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1.  A correlative analysis of epidemiologic and molecular characteristics of methicillin-resistant Staphylococcus aureus clones from diverse geographic locations with virulence measured by a Caenorhabditis elegans host model 
Methicillin-resistant Staphylococcus aureus (MRSA) strains from different geographic areas have different genetic backgrounds, suggesting independent clonal evolutions. To better understand the virulence of MRSA strains and the relationship to their clonal and geographic origins, we undertook an analysis of epidemiologic, molecular, and virulence characteristics of a large number of MRSA isolates from geographically diverse origins, in a Caenorhabditis elegans infection model. A total of 99 MRSA isolates collected between 1993 and 2010 at the Geneva University Hospitals from diverse global origins were characterized with Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (TSST), accessory gene regulator (agr) group, staphylococcal cassette chromosome mec (SCCmec), S. aureus protein A (spa), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) typing. Epidemiologic data were provided from clinical records. The bacterial virulence was tested in a C. elegans host model. The inter-relationships of epidemiological/molecular characteristics in association with nematocidal activities were analyzed with univariate and two-factor analysis of variance (ANOVA). Community-associated MRSA (CA-MRSA) strains were more virulent than hospital-associated MRSA (HA-MRSA), with higher nematocidal activities in CA-MRSA strains (0.776 vs. 0.506, p = 0.0005). All molecular characteristics (PVL, TSST, spa, SCCmec, MLST, and PFGE types) showed a significant association with nematocidal activities on univariate analysis (p < 0.005). PVL was not a significant predictor after adjusting for genomic backgrounds using spa, MLST, or PFGE typing. The dominant CA-MRSA strains in North America showed higher nematocidal activities than strains from other regions (p < 0.0001). Strains with global origins containing distinct genetic backgrounds have different virulence in the C. elegans model. Nematocidal activities were most highly correlated with SCCmec, spa, MLST, and PFGE typing, suggesting that genomic background rather than a single exotoxin characteristic was the most discriminating predictor of virulence.
doi:10.1007/s10096-012-1711-x
PMCID: PMC3545200  PMID: 22898726
2.  Extensive Dissemination of Methicillin-Resistant Staphylococcus aureus (MRSA) between the Hospital and the Community in a Country with a High Prevalence of Nosocomial MRSA 
PLoS ONE  2013;8(4):e59960.
According to the EARS-Net surveillance data, Portugal has the highest prevalence of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) in Europe, but the information on MRSA in the community is very scarce and the links between the hospital and community are not known. In this study we aimed to understand the events associated to the recent sharp increase in MRSA frequency in Portugal and to evaluate how this has shaped MRSA epidemiology in the community. With this purpose, 180 nosocomial MRSA isolates recovered from infection in two time periods and 14 MRSA isolates recovered from 89 samples of skin and soft tissue infections (SSTI) were analyzed by pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec) typing, spa typing and multilocus sequence typing (MLST). All isolates were also screened for the presence of Panton Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) by PCR. The results showed that ST22-IVh, accounting for 72% of the nosocomial isolates, was the major clone circulating in the hospital in 2010, having replaced two previous dominant clones in 1993, the Iberian (ST247-I) and Portuguese (ST239-III variant) clones. Moreover in 2010, three clones belonging to CC5 (ST105-II, ST125-IVc and ST5-IVc) accounted for 20% of the isolates and may represent the beginning of new waves of MRSA in this hospital. Interestingly, more than half of the MRSA isolates (8/14) causing SSTI in people attending healthcare centers in Portugal belonged to the most predominant clones found in the hospital, namely ST22-IVh (n = 4), ST5-IVc (n = 2) and ST105-II (n = 1). Other clones found included ST5-V (n = 6) and ST8-VI (n = 1). None of the MRSA isolates carried PVL and only five isolates (ST5-V-t179) carried ACME type II. The emergence and spread of EMRSA-15 may be associated to the observed increase in MRSA frequency in the hospital and the consequent spillover of MRSA into the community.
doi:10.1371/journal.pone.0059960
PMCID: PMC3617237  PMID: 23593155
3.  Use of a Single-Nucleotide Polymorphism Genotyping System To Demonstrate the Unique Epidemiology of Methicillin-Resistant Staphylococcus aureus in Remote Aboriginal Communities 
Journal of Clinical Microbiology  2006;44(10):3720-3727.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.
doi:10.1128/JCM.00836-06
PMCID: PMC1594797  PMID: 17021102
4.  Molecular Characterization of Methicillin-Resistant Staphylococcus aureus with Emergence of Epidemic Clones of Sequence Type (ST) 22 and ST 772 in Mumbai, India▿  
Journal of Clinical Microbiology  2010;48(5):1806-1811.
A total of 412 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated between October 2006 and June 2009, representing a mixed hospital- and community-associated patient population from Mumbai, India, were evaluated. MRSA was characterized by multiplex PCR amplification of the Panton-Valentine leukocidin (PVL) gene and the mecA gene, staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST). PCR results were compared with patient risk factors (CDC guidelines) and antimicrobial susceptibility profiles. A total of 395 MRSA strains were mecA positive, and 224 were PVL gene positive. A total of 97 mecA-positive strains were SCCmec III (25%), 136 were SCCmec IV (34%), and 162 were SCCmec V (41%). All SCCmec III strains were multidrug resistant, and all patients had risk factors. Of the SCCmec IV and V strains, 73% were multidrug susceptible and 72% of the associated patients had no risk factors. The multidrug susceptibility and absence of patient risk factors in 72% of cases with SCCmec IV and SCCmec V MRSA demonstrate the presence of community-associated MRSA (CA-MRSA) in Mumbai. Twenty-one percent of these patients had risk factors, signifying CA-MRSA infiltration into hospitals. MLST showed clonal expansion of multidrug-susceptible sequence type (ST) 22 (SCCmec IV) and ST 772 (SCCmec V), both of which feature in Asian studies and may be slowly replacing the multidrug-resistant ST 239 (SCCmec III) in hospitals. The PVL gene-positive methicillin-sensitive S. aureus (MSSA) strains were ST 30 and were postulated to be related to the penicillin-resistant S. aureus phage type 80/81, notorious for its virulence in the 1950s.
doi:10.1128/JCM.01867-09
PMCID: PMC2863868  PMID: 20351212
5.  Population Structure of Staphylococcus aureus Strains Isolated from Intensive Care Unit Patients in The Netherlands over an 11-Year Period (1996 to 2006) ▿  
Journal of Clinical Microbiology  2009;47(12):4090-4095.
The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.
doi:10.1128/JCM.00820-09
PMCID: PMC2786662  PMID: 19812275
6.  Presence and Molecular Epidemiology of Virulence Factors in Methicillin-Resistant Staphylococcus aureus Strains Colonizing and Infecting Soldiers▿  
Journal of Clinical Microbiology  2009;47(4):940-945.
Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as an important cause of skin and soft-tissue infections (SSTI). The understanding of the molecular epidemiology and virulence of MRSA continues to expand. From January 2005 to December 2005, we screened soldiers for MRSA nasal colonization, administered a demographic questionnaire, and monitored them prospectively for SSTI. All MRSA isolates underwent molecular analysis, which included pulsed-filed gel electrophoresis (PFGE) and PCR for Panton-Valentine leukocidin (PVL), the arginine catabolic mobile element (ACME), and the staphylococcal cassette chromosome mec (SCCmec). Of the 3,447 soldiers screened, 134 (3.9%) had MRSA colonization. Of the 3,066 (89%) who completed the study, 39 developed culture-confirmed MRSA abscesses. Clone USA300 represented 53% of colonizing isolates but was responsible for 97% of the abscesses (P < 0.001). Unlike colonizing isolates, isolates positive for USA300, PVL, ACME, and type IV SCCmec were significantly associated with MRSA abscess isolates. As determined by multivariate analysis, risk factors for MRSA colonization were a history of SSTI and a history of hospitalization. Although various MRSA strains may colonize soldiers, USA300 is the most virulent when evaluated prospectively, and PVL, ACME, and type IV SCCmec are associated with these abscesses.
doi:10.1128/JCM.02352-08
PMCID: PMC2668321  PMID: 19213694
7.  A Panton-Valentine Leucocidin (PVL)-Positive Community-Acquired Methicillin-Resistant Staphylococcus aureus (MRSA) Strain, Another Such Strain Carrying a Multiple-Drug Resistance Plasmid, and Other More-Typical PVL-Negative MRSA Strains Found in Japan 
Journal of Clinical Microbiology  2005;43(7):3356-3363.
Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL+ CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing, spa typing, agr typing, coagulase typing, staphylococcal cassette chromosome mec (SCCmec) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL+ CA-MRSA strains belonged to the globally extant (“pandemic”) sequence type 30 (ST30) with SCCmec IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL− CA-MRSA strains (“domestic” CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL− CA-MRSA strain.
doi:10.1128/JCM.43.7.3356-3363.2005
PMCID: PMC1169136  PMID: 16000460
8.  Panton–Valentine Leukocidin-Positive Methicillin-Resistant Staphylococcus aureus Infections in Children With Cancer 
Pediatric blood & cancer  2009;53(7):1216-1220.
Background
New strains of methicillin-resistant Staphylococcus aureus (MRSA) which frequently carry the Panton–Valentine leukocidin (PVL) genes have been recognized to cause invasive infections in otherwise healthy children and adults. However, the epidemiology of PVL-positive MRSA infections has not been described in children or adults with cancer.
Procedure
The epidemiology of MRSA infections in patients with cancer was retrospectively studied from 2000 to 2007. Molecular typing was performed by polymerase chain reaction (PCR) for the detection of the PVL genes. Staphylococcus cassette chromosome (SCC) mec and spa typing was performed on all PVL-positive isolates.
Results
A total of 88 MRSA isolates from clinically distinct infectious episodes were collected from 88 patients with cancer during the 8-year study period. Infections were predominant in the skin and soft tissues (SSTI; P =0.0003). PVL-positive isolates, bearing the type IV SCCmec element, encoding the gene for methicillin resistance, increased significantly during this period (P =0.043) and comprised 35 of 88 (40%) MRSA isolates. Of these 35 isolates, 32 belonged to spa type 8 and were USA300 genotype. Patients infected with PVL-positive strains did not have more SSTI (P =0.166) or bacteremia (P =0.510) as compared to patients with PVL-negative strains. A greater percentage of PVL-positive isolates were susceptible to ciprofloxacin (P =0.006).
Conclusions
PVL-positive MRSA infections are not associated with a higher morbidity as compared to PVL-negative MRSA infections in children with cancer.
doi:10.1002/pbc.22254
PMCID: PMC3075015  PMID: 19731325
cancer; children; methicillin-resistant Staphylococcus aureus; Panton-Valentine leukocidin
9.  Rapid Increase of Genetically Diverse Methicillin-Resistant Staphylococcus aureus, Copenhagen, Denmark 
Emerging Infectious Diseases  2007;13(10):1533-1540.
Community-onset MRSA with diverse genetic backgrounds is rapidly emerging in this previously low MRSA prevalence area.
In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for <15 isolates per year during 1980–2002. However, since 2003 an epidemic increase has been observed, with 33 MRSA cases in 2003 and 110 in 2004. We analyzed these 143 cases epidemiologically and characterized isolates by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had no identified risk factors. We identified 29 spa types (t) and 16 sequence types (STs) belonging to 8 clonal complexes and 3 ST singletons. The most common clonal types were t024/ST8-IV, t019/ST30-IV, t044/ST80-IV, and t008/ST8-IV (USA300). A total of 86% of isolates harbored SCCmec IV, and 44% had PVL. Skin and soft tissue infections dominated. CO-MRSA with diverse genetic backgrounds is rapidly emerging in a low MRSA prevalence area.
doi:10.3201/eid1310.070503
PMCID: PMC2851516  PMID: 18258003
MRSA; Panton-Valentine leukocidin; MLST; spa typing; Staphylococcus aureus; Community-onset MRSA; SCCmec; USA300; PFGE; Nursing home; research
10.  Short Communication: Methicillin-Resistant Staphylococcus aureus Infections in Children and Young Adults Infected with HIV 
AIDS Research and Human Retroviruses  2009;25(12):1219-1224.
Abstract
The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) infections, in particular with Panton-Valentine leukocidin (PVL)-positive strains, has not been well characterized in children and young adults with HIV infection. It is not known if PVL-positive strains of MRSA cause an increased morbidity in this population compared to PVL-negative strains. The purpose of this study was to retrospectively analyze the epidemiology of PVL-positive and PVL-negative MRSA infections in children and young adults with HIV from 2000 to 2007. Molecular typing was performed by polymerase chain reaction (PCR) for detection of the PVL genes. Staphylococcus Cassette Chromosome (SCC) mec and spa typing were performed on all PVL-positive isolates. The number of HIV patients with MRSA infection increased significantly between 2000 and 2007 (p = 0.0015). Twenty seven (87%) of the 31 MRSA isolates were from skin and soft tissue infections (SSTI). Clindamycin resistance was observed in 19% of the MRSA isolates. PVL-positive isolates bearing the type IV SCC mec element comprised 16 of 31 (52%) MRSA isolates. All the PVL-positive isolates belonged to the USA300 pulsed-field type. There was no difference in the mean CD4 count and HIV viral load between patients with PVL-positive and PVL-negative MRSA infections. PVL-positive MRSA infections were associated with more SSTI (p = 0.043) but not with increased morbidity or a higher risk of complications compared to PVL-negative MRSA infections in children and young adults with HIV.
doi:10.1089/aid.2009.0040
PMCID: PMC2858900  PMID: 20001313
11.  Predominance of Community-Associated Methicillin-Resistant Staphylococcus aureus Strains Carrying Staphylococcal Chromosome Cassette mec Type IVA in South Korea▿  
Journal of Clinical Microbiology  2007;45(12):4021-4026.
Studies on the molecular epidemiologic characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains have demonstrated their genetic and geographical diversity. In addition, it has been reported that there are genetic differences between community-associated (CA) and health care-associated (HA) MRSA strains. Therefore, we investigated the major epidemiologic characteristics of CA MRSA isolates in South Korea and compared them with those of HA MRSA strains. Distributions of staphylococcal chromosome cassette mec (SCCmec) types and other molecular features, including the Panton-Valentine leukocidin (PVL) gene, were studied in 138 invasive MRSA isolates. Multiplex type IVA SCCmec was identified as the major CA MRSA infection type (53.1%), with a significantly higher prevalence than in HA MRSA (P < 0.001). One major group of type IVA strains carried a larger atypical class B mec element and new subtypes of ccrA2 (96% amino acid homology). The PVL gene was detected in one USA300-like isolate only. Seven major clone types determined by combinational grouping (genetic background SCCmec typing) showed representative patterns of antimicrobial susceptibilities. We concluded that less multi-drug-resistant strains of clone types B-I and D-1 (genetic background, B and D complexes; type IVA SCCmec) predominate in CA MRSA and that international PVL-positive strains have not spread in South Korea as yet.
doi:10.1128/JCM.01147-07
PMCID: PMC2168574  PMID: 17942660
12.  Antimicrobial Susceptibility Patterns and Staphylococcal Cassette Chromosome mec Types of, as Well as Panton-Valentine Leukocidin Occurrence among, Methicillin-Resistant Staphylococcus aureus Isolates from Children and Adults in Middle Tennessee▿  
Journal of Clinical Microbiology  2006;44(12):4436-4440.
Antimicrobial susceptibility patterns, Panton-Valentine leukocidin (PVL) occurrence, and staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from children and adults at Vanderbilt University Medical Center during a 12-month period were evaluated. A total of 1,315 MRSA isolates were collected, of which 748 (36.7%) were recovered from children. Among all isolates, 448 (34.1%) were SCCmec-II, and 847 (64.4%) were SCCmec-IV. More SCCmec-IV isolates were recovered from children than SCCmec-II isolates (424 [50.1%] versus 50 [11.2%]; odds ration [OR] = 7.98; P < 0.000001). The PVL gene was detected in 93.6% of SCCmec-IV isolates, in contrast to 0.2% in SCCmec-II isolates. Within SCCmec-IV isolates, a statistically higher PVL occurrence was noticed in children (98.1%) than in adults (89.1%) (OR = 6.34; P < 0.000001). Overall, SCCmec-II strains showed greater resistance than SCCmec-IV strains to clindamycin, erythromycin, levofloxacin, gentamicin, rifampin, minocycline, and trimethoprim-sulfamethoxazole. Both SCCmec-II and SCCmec-IV strains recovered from adults were more resistant to these antibiotics than those recovered from children. SCCmec-II strains were predominantly recovered from the respiratory tract, whereas SCCmec-IV strains were predominantly recovered from skin, soft tissue, abscesses, and surgical wounds. These data indicate that SCCmec-IV MRSA isolates frequently infect children in middle Tennessee and are likely to harbor the PVL gene.
doi:10.1128/JCM.01546-06
PMCID: PMC1698407  PMID: 17065272
13.  Methicillin-Resistant Staphylococcus aureus Carriage among Students at a Historically Black University: A Case Study 
Background. Black people in the USA is afflicted with a higher rate of methicillin-resistant Staphylococcus aureus (MRSA) infection. This study determined the prevalence of MRSA carriage among black college students at a university setting. Methods. Hand and nasal swabs were collected and screened for MRSA by mannitol fermentation, coagulase, and DNase activities and their resistance to oxacillin. MRSA isolates were analyzed for antimicrobial resistance pattern, genetic profile for staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field type, multilocus sequence type (ST), and the presence of Panton-Valentine leukocidin (PVL) gene. Results. MRSA was isolated from 1 of the 312 (0.3%) hand swabs and 2 of the 310 (0.65%) nasal swabs, respectively. All isolates lack multidrug resistance and have type IV SCCmec, characteristic of community-associated MRSA. These isolates were a ST8-MRSA-IVa-PVL(+) (USA300 strain), a ST8-MRSA-IVb-PVL(−), and a new MLST, ST2562-MRSA-IV-PVL(−), identified in this study. These isolates were thus not transmitted among students. Conclusion. We found a low rate of MRSA carriage among students in a black university. Our finding highlights the need of future study which involves multiinstitutions and other ethnic group to assess the association of black race with MRSA carriage.
doi:10.1155/2013/979734
PMCID: PMC3563209  PMID: 23401691
14.  Multilocus Sequence Typing of Methicillin-Resistant Staphylococcus aureus from an Area of Low Endemicity by Real-Time PCR 
Journal of Clinical Microbiology  2005;43(9):4448-4454.
A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.
doi:10.1128/JCM.43.9.4448-4454.2005
PMCID: PMC1234089  PMID: 16145090
15.  Recombination between ccrC Genes in a Type V (5C2&5) Staphylococcal Cassette Chromosome mec (SCCmec) of Staphylococcus aureus ST398 Leads to Conversion from Methicillin Resistance to Methicillin Susceptibility In Vivo▿ †  
Various types of the staphylococcal cassette chromosome mec (SCCmec) are known to confer methicillin resistance on the human pathogen Staphylococcus aureus. Such cassettes are not always stably maintained. The present studies were aimed at identifying the mechanism underlying the in vivo conversion of methicillin-resistant S. aureus (MRSA) to methicillin-susceptible S. aureus (MSSA) derivatives as encountered in two patients suffering from pneumonia and an umbilicus infection, respectively. All MRSA and MSSA isolates identified belong to multilocus sequence type (MLST) 398, have spa type t034, and are Panton-Valentine leukocidin positive. Sequencing of 27,616 nucleotides from the chromosomal SCCmec insertion site in orfX to the hsdR gene for a restriction enzyme revealed a type V (5C2&5) SCCmec. Sequence comparisons show that parts of the cassette are highly similar to sequences within SCCmec elements from coagulase-negative staphylococci, indicating a possible common origin. The cassette investigated contains ccrC-carrying units on either side of its class C2b mec gene complex. In vivo loss of the mec gene complex was caused by recombination between the recombinase genes ccrC1 allele 8 and ccrC1 allele 10. In vitro, the SCCmec was very stable, and low-frequency MRSA-to-MSSA conversion was only observed when MRSA isolates were cultivated at 41°C for prolonged periods of time. In this case also, loss of the mec complex was due to ccrC gene recombination. Interestingly, the MRSA and MSSA isolates studied displayed no detectable differences in competitive growth and virulence, suggesting that the presence of the intact type V (5C2&5) SCCmec has no negative bearing on staphylococcal fitness under the conditions used.
doi:10.1128/AAC.00696-09
PMCID: PMC2812175  PMID: 19995931
16.  Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI 
In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton–Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States.
doi:10.1007/s10096-010-0892-4
PMCID: PMC2854357  PMID: 20229224
17.  Carriage of Community-Associated Methicillin-Resistant Staphylococcus aureus in a Cohort of Infants in Southern Israel: Risk Factors and Molecular Features▿  
Journal of Clinical Microbiology  2009;48(2):531-538.
There are few data about the epidemiology of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among children in Israel. This study was intended to identify risk factors for CA-MRSA colonization in healthy infants, to characterize the molecular features of colonizing organisms, and to determine whether they are responsible for health care-associated (HA) infections. Nasal cultures and demographic details were collected from a cohort of healthy infants at 5 visits between the ages of 2 and 12 months. Clinical characteristics of pediatric MRSA bloodstream infections (2001 to 2006) and wound cultures collected over 6 months were also studied. Clonal structure was evaluated by multilocus sequence typing. Isolates were studied for the staphylococcal cassette chromosome mec (SCCmec) type and for the presence of Panton-Valentine leukocidin (PVL) genes. MRSA was cultured at least once from 45 of 659 infants (346 Jewish and 313 Bedouin infants). Forty of 45 (89%) isolates were from Bedouin infants. Twenty-nine of 45 (64.4%) belonged to a new clonal complex, designated CC913, that carries SCCmec IV but not the PVL genes. CC913 was also isolated from 9/14 blood cultures and 7/8 wounds. All CC913 infections occurred in Bedouin children, and all but two were HA. In conclusion, Bedouin origin was the main risk factor for carriage of CA-MRSA. CC913 was dominant both in healthy carriers and as a cause of pediatric HA-MRSA bloodstream infections.
doi:10.1128/JCM.02290-08
PMCID: PMC2815591  PMID: 20007386
18.  Nasal Carriage of Staphylococcus aureus in Botucatu, Brazil: A Population-Based Survey 
PLoS ONE  2014;9(3):e92537.
Recent increases in the incidence and severity of staphylococcal infections renewed interest in studies that assess the burden of asymptomatic carriage of Staphylococcus aureus in the community setting. We conducted a population-based survey in the city of Botucatu, Brazil (122,000 inhabitants), in order to identify the prevalence of nasal carriage of Staphylococcus aureus (including methicillin-resistant strains). Nasal swabs were obtained from 686 persons over one year of age. Resistance to methicillin was assessed through phenotypic methods, identification of the mecA gene and typing of the Staphylococcal Chromosome Cassette mec (SCCmec). Methicillin-resistant S. aureus (MRSA) isolates were characterized using Pulsed-Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and spa typing. Polymerase chain reaction was applied to identify genes coding for Panton-Valentine Leukocidin (PVL) in isolates. The prevalence of overall S. aureus carriage was 32.7% (95%CI, 29.2%–36.2%). Carriers were significantly younger (mean age, 28.1 versus 36.3 for non-carriers; OR for age, 0.98; 95%CI, 0.97–0.99) and likely to report recent skin infection (OR, 1.85; 95%CI, 1.03–3.34). Carriage of methicillin-resistant S. aureus (MRSA) was found in 0.9% of study subjects (95%CI, 0.4%–1.8%). All MRSA isolates harbored SCCmec type IV, and belonged to spa types t002 or t021, but none among them harbored genes coding for PLV. In MLST, most isolates belonged to clones ST5 or ST1776. However, we found one subject who carried a novel clone, ST2594. Two out of six MRSA carriers had household contacts colonized with isolates similar to theirs. Our study pointed to dissemination of community-associated MRSA among the Brazilian population.
doi:10.1371/journal.pone.0092537
PMCID: PMC3963891  PMID: 24663818
19.  The dissemination of ST80-SCCmec-IV community-associated methicillin resistant Staphylococcus aureus clone in Kuwait hospitals 
Background
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a global healthcare problem. The purpose of this study was to characterize CA-MRSA clones and their distribution in Kuwait hospitals.
Methods
In total, 135 CA-MRSA isolates, carrying the SCCmec IV or V genetic elements, isolated in eight hospitals were characterized using antibiogram, pulsed-field gel electrophoresis, multilocus sequence typing, and carriage of genes for Panton-Valentine Leukocidin (PVL), capsular polysaccharides types (cap) 5 and 8, accessory genes regulators (agr), Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (tst).
Results
They were susceptible to vancomycin, teicoplanin and linezolid but resistant to kanamycin (62%), fusidic acid (42.2%), tetracycline (39.3%), erythromycin and clindamycin (21.5%), gentamicin (5.9%), streptomycin (6.7%), trimethoprim (5.9%), mupirocin (6.6%) and cadmium acetate (82.2%). They consisted of 10 pulsotypes with the majority belonging to PFGE type I (51.1%), type II (22.2%), type IV (13.3%) and type III (3.7%). They belonged to 10 sequence types (ST) comprising ST80 (51.1%), ST30 (22.2%), ST5 (14.1%), ST1 (4.45), ST6 (3.7%), ST88 (1.5%), ST834 (1.5%), ST8 (0.7%), ST46 (0.7%) and ST950 (0.7%). Genes for PVL, cap 8, cap 5 and agr III, agr I and agr II were detected in 61.5%, 77.3%, 20.7% and 62.2%, 17% and 8.1% of the isolates respectively. Nine (6.7%) isolates contained tst while 103 isolates were positive for SE genes with sei (63.0%), seg (41.5%) and sed (29.6%) as the common SE genes.
Conclusions
ST80-SCCmecIV was the most common CA-MRSA clone in Kuwait hospitals presenting new challenges for infection control.
doi:10.1186/1476-0711-9-31
PMCID: PMC2989929  PMID: 21050425
20.  Characterization of Methicillin-Resistant Staphylococcus aureus Strains Recovered from a Phase IV Clinical Trial for Linezolid versus Vancomycin for Treatment of Nosocomial Pneumonia 
Journal of Clinical Microbiology  2012;50(11):3694-3702.
A total of 434 methicillin-resistant Staphylococcus aureus (MRSA) baseline isolates were collected from subjects enrolled in a prospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia. Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, and screened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCCmec, pulsed-field gel electrophoresis (PFGE), and spa typing. Selected strains were evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the spa and/or MLST results. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCCmec II/IV; 70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent clones were CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominated in Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively). The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S. MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7% (56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediate to vancomycin (MIC, 4 μg/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin. hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, each region had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300 strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.
doi:10.1128/JCM.02024-12
PMCID: PMC3486224  PMID: 22972817
21.  Detection of Staphylococcal Cassette Chromosome mec-Associated DNA Segments in Multiresistant Methicillin-Susceptible Staphylococcus aureus (MSSA) and Identification of Staphylococcus epidermidis ccrAB4 in both Methicillin-Resistant S. aureus and MSSA▿  
Antimicrobial Agents and Chemotherapy  2008;52(12):4407-4419.
Methicillin-susceptible Staphylococcus aureus (MSSA) can arise from methicillin-resistant S. aureus (MRSA) following partial or complete excision of staphylococcal cassette chromosome mec (SCCmec). This study investigated whether multiresistant MSSA isolates from Irish hospitals, where MRSA has been endemic for decades, harbor SCCmec DNA. Twenty-five multiresistant MSSA isolates recovered between 2002 and 2006 were tested for SCCmec DNA by PCR and were genotyped by multilocus sequence typing and spa typing. All isolates lacked mecA. Three isolates (12%) harbored SCCmec DNA; two of these (genotype ST8/t190) harbored a 26-kb SCCmec IID (II.3.1.2) remnant that lacked part of mecI and all of mecR1, mecA, and IS431; the third isolate (ST8/t3209) harbored the SCCmec region from dcs to orfX. All three isolates were detected as MRSA using the BD GeneOhm and Cepheid's Xpert MRSA real-time PCR assays. Six isolates (ST8/t190, n = 4; ST5/t088, n = 2), including both isolates with the SCCmec IID remnant, harbored ccrAB4 with 100% identity to ccrAB4 from the Staphylococcus epidermidis composite island SCC-CI. This ccrAB4 gene was also identified in 23 MRSA isolates representative of ST8/t190-MRSA with variant SCCmec II subtypes IIA to IIE, which predominated previously in Irish hospitals. ccrAB4 was located 5,549 bp upstream of the left SCCmec junction in both the MRSA and MSSA isolates with SCCmec elements and remnants and 5,549 bp upstream of orfX in the four MSSA isolates with ccrAB4 only on an SCC-CI homologous region. This is the first description of a large SCCmec remnant with ccr and partial mec genes in MSSA and of the S. epidermidis SCC-CI and ccrAB4 genes in S. aureus.
doi:10.1128/AAC.00447-08
PMCID: PMC2592854  PMID: 18852274
22.  Staphylococcal Cassette Chromosome mec and Panton-Valentine Leukocidin Characterization of Methicillin-Resistant Staphylococcus aureus Clones▿  
Journal of Clinical Microbiology  2006;45(3):1019-1021.
Staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant Staphylococcus aureus MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCCmec type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCCmec type II complex and did not harbor the PVL genes.
doi:10.1128/JCM.01706-06
PMCID: PMC1829128  PMID: 17192420
23.  Molecular Epidemiology of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolated from the Eye 
Current Eye Research  2010;36(2):94-102.
Purpose:
Methicillin-resistant Staphylococcus aureus (MRSA) strains are commonly classified as hospital-acquired (HA) or community-acquired (CA). Typical HA-MRSA isolates are characterized by multidrug resistance and the SCCmec type II cassette, while CA-MRSA isolates are generally susceptible to more drug classes, are often of SCCmec type IV, and frequently carry the Panton-Valentine leukocidin (PVL) genes. This study determined the presence of traits characteristic for CA and HA strains in ocular MRSA isolates.
Materials and Methods:
Fifty-six recent ocular isolates, consisting of 40 MRSA and 16 methicillin-susceptible Staphylococcus aureus (MSSA) comparator strains, were characterized. Minimum inhibitory concentration (MIC) testing was done according to current Clinical and Laboratory Standards Institute guidelines. Detection of the PVL encoding genes and determination of the SCCmec type was done by polymerase chain reaction (PCR), while spa typing and cluster analysis was performed following DNA sequencing.
Results:
Of the 38 typeable MRSA isolates, 22 were of SCCmec type II and 16 were of SCCmec type IV. All SCCmec type II isolates were multidrug-resistant, lacked the PVL genes, and were of spa type t002 or closely related spa types. In contrast, the SCCmec type IV isolates were resistant to fewer classes of antimicrobial agents, often possessed the PVL genes (75.0%), and were of spa type t008 or closely related spa types.
Conclusions:
While the majority of ocular MRSA strains in this study fit the classical profile of HA-and CA-MRSA, some CA-MRSA isolates exhibited higher levels of antimicrobial resistance, which should be of particular concern to eye-care professionals. Furthermore, the apparent association of spa types and SCCmec types observed here warrants further investigation and suggests that spa typing may be useful in future HA- and CA-MRSA characterization studies.
doi:10.3109/02713683.2010.534229
PMCID: PMC3021952  PMID: 21158584
Bacterial infection; Community-acquired MRSA; Multi-drug resistance; spa typing; Staphylococcal virulence factors
24.  Differences in Clinical and Molecular Characteristics of Skin and Soft Tissue Methicillin-Resistant Staphylococcus aureus Isolates between Two Hospitals in Northern California▿  
Journal of Clinical Microbiology  2007;45(6):1798-1803.
Community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) skin and soft tissue infections (SSTI) are associated with SCCmec IV and Panton-Valentine leukocidin (PVL) genes. CO-MRSA epidemiologic studies suggest that genotypic variation exists within one geographic region. We compared MRSA genotypes and demographic and clinical characteristics of patients with CO-MRSA SSTI between two regional medical centers. We also examined factors associated with SCCmec IV and PVL carriage. A total of 279 MRSA SSTI isolates from 2000 to 2002 at San Francisco General Hospital (SFGH) and Stanford University Hospital (SUH) were genotyped by pulsed-field gel electrophoresis and PCR for SCCmec and PVL genes. Medical records were reviewed for clinical characteristics. Ninety-three percent and 69% of MRSA SSTI were caused by CO-MRSA at SFGH and SUH, respectively. Patients with CO-MRSA SSTI at SFGH were more likely to be nonwhite, younger, homeless, and have no previous exposure to health care (P < 0.01). SFGH CO-MRSA strains were more likely to carry SCCmec type IV and PVL genes (90% and 55%, respectively) than SUH strains (29% and 16%, respectively). In multivariate analyses, nonwhite ethnicity was associated with both SCCmec type IV and PVL carriage (odds ratio [OR] of 2.65 and 95% confidence interval [CI] of 1.19 to 5.95 and OR of 1.94 and 95% CI of 1.03 to 3.65, respectively). ST8:USA300:IV became the dominant clone at SFGH, but not at SUH, by 2002. Despite geographic proximity, CO-MRSA SSTI exhibited differing SCCmec types, PVL carriage, and clonal dynamics. CO-MRSA SSTI at SUH were more likely to represent feral isolates of nosocomial origin. Clinicians should assess for nosocomial and community risk factors, realizing that different populations with CO-MRSA SSTI may require separate antimicrobial strategies.
doi:10.1128/JCM.01747-06
PMCID: PMC1933023  PMID: 17409211
25.  Genetically restricted, methicillin-susceptible strains contribute to the ongoing epidemic of community-acquired Staphylococcus aureus infections 
Background
Within the current worldwide epidemic of community-acquired Staphylococcus aureus infections, attention has focused on the role of methicillin-resistant strains. We characterized methicillin-susceptible strains that also contribute.
Methods
We tracked cultures from abscesses submitted to the microbiology laboratory at St. Louis Children’s Hospital. We also sought Panton-Valentine leukocidin (PVL) genes in methicillin-susceptible Staphylococcus aureus (MSSA) isolates, and we further characterized some isolates by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), antibiotic susceptibility, accessory gene regulator (agr) allele, and presence of the arcA gene of the arginine catabolic mobile element (ACME).
Results
From 1999 to 2007, we detected a 250-fold increase in cultures of abscesses yielding methicillin-resistant Staphylococcus aureus (MRSA) and a 5-fold increase in abscess cultures yielding MSSA. MSSA isolates from abscesses and wounds were more likely to encode PVL than isolates from other sources. In contrast to PVL-negative isolates of MSSA which were genetically diverse, PVL-positive isolates were predominantly MLST 8, Agr type 1. More than half of PVL-positive MSSA isolates were resistant to erythromycin and susceptible to clindamycin with absence of inducible resistance, a pattern uncommon in PVL-negative MSSA but frequent in the USA300 clone of MRSA. In addition, PFGE of PVL-positive MSSA strains revealed the USA300 pattern.
Conclusions
In addition to methicillin-resistant strains, the current epidemic of Staphylococcus aureus infections includes infections caused by methicillin-susceptible strains that are closely related genetically and share phenotypic characteristics other than susceptibility to methicillin. These findings suggest that factors other than methicillin resistance are driving the epidemic.
doi:10.1086/600881
PMCID: PMC2965061  PMID: 19589082
Staphylococcus aureus; Panton-Valentine leukocidin; methicillin resistance

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