method Nasal carriage Clinical MSSA Nasal carriage 71 (63) 18 (16) 41 (36) 52 (46) MSSA (113) (103) MSSA (113) spa typing 0.963 0.973 59 (57) 17 (17) 38 (37) 64 (62) Clinical MSSA MLST 0.931 0.897 (103) PFGE 0.988 0.946 CA-MRSA (77) 9 (12) 1 (1) 1 (1) 6 (7.8)

     data in Table 5 and found that the results for genes sej, hlb, MSSA isolates were 0.988 and 0.946 by PFGE, 0.963 and 0.973 can, fnbA, and sdrE were no longer statistically signicant in by spa typing, and 0.931 and 0.897 by MLST, respectively each paired comparison among the three groups. (Table 4). Nasal carriage isolates were genotypically more di- We also identied six nasal carriage and eight clinical MSSA verse by MLST and PFGE than were clinical MSSA isolates. isolates whose genotypes were similar to that of the USA400 Differential distribution of known virulence genes in CA- clone. While the six nasal carriage MSSA strains were isolated MRSA USA400 compared to clinical and nasal carriage MSSA in the year 2000 or later, two of the clinical MSSA strains isolates. Representative agarose gels of the four multiplex (WSLH-6 and MNDH-77) in this group were recovered in PCRs are shown in Fig. S1A to D in the supplemental material. 1987 and 1998, respectively, a period that either predated most Overall, the rate of positivity of the 40 virulence genes showed of the CA-MRSA outbreaks or overlapped with the early out- a wide range, from 0% to 100% across the three groups. Some break. The virulence proles of these 14 isolates were com- genes were preferentially present in one group versus the two pared against those of the three representative CA-MRSA other groups. Twelve of the 40 genes (ssl1, hla, hlb, hld, hlgB, isolates of the USA400 lineage (Table 6). Notably, both clfB, cna, fnbA, icaA, sdrD, sdrE, and bsa) were more fre- WSLH-6 and MNDH-77 isolates harbored lukSF-PV, lpl10, quently ( 85%) present across all three groups than were sed, ssl1, sea, sek, seh, sec4, ear, and sel2, some of the genes more see, sej, etb, and fnbB, which were present in the range of 0% frequently present in USA400 CA-MRSA strains (1, 40). Un- to 24%. In addition, the rate of positivity of genes seg, sei, sek, like the WSLH-6 isolate, MNDH-77 also harbored two of the sel, sen, seo, and lukSF-PV varied widely and ranged from egc genes (seg and sei) besides eta, etb, and tst. 10% to 80% in the three groups. Several enterotoxin genes (sea, sec, sec4, seg2, sek, sel, and

    sel2), leukocidin genes (lukD, lukE, and lukSF-PV), adhesion DISCUSSION genes (sdrD and clfA), and putative virulence genes (lpl10 and This study presents the comparative analysis of genotypes ear) were signicantly associated with CA-MRSA compared to

    nasal carriage or clinical MSSA strains (P value of 0.05). and virulence proles of 293 S. aureus isolates representing

    Enterotoxin genes sed and see, including the enterotoxin gene three major phenotypic groups of S. aureusnasal carriage

    MSSA, clinical MSSA, and clinical CA-MRSA strainscol- cluster (egc), seg, sei, sem, sen, and seo; exotoxin genes (tst, eta,

    and etb); and the bronectin binding protein gene (fnbB) were lected over a 19-year period. Specically, we determined the

    present in less than 1% of the CA-MRSA isolates. The toxic breadth of genotypic diversity for each group and assessed shock syndrome toxin gene (tst) and exfoliative toxin genes (eta whether or not the virulence prole of CA-MRSA strains was

    and etb) were not present in the CA-MRSA group. Entero- signicantly different from that of the MSSA groups. Geno- toxin genes sed and sej were present at a low frequency, prob- typing using PFGE, MLST, and spa typing showed that the

    ably because the plasmid pIB485, which harbors these genes, clinical and nasal carriage MSSA strains were highly diverse may not be common in these groups of isolates. Genes sea and compared to the CA-MRSA group, which was found to be sek, which reside on Sa3mw, were almost always present in highly clonal. All CA-MRSA isolates belonged to the USA400 CA-MRSA isolates but infrequently present in MSSA isolates. lineage only. This result was not entirely surprising as USA400 Genes such as staphylococcal superantigen protein-like genes clones have primarily been reported from the midwestern (ssl1), hemolysin genes (hla, hlb, hld, and hlgB), surface protein United States during the collection period of 1990 to 1999 and genes (clfB, cna, fnbB, icaA, and sdrE), and the bacteriocin continued to be reported from the midwestern United States gene (bsa) were present in nearly all three groups of the iso- and Alaska (8, 17, 40, 50, 52; S. K. Shukla et al., unpublished lates. At the same time fnbB, although at a lower rate (14% to data). Unfortunately, we did not have USA300 strains in the 24%), was present exclusively in MSSA strains. study sample due to the time period (1990 to 1999) in which In general, the MSSA clinical and nasal carriage strains have the gCA-MRSA isolates were collected. It should be noted overall virulence proles similar to those of the CA-MRSA that USA300, which was rst identied in 2000, has now be-

    group, except for sel2, tst, hlb, cna, lpl10, and sdrC, which were come the predominant CA-MRSA strain in most geographic statistically different (P value, 0.05) when one was used as a areas in the United States, including the Midwest. Equally reference group (Table 5). All of these genes except lpl10 had important was that we did not identify any clinical MSSA a signicantly higher rate of positivity in nasal carriage isolates strains belonging to the USA300 clone in our collection. than in the clinical MSSA strains. To address the issue of Our study demonstrated that the virulence prole of the

    multiple comparisons, we applied the Bonferroni correction to CA-MRSA USA400 strains was signicantly different from

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TABLE 5. Summary of the ranges of virulence gene positivity for each gene and percentages and numbers of gene positivity in each of the

    three groups along with the P values within each cohort

    % of positivity (no. of positives); P value cGene % of positivity range P value abClinical MSSA CA-MRSA Nasal carriage Enterotoxin genes 58 (66); 0.0001 54 (56); 0.0001 95 (73) 5495 sea seb 330 26 (29); 0.0001 30 (31); 0.0001 3 (2) sec 2595 27 (30); 0.0001 25 (26); 0.0001 95 (73) sec4 2597 29 (33); 0.0001 25 (26); 0.0001 97 (75) sed 013 8 (9); 0.0116 13 (13); 0.0007 0 (0) see 03 0 (0); 1.000 3 (3); 0.2615 0 (0) seg 090 90 (102); 0.0001 89 (92); 0.0001 0 (0) seg2 2796 27 (31); 0.0001 31 (32); 0.0001 96 (74) seh 15100 15 (17); 0.0001 17 (17); 0.0001 100 (77) sei 191 89 (101); 0.0001 91 (94); 0.0001 1 (1) sej 19 9 (10); 0.0300 9 (9); 0.0452 1 (1) sek 896 12 (14); 0.0001 8 (8); 0.0001 96 (74) sel 996 9 (10); 0.0001 9 (9); 0.0001 96 (74) sel2 2597 0.0294 40 (45); 0.0001 25 (26); 0.0001 97 (75) sem 047 42 (47); 0.0001 44 (45); 0.0001 0 (0) sen 081 75 (85); 0.0001 81 (83); 0.0001 0 (0) seo 082 76 (86); 0.0001 82 (84); 0.0001 0 (0)

     Exotoxin genes 86100 91 (103); 0.0061 86 (89); 0.0003 100 (77) ssl1 tst 078 0.0021 78 (88); 0.0001 58 (60); 0.0001 0 (0) eta 056 54 (61); 0.0001 56 (58); 0.0001 0 (0) etb 022 22 (25); 0.0001 18 (19); 0.0001 0 (0)

     Leukocidin genes 0100 0 (0); 0.0001 3 (3); 0.0001 100 (77) lukSF-PV lukD 44100 0.0010 66 (75); 0.0001 44 (45); 0.0001 100 (77) lukE 62100 0.0279 77 (87); 0.0001 62 (64); 0.0001 100 (77)

     Hemolysin genes 100 100 (113); 1.0 100 (103); 1.0 100 (77) hla hlb 91100 0.0279 98 (111); 0.5154 91 (94); 0.0109 100 (77) 100 100 (113); 1.0 100 (103); 1.0 100 (77) hld hlgB 100 100 (113); 1.0 100 (103); 1.0 100 (77)

     Surface protein genes 7099 76 (86); 0.0001 70 (72); 0.0001 99 (76) clfA clfB 97100 97 (110); 0.2731 99 (102); 1.0 100 (77) cna 95100 0.0234 100 (113); 1.0 95 (98); 0.0721 100 (77) fnbA 92100 92 (104); 0.0116 95 (98); 0.0721 100 (77) fnbB 024 24 (27); 0.0001 14 (14); 0.0003 0 (0) icaA 99100 100 (113); 0.4053 100 (103); 0.4278 99 (76) lpl10 55100 0.0171 55 (62); 0.0001 71 (73); 0.0001 100 (77) sdrC 1761 0.0008 82 (93); 0.0001 61 (63); 0.0001 17 (13) sdrD 85100 88 (100); 0.0010 85 (88); 0.0002 100 (77) sdrE 90100 96 (109); 0.1481 90 (93); 0.0054 100 (77)

     Other genes 100 100 (113); 1.0 100 (103); 1.0 100 (77) bsa 1897 18 (20); 0.0001 ear 21 (22); 0.0001 97 (75) avalues were determined using CA-MRSA (reference) versus clinical MSSA. Pbvalues were determined using CA-MRSA (reference) versus nasal carriage isolates. Pcvalues were determined using clinical MSSA (reference) versus nasal carriage isolates. , data not shown. P

    those of the nasal carriage and clinical MSSA isolates based on hld, and hlgB; and surface protein genes clfB, cna, and icaA

    (Table 5) had a higher rate of positivity in CA-MRSA USA400 the presence of a number of pyrogenic superantigens and sur-

    face proteins in addition to the putative toxin genes identied isolates than in either clinical or nasal carriage MSSA isolates.

    The presence of additional virulence genes, particularly those in MW2. Fourteen of the 40 genes were signicantly associated

    that seem to provide redundant functions, could help to ex- with the CA-MRSA group (Table 5). These genes included

    plain why strains of CA-MRSA USA400 are found to be more multiple enterotoxin genes, leukocidin genes, adhesion genes

    sdrC and clfA, and a few putative virulence genes. Specically, virulent than S. aureus strains in general. While expression of all genes except the enterotoxin gene see; hemolysin genes hla, virulence genes is likely to be regulated by several factors,

     VOL. 48, 2010 GENOTYPIC AND VIRULENCE GENE ASSOCIATIONS IN S. AUREUS 3589

     CA-MRSA Clinical Nasal Isolate a including host factors, one can speculate that additional genes 19941993 1992 20041997 2004 2003 2002 2004 1987 1998 20002001 2005 2003 2004 2003 and TABLE within the same functional class (e.g., toxins, adhesion factors, , MSSA PCR MSSA group yr etc.) could provide a synergistic and/or complementary effect. negative; Several studies have described the evolution and virulence 16988-R-34R -R MCMNDH-118MCWSLH--S127- WSLH31MC--SS- 35 WSLH--112-32MNDHS --SS- 6-S-MNDH77 MNDH-S UWL-UWL97-UWL-125-92S UWL--33-SS- -83S- 57-S -S 6. factor genes of HA-MRSA strains (15, 18); however, little has Comparison Sample been done to examine these genes in CA-MRSA strains. Of the toxin-related virulence factor genes that have been exam- , PCR ined in CA-MRSA strains, much has been done to characterize seh lukSF-PV, encoding the PVL. lukSF-PV has been shown to be positive. of conserved among most CA-MRSA isolates tested, but these ssl1 virulence same genes have been shown to be present in a low number of lpl10 other S. aureus isolates (13, 30, 31, 47, 50, 57). Since this gene sdrC was present in only 1 to 5% of S. aureus strains in general but proles almost always in CA-MRSA USA400 strains isolated in the sdrD 1990s from the United States, it had become a molecular sdrE marker for USA400 isolates isolated during that time period of (40, 50, 57). However, some recent studies from the United nasal ear States and other parts of the world showed that the presence of sec carriage PVL may not be the most denitive marker for recent CA- sec4 MRSA strains, including USA400 (32, 48, 55, 61). The results sel from our study found that 100% of CA-MRSA USA400 iso- MSSA sel2 lates and less than 1% of MSSA isolates possessed lukSF-PV,

     supporting previous studies (50, 57). The PVL has been shown clfa to cause severe tissue damage and has been implicated in lung and hla damage associated with strains causing severe necrotizing clinical bsa pneumonia (11, 19, 28, 31, 60). However, both a sepsis and an lukSF-PV abscess model of infection with isogenic strains demonstrated MSSA that PVL-negative strains were as virulent as the PVL-positive

    parental strains (58). Thus, the role of PVL as a virulence lukD factor is still under investigation. Besides PVL, -hemolysin, USA400 -type phenol-soluble modulins (PSMs), and the arginine cat- lukE abolic mobile element (ACME) have been studied for their sea roles in the pathogenesis of CA-MRSA (9, 11). Lethal pneu- pulsed-eld seg2 monia may be tied to levels of -hemolysin expressed in

    USA300 and USA400 strains (5). The PSMs expressed by sek USA300 and USA400 strains are thought to attack human hlb pulsotypes neutrophils (59). Montgomery et al. (37), using pneumonia hld and the skin infection model in rabbits, showed that ACME is hlgB not associated with enhanced virulence. In contrast, Diep et al. (11) showed that deletion of ACME affects the pathogenicity fnbB with and tness of USA300. ACME, which was identied in the fnbA USA300 lineage, was not detected in 10 random samples of three

     CA-MRSA strains from this study (data not shown). clfB representative Other toxins produced by S. aureus can also cause damage to icaA host tissues. One class includes the staphylococcal pyrogenic exotoxins that can cause direct as well as indirect damage cna through overstimulation of the immune system (49). These seb exotoxins function as superantigens that keep the immune sed strains system from recognizing and attacking the invading S. aureus. see A wide variety of toxins produced by S. aureus are considered seg o to be pyrogenic exotoxins, including staphylococcal enterotox- f sei USA400 ins and toxic shock syndrome toxin (4). sej Fourteen classical enterotoxin genes (sea, seb, sec, sed, see, sem seg, seh, sei, sej, sek, sel, sem, sen, and seo) have been previously CA-MRSA sen identied in S. aureus and were screened for in this study (12, 23, 42, 43, 46, 62). The sea, seb, sec, seh, and sek genes have seo been reported to be found in the majority of CA-MRSA eta USA400 isolates from the United States collected prior to 2003 a etb (17, 40), in contrast to USA400 isolates recovered during 2005- tst 2006 (32). However, our results demonstrated that the CA-

     3590 SHUKLA ET AL. J. CLIN. MICROBIOL.

    MRSA isolates harbored a signicantly higher percentage not that virulence in S. aureus is not likely to be associated with one

    toxin or surface protein but a cumulative or synergistic effect of only of the sea, sec, seh, and sek genes as expected but also of

    several virulence factors to initiate, maintain, and prolong an the sec4, seg2, sel, sel2, lukSF-PV, lukD, lukE, clfA, and sdrD

    infection. It is also very likely that the genomic background of genes than did either the nasal carriage or clinical MSSA

    groups. Interestingly, the clinical and nasal carriage MSSA a strain contributes to the overall virulence of an S. aureus

    strain (29). It was interesting that the nasal carriage and clin- groups possessed a signicantly greater frequency of the seb,

    ical MSSA isolates belonging to the USA400 genotype har- sed, seg, sei, sej, sem, sen, seo, tst, eta, etb, fnbB, and sdrC genes

    than did the CA-MRSA group. The higher frequency of egc in bored one or more of the genes egc, eta, and etb, unlike the

    the MSSA cohorts and not in CA-MRSA strains suggests that CA-MRSA isolates (Table 6). Only one of the USA400 MSSA they are acquired as part of egc as shown previously (24). A strains harbored fnbB, typically found in USA300 strains. It is

    possible that a lack of these genes in the USA400 CA-MRSA study by van Belkum et al. (56) determined that the egc genes

    were present in 63.7% of carriage isolates and 52.9% of inva- strains, along with the gain of the SCCmec element, could have

    sive isolates. It is generally believed that the presence of this aided in the success of the CA-MRSA USA400 clone at the

    time in the midwestern United States. It is worth mentioning gene cluster is not associated with severe infections but prob-

    ably helps in the carriage potential of an S. aureus strain (16, that now USA300 is the predominant CA-MRSA clone in most 22, 56). However, a case report of a severe case of necrotizing parts of the United States (54). Interestingly, introduction of fasciitis in a leg of a diabetic patient that resulted in the USA300 in a correctional facility resulted in a clonal shift in amputation of the leg was caused by an MSSA strain that the CA-MRSA clone, i.e., from USA400 to USA300 in that lacked most of the common virulence genes except the egc facility (51). This displacement of ST1:USA400 by the ST8: genes, suggesting that the products of egc may play a role, in USA300 clone in different parts of the United States may be some cases, in severe infections, especially in compromised due to ACME in its genome, which allows enhanced survival patients (38). However, the proteins encoded by the egc genes on skin (9).

    seem to be produced in smaller amounts than are the other Although the roles that enterotoxins play in skin and soft well-studied enterotoxins, inducing a lower immunologic re- tissue infections are not known, they have been shown to in- sponse among human hosts (22) that may allow strains pos- uence the development of secondary staphylococcal infec- sessing the egc element to live a commensal lifestyle in healthy tions due to their proinammatory effects that trigger in- humans. In our study, we did not see any signicant difference creased inammation (36). Although the other toxin genes in the prevalence of egc between the carriage and clinical have been described in S. aureus, their presence has not been

    MSSA groups. The three other enterotoxin genes (seb, sed, and tied to virulence. An exciting new nding that may offer some

    sej) were found in a much smaller percentage of the MSSA signicant clues to the pathogenicity of the USA400 type of isolates but were almost always absent in CA-MRSA isolates. CA-MRSA was the presence of several new putative toxin The seb gene encodes an enterotoxin that is known to be a genes (for example, seg2, sel2, sec4, ear, lpl10, and ssl1) in the

    potent superantigen (4, 49) that can overstimulate the hosts CA-MRSA isolates that were present in lower percentages in immune system and be detrimental to a commensal lifestyle for both cohorts of MSSA strains. Each of the genes was found on the MSSA isolates. The function of sed and sej is not well pathogenicity islands on the genome of MW2 (1). Pathogenic- established. ity islands are distinct genetic elements that encode virulence The success of the USA400 lineage of CA-MRSA in the factors found on bacterial genomes (21). The seg2 gene was

    midwestern United States and its enhanced virulence could be found on the Sa3 pathogenicity island, while the lpl10 and

    due to a number of reasons. First of all, this group of isolates ssl1 genes were found on vSa , and the sel2, sec4, and ear genes

    harbors more enterotoxin and surface protein genes than do were found on a separate pathogenicity island labeled vSa3.

    the clinical MSSA strains or the nasal carriage strains in the The functions of these genes have not yet been determined, study. It has been shown that sea and sec are more frequently and yet some sequence homology to the staphylococcal ente- present in CA-MRSA than in HA-MRSA strains (17, 40), and rotoxins may help to elucidate the functions of the proteins septic shock appears to be associated with the presence of sea encoded by the new genes (1). Furthermore, a proteomic study (16). Enterotoxins A and J along with virulence genes fnbA, has shown antibodies to the Sel2 and Ear proteins in patients can, sdrE, sea, sej, eta, hlg, and ica tend to be more common in infected with CA-MRSA, suggesting a role for both proteins in invasive isolates (44). In a report by Kravitz et al., three of the CA-MRSA pathogenicity (6).

    ve cases of purpura fulminans due to either MSSA or MRSA Acquisition of one or more of the new pathogenicity islands strains produced staphylococcal enterotoxin C (SEC) and PVL may play a role in CA-MRSA infections. Until now, there have (27). SEA and SEC tend to produce higher immunological been no studies that have looked at the distribution of these responses leading to host tissue damage (4, 49) than do other pathogenicity islands in CA-MRSA or any other S. aureus

    enterotoxins (16). In this context, the presence of the sea and isolates. Our results show that the newly discovered toxin genes sec genes along with other genes such as seh, sek, sel, sel2, were found in most CA-MRSA isolates from the USA400 lukSF-PV, lukD, lukE, clfA, and sdrD suggests that several lineage. The MSSA cohorts displayed much smaller detection virulence genes may be working in unison to facilitate or pro- percentages for the seg2, sel2, sec4, and ear genes, while per-

    long an infection. It is also possible that the presence of mul- centages for ssl1 and lpl10 were relatively higher in these tiple toxin and adhesion proteins could afford a redundancy of groups but signicantly less than those in the CA-MRSA co- virulence arsenal to the pathogen to mount a more potent hort. These data suggest that the vSa pathogenicity island

    infection. Contradictory results of the virulence studies in an- (ssl1 and lpl10) seems to be widely dispersed throughout S.

    imal models for PVL and ACME (10, 11, 19, 31, 37, 58) suggest aureus isolates; therefore, it is unlikely that the presence of this

VOL. 48, 2010 GENOTYPIC AND VIRULENCE GENE ASSOCIATIONS IN S. AUREUS 3591

sabaugh, F. R. DeLeo, and H. F. Chambers. 2008. The arginine catabolic island alone is responsible for the pathogenicity of CA-MRSA mobile element and staphylococcal chromosomal cassette mec linkage: con- USA400 isolates. It seems more likely that the other islands are vergence of virulence and resistance in the USA300 clone of methicillin- acting in unison to affect the pathogenicity of CA-MRSA iso- resistant Staphylococcus aureus. J. Infect. Dis. 197:15231530. 12. Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of lates compared to the MSSA populations. Staphylococcus aureus. Clin. Microbiol. Rev. 13:1634. The presence of these genes among the CA-MRSA isolates 13. Dufour, P., Y. Gillet, M. Bres, G. Lina, F. Vandenesch, D. Floret, J. Etienne, and their absence among MSSA isolates suggest that the vir- and H. Richet. 2002. Community-acquired methicillin-resistant Staphylococ- cus aureus infections in France: emergence of a single clone that produces ulence of the CA-MRSA USA400 pulsed-eld type may be Panton-Valentine leukocidin. Clin. Infect. Dis. 35:819824. highly dependent on this group of new putative toxin genes and 14. Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. that the virulence of this CA-MRSA type could be tied to the Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. synergistic activities of the proteins encoded by the variety of 38:10081015. these genes. Peacock et al. (44) postulated that the presence of 15. Enright, M. C., D. A. Robinson, G. Randle, E. J. Feil, H. Grundmann, and B. G. Spratt. 2002. The evolutionary history of methicillin-resistant Staphy- certain virulence genes among S. aureus isolates is dependent lococcus aureus (MRSA). Proc. Natl. Acad. Sci. U. S. A. 99:76877692. upon the rates of gene acquisition and the cost to biological 16. Ferry, T., D. Thomas, A. L. Genestier, M. Bes, G. Lina, F. Vandenesch, and tness of the organism itself. J. Etienne. 2005. Comparative prevalence of superantigen genes in Staphy- lococcus aureus isolates causing sepsis with and without septic shock. Clin. In summary, our study showed that isolates belonging to the Infect. Dis. 41:771777. CA-MRSA USA400 lineage were genetically less diverse than 17. Fey, P. D., B. Said-Salim, M. E. Rupp, S. H. Hinrichs, D. J. Boxrud, C. C. the clinical and carriage MSSA isolates and harbored several Davis, B. N. Kreiswirth, and P. M. Schlievert. 2003. Comparative molecular analysis of community-or hospital-acquired methicillin-resistant Staphylococ- additional virulence genes which may have helped them in cus aureus. Antimicrob. Agents Chemother. 47:196203. efcient host acquisition and subsequent infection. 18. Fitzgerald, J. R., D. E. Sturdevant, S. M. Mackie, S. R. Gill, and J. M. Musser. 2001. Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epi- ACKNOWLEDGMENTS demic. Proc. Natl. Acad. Sci. U. S. A. 98:88218826. 19. Gillet, Y., B. Issartel, P. Vanhems, J. C. Fournet, G. Lina, F. Vandenesch, Y. We thank the Network on Antibiotic Resistance in Staphylococcus Piedmont, N. Brousse, D. Floret, and J. Etienne. 2002. Association between aureus and Jean Lee for supplying strains used as controls in this study. Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin We also acknowledge Kathy Como-Sabetti, Jessica Nerby, and Dave and highly lethal necrotizing pneumonia in young immunocompetent pa- Boxrud at the Minnesota Department of Health for their assistance tients. Lancet 359:753758. with the MSSA isolates. 20. Grundmann, H., S. Hori, M. C. Enright, C. Webster, A. Tami, E. J. Feil, and This work was supported by NIH grant AI061385 to S.K.S., the T. Pitt. 2002. Determining the genetic structure of natural population of Staphylococcus aureus: a comparison of multilocus sequence typing with Marsheld Clinic Research Foundation, and a Graduate Student Re- pulsed-eld gel electrophoresis, randomly amplied polymorphic DNA anal- search, Service, and Education Leadership Grant to M.E.K. ysis, and phage typing. J. Clin. Microbiol. 40:45444546. 21. Hacker, J., and J. B. Kaper. 2000. Pathogenicity islands and the evolution of REFERENCES microbes. Annu. Rev. Microbiol. 54:641679. 1. Baba, T., F. Takeuchi, M. Kuroda, H. Yuzawa, K. Aoki, A. Oguchi, Y. Nagai, 22. Holtfreter, S., K. Bauer, D. Thomas, C. Feig, V. Lorenz, K. Roschack, E. N. Iwama, K. Asano, T. Naimi, H. Kuroda, L. Cui, K. Yamamoto, and K. Friebe, K. Selleng, S. Lovenich, T. Greve, A. Greinacher, B. Panzig, S. Engelmann, G. Lina, and B. M. Broker. 2004. egc-encoded superantigens Hiramatsu. 2002. 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