PVL-positive Isolates Research Articles

Molecular characterization of Staphylococcus aureus from surgical site infections in orthopedic patients in an orthopedic trauma clinical medical center in Shanghai.

Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of surgical site infections (SSIs). The aim of our study was to characterize molecularly S. aureus isolates from SSIs in orthopedic patients in Shanghai, China. Eighty-two S. aureus isolates (46 methicillin-susceptible Staphylococcus aureus [MSSA] and 36 MRSA) were collected from SSIs in orthopedic patients. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and a variety of clinically important toxin genes were detected. The sequence type, spa type, and agr group were determined to analyze the genotypes of all the isolates collected. In addition, MRSA isolates were characterized by staphylococcal cassette chromosome mec (SCCmec) type as well. The strains showed susceptibility to antibiotics such as teicoplanin, minocycline, quinupristin-dalfopristin, linezolid, mupirocin, and vancomycin. Ten pvl-positive isolates (three MRSA and seven MSSA) were found among all isolates. Eight community-associated MRSA (CA-MRSA) isolates were found, six of which belonged to ST59-MRSA-IV but most MRSA isolates (20/36, 55.6%) belonged to ST239-MRSA-III-t030/t037 with a wide range of antibiotic resistance. By contrast, MSSA isolates were more diverse in both molecular characterizations and virulence factors, with eight MSSA isolates harboring more than six toxin genes detected. ST239-MRSA-III-t030/t037 was the epidemic clone, and healthcare-associated MRSA (HA-MRSA) strains might be the major pathogen causing S. aureus SSIs in orthopedic patients.

A 12-year survey of methicillin-resistant Staphylococcus aureus infections in Greece: ST80-IV epidemic?

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) infections. Severe MRSA infections have been associated with the virulence factor Panton–Valentine leukocidin (PVL). The aim of this study was to investigate susceptibility patterns, the presence of toxin genes, including that encoding PVL, and clonality among MRSA isolates collected from patients in Greece over a 12-year period. MRSA isolates were collected from January 2001 to December 2012 from six different hospitals. Antibiotic susceptibility was determined with the disk diffusion method and the Etest. The presence of the toxic shock syndrome toxin-1 gene (tst), the enterotoxin gene cluster (egc) and the PVL gene was tested with PCR. The genotypic characteristics of the strains were analysed by SCCmec and agr typing, and clonality was determined with pulsed-field gel electrophoresis and multilocus sequence typing. An increasing rate of MRSA among S. aureus infections was detected up to 2008. The majority of PVL-positive MRSA isolates belonged to a single clone, sequence type (ST)80-IV, which was disseminated both in the community and in hospitals, especially during the warmest months of the year. Carriage of tst was associated with ST30-IV, whereas egc was distributed in different clones. CA-MRSA isolates were recovered mainly from skin and soft tissue infections, whereas HA-MRSA isolates were associated with surgical and wound infections. During the period 2001–2012, ST80-IV predominated in the community and infiltrated the hospital settings in Greece, successfully replacing other PVL-positive clones. The predominance of ST239-III in HA-MRSA infections was constant, whereas new clones have also emerged. Polyclonality was statistically significantly higher among CA-MRSA isolates and isolates from adult patients.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA.

Molecular epidemiology of Staphylococcus aureus in post-earthquake northern Haiti

Knowledge of nasal carriage is important in predicting staphylococcal infection, and no information exists regarding the endemicity of Staphylococcus aureus in Haiti. We performed a cross-sectional analysis of S. aureus nasal screening in an acute care, a subacute rehabilitation, and a community setting, with a brief medical and epidemiological history. PCR-positive S. aureus screening nasal cultures underwent molecular analysis for spa type, SCCmec type, and virulence genes (Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin (TSST), and arginine catabolic mobile element (ACME)), and were evaluated for antibiotic susceptibility using commercial tests. Overall carriage rates of 8.4% methicillin-susceptible S. aureus (MSSA) and 2.8% methicillin-resistant S. aureus (MRSA) were identified, with a high rate of tetracycline resistance. TSST and PVL genes were identified in MSSA. MRSA isolates contained no virulence markers. Unique MSSA phenotypes (i.e., linezolid-resistant, vancomycin-sensitive/daptomycin non-susceptible) were identified, as were two PVL-positive ST152 MSSA colonization isolates, previously geographically limited to Africa. We found a low S. aureus carriage rate with complete vancomycin susceptibility and high tetracycline resistance, which has important public health implications with regard to treatment. Additionally, the finding of PVL-positive MSSA isolates, including the expansion of a previously described limited 'divergent' clone, ST152, warrants further evaluation.

Panton-Valentine leukocidin (PVL)-positive health care-associated methicillin-resistant Staphylococcus aureus isolates are associated with skin and soft tissue infections and colonized mainly by infective PVL-encoding bacteriophages.

The emergence of Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) is a public health concern worldwide. PVL is associated with community-associated MRSA and is linked to skin and soft tissue infections (SSTIs). However, PVL genes have also been detected in health care-associated (HA) MRSA isolates. The diseases associated with PVL-positive HA-MRSA isolates and the distributions of PVL-encoding bacteriophages in HA-MRSA have not been determined. In this study, a total of 259 HA-MRSA strains isolated between 2009 and 2012 in China from inpatients with SSTIs, pneumonia, and bacteremia were selected for molecular typing, including staphylococcal cassette chromosome mec typing, multilocus sequence typing, and staphylococcal protein A gene typing. The PVL genes and PVL bacteriophages in the MRSA isolates were characterized by PCR. Among the tested MRSA isolates, 28.6% (74/259) were PVL positive. The high prevalence of PVL-carrying HA-MRSA was observed to be associated with SSTIs but not with pneumonia or bacteremia. The PVL-positive HA-MRSA isolates were colonized mainly by infective PVL phages, namely, Φ7247PVL, ΦSLT, and ΦSa2958. The distribution of PVL-carrying bacteriophages differed geographically. Our study highlights the potential risk of the emergence of multidrug-resistant HA-MRSA strains with increased virulence.

Molecular epidemiology of community-associated methicillin-resistant Staphylococcus aureus in Spain: 2004-12.

In Spain, despite the high rates of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA), the incidence of community-associated (CA) MRSA seems to be low on the basis of a small number of studies. We analysed the evolution of CA-MRSA in Spain from 2004 to 2012, and identified the clonal lineages and population structure. The study included 8326 MRSA strains. Susceptibility to 18 antimicrobials was determined. Isolates were tested for the presence of mecA, Panton-Valentine leucocidin (PVL) and the arginine catabolic mobile element (ACME) by PCR, and typed by staphylococcal cassette chromosome mec, PFGE, spa, multilocus sequence typing and agr. Among the 8326 isolates, 246 (2.9%) were CA-MRSA. We identified genotypically 226 PVL-positive CA-MRSA isolates (88% agr type I, 10.2% agr type III and 1.8% agr type II) and 20 PVL-negative CA-MRSA isolates (all agr type I) from children and adults (82.1% from wounds) from 13 different geographical areas. A significant increase in the rates of CA-MRSA was observed when comparing 2004-07 (0.43%) with 2008-12 (5.44%). Resistance rates were as follows: only β-lactams, 84.5%; erythromycin, 12.8%; tetracycline, 8.8%; clindamycin, 4.9%; ciprofloxacin, 3.1%; fusidic acid, 2.0%; others, 0.4%; and multiresistant, 6.2% (six isolates USA300). The strains belonged to the PVL-positive clones ST8-IVc (69.9%), ST8-IVa-ACME-positive (USA300, 8.9%), ST8-IVa-ACME-negative (0.8%), ST30-IVc (4.5%), ST80-IVc (2.0%), ST5-IVc (1.2%) and others (ST59, ST72, ST88, ST642, ST1472 and ST1829; 4.5%) and to the PVL-negative ST398-V (8.1%). We confirm an increase in CA-MRSA in Spain, the predominance of the ST8-IVc clone, the emergence of the USA300 clone, a high genetic diversity among PVL-positive CA-MRSA isolates and the recent emergence of the pig-associated ST398-V clone.

In vitro characterization of representative clinical South African Staphylococcus aureus isolates from various clonal lineages.

Data concerning the virulence and pathogenesis of South African strains of Staphylococcus aureus are limited. We investigated host–pathogen interactions of randomly selected clinical S. aureus isolates representing various clones. We characterized the ability of isolates to adhere to fibronectin, fibrinogen, collagens IV and VI, to invade host cells and to induce cell death in vitro. We analysed the possible association of these results with characteristics such as methicillin resistance, Panton–Valentine leucocidin (PVL) positivity and clonality. The S. aureus isolates displayed diversity in their abilities to adhere to various human ligands. All isolates were highly invasive except for ST121. PVL-negative isolates were significantly more invasive than the PVL-positive isolates (p 0.004). Isolates of CC5, CC30 and CC121 were non-cytotoxic, whereas isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic. No statistical association was identified between cell death and methicillin resistance, bacterial PVL status, clonality or patient HIV status. The vast majority of isolates were invasive and induced significant cell death. PVL-negative isolates were more invasive than PVL-positive isolates, while methicillin-resistant isolates were not found to be more invasive or cytotoxic than methicillin-susceptible isolates.

Staphylococcus aureus nasal and pharyngeal carriage in Senegal

Nasal and pharyngeal swabs were collected from 132 patients admitted to the Principal Hospital in Dakar (Senegal), in January and February 2012. The prevalence of Staphylococcus aureus carriage was 56.1% (n = 74): 40.2% for pharyngeal samples and 36.4% for nasal samples. None of the isolates was methicillin-resistant. Carriage was independently associated with being female (p <0.01) and large households (≥15 members) (p 0.04). The luk-PV genes encoding Panton–Valentine leukocidin (PVL) were present in 26.2% of the isolates. These data highlight the importance of the oropharynx as a site of colonization, and the high prevalence of PVL-positive isolates in Senegal as compared with industrialized countries.

Antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus: An international survey.

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was <1%. Among 1798 MRSA isolates from 13 countries that were tested for the requested 10 non-β-lactam antibiotics, 49.4% were multisusceptible. However, multiresistant isolates (resistant to at least three classes of non-β-lactam antibiotics) were reported from all regions. Sequence type 30 (ST30) was reported worldwide, whereas ST80 and ST93 were exclusive to Europe and Australia, respectively. USA300 and related clones (ST8) are progressively replacing the ST80 clone in several European countries. Eight major clusters were discriminated by multilocus variable-number tandem repeat assay (MLVA), showing a certain geographic specificity. PVL-positive MRSA isolates frequently remain multisusceptible to non-β-lactam agents, but multiresistance is already prevalent in all regions. Surveillance of MRSA susceptibility patterns should be monitored to provide clinicians with the most current information regarding changes in resistance patterns.

Epidemiological and molecular characterization of community and hospital acquired Staphylococcus aureus strains prevailing in Shenyang, Northeastern China

In order to obtain adequate information for the treatment of methicillin resistant Staphylococcus aureus (MRSA) infections, it is crucial to identify trends in epidemiological and antimicrobial resistance patterns of local S. aureus strains. Community and hospital acquired S. aureus isolates (n=202) were characterized using staphylococcal cassette chromosome mec (SCCmec) typing, pulse field gel electrophoresis (PFGE) analysis, spa typing and minimal inhibitory concentration (MIC) determination. The prevalence of the Panton-Valentine leukocidine (pvl) and several antibiotic resistance genes among the isolates were also detected by PCR. All of the S. aureus isolates were susceptible to vancomycin, daptomycin and linezolid. Three hospital isolates were resistant to teicoplanin while 14 showed intermediate resistance to teicoplanin. The resistance patterns of community-acquired MRSA (CA-MRSA) isolates to other antimicrobials were similar to those of hospital-acquired MRSA (HA-MRSA) isolates except for clindamycin and gentamicin. There was excellent correlation between phenotypes and genotypes in the determination of S. aureus resistance to erythromycin, gentamicin, and tetracycline. The SCCmec type II and SCCmec type IV were the predominant types detected in hospital and community isolates, respectively. The most frequently encountered spa types were t002 and t030 both in HA- and CA-MRSA isolates. Pulsotype A was the most predominant pulsotype identified among the isolates tested, followed by pulsotype B. Seventy-two hospital isolates (19 HA-MRSA and 53 HA-MSSA) and 10 CA-MRSA were positive for the pvl gene. This study shows that the combination of susceptibility testing and various molecular methods has provided useful information on the antibiotic resistance and molecular diversity of S. aureus in a specific region of China. The high proportion of pvl positive MSSA and MRSA isolates observed in this study indicates that adequate measures are needed to curtail the spread of those MRSA and MSSA clones prevailing both in hospital and the community.

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.

The expression of Panton-Valentine leukocidin gene and its correlation with agrA among different clinical isolates of Staphylococcus aureus

Objective To investigate the correlation between expression of Panton-Valentine leukocidin gene and accessory gene regulator among different clinical isolates of Staphylococcus aureus. Methods All non-duplicate Staphylococcus aureus clinical isolates were isolated from various clinical specimens of the patients at 4 hospitals from January 2003 to December 2010.Panton-Valentine leukocidin genes among Staphy lococcus aureus clinical isolates were detected by PCR and DNA sequencing.The expressions of lukS-PV and agr A were determined by real-time PCR. Results Nintysix S.aureus isolates including 58 hospital-acquired and 28 community-acquired isolates were positive for PVL genes,among which 54 from blood,33 from pus and 9 from sputum.Ten isolates cannot be classified due to lack of information.Sixty-seven and 29 PVL-positive isolates were isolated from the specimens of adults and children.The median relative quantities of luk SmRNA of the isolates from pus and blood were 1.500 and 0.818.The quantity of lukSmRNA among the isolates from pus was significantly higher than that from blood (U=634,P=0.025).The median relative quantities of luk SmRNA of the isolates from children and adults were 1.292 and 0.540,respectively.The quantity of lukSmRNA among the isolates from children was significantly higher than that from adults (U=660,P=0.013).The median relative quantities of lukSmRNA among community-acquired and hospital-acquired isolates were 1.034 and 0.536,respectively.The quantity of lukSmRNA among community-acquired isolates was significantly higher than that from hospital-acquired isolates (U=338,P=0.012).The correlation coefficients between lukSmRNA and agr Am RNA of total isolates,pus isolates and blood isolates were 0.592 (P<0.01),0.810 (P<0.01) and 0.543 (P<0.01),respectively.While the correlation coefficients of those among the isolates from children and adults were 0.804 (P<0.01) and 0.476 (P<0.01).The correlation coefficients of those among the isolates from community-acquired and hospital-acquired isolates were 0.767 (P<0.01) and 0.556 (P<0.01). Conclusions The quantity of lukSmRNA of Staphylococcus aureus isolates from pus was significantly higher than that from blood.The agr may have positive regulation effect on the expression of lukS/F-PV,especially among the isolates from pus and children.(Chin J Lab Med,2013,36:313-317) Key words: Staphylococcus aureus; Bacterial toxins; Exotoxins; Leukocidins; Bacterial proteins

Meticillin-resistant Staphylococcus aureus in elderly residents of care homes: colonization rates and molecular epidemiology

Meticillin-resistant Staphylococcus aureus (MRSA) is a significant cause of mortality and morbidity in healthcare and community settings; however, there is a paucity of large-scale, longitudinal studies monitoring the occurrence of MRSA in the care home setting. To determine the molecular epidemiology of MRSA colonizing elderly residents of care homes. Residents in 65 care homes in Leeds, UK, were screened for MRSA nasal colonization in four consecutive years (2006-2009). Isolates were characterized using antibiotic susceptibility testing, detection of the Panton-Valentine leucocidin (PVL) locus, accessory gene regulator allotyping, characterization of the staphylococcal cassette chromosome mec element, spa-typing and pulsed-field gel electrophoresis. MRSA was recovered from 888 nasal swabs of 2492 residents and prevalence was similar (19-22%) throughout the study. Resistance to ≥3 antibiotic classes was common (34%), but resistance to only β-lactam agents was rare (3%); no PVL-positive isolates were identified. Most isolates were related to healthcare-associated epidemic-MRSA type 15 (EMRSA-15, ST22-IV); such isolates decreased in prevalence during the study (86-72%; P < 0.0001, χ(2)-test). The remainder belonged to five different multi-locus sequence type clonal complexes (CC). Most notably, CC59 strains increased in prevalence (10-25%; P < 0.0001, χ(2)-test) and were associated with high-level mupirocin resistance. The molecular epidemiology of MRSA in care homes is complex and dynamic. There was a high, consistent prevalence of MRSA nasal colonization, dominated by healthcare-associated strains. Vigilance is recommended; however, as high-level mupirocin resistance was associated with a single clonal group (CC59) that significantly increased in prevalence during the study.

Current status of Staphylococcus aureus infection in a central teaching hospital in Shanghai, China

BackgroundTo control the spread of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals, infection control measures such as hand hygiene practices were introduced into the teaching hospitals in Shanghai, China, in 2008. Currently, there is limited information characterizing the latest hospital-acquired S. aureus infections in this area. Therefore, we sought to determine the prevalence, molecular characteristics, and genotype-phenotype correlation of hospital-acquired S. aureus infections in Huashan Hospital, one of the largest teaching hospitals in Shanghai.ResultsAmong 608 hospital-acquired S. aureus clinical isolates obtained from January to December of 2011 in Huashan Hospital, 68.1% were MRSA. The predominant MRSA clones were ST239-SCCmecIII and ST5-SCCmecII. ST239 was mainly recovered from respiratory specimens and sterile body fluids, ST5 was associated with respiratory specimens and blood, and ST1 was most prevalent in urine samples. In this study, 31 dispersed sequence types (STs) of methicillin-susceptible S. aureus (MSSA) were identified, most of which caused skin/soft tissue infection and bacteremia. The frequencies of pvl-, muPA-, and qacA/B-positive isolates were 1.6, 9.9, and 11.8% respectively. MuPA was more frequently identified in ST1 and ST5, and qacA/B was more prevalent in ST239 and ST5. Most of the pvl-positive isolates were MSSA, whereas the majority of muPA- and qacA/B-positive isolates were MRSA. ST239 and ST5 had higher resistance rates to multiple antibiotics. In Huashan Hospital, the infection rate in the intensive care unit (ICU) was 3.9 per 1000 hospitalized days, but only 1.2 per 1000 hospitalized days in the other wards. Each ward harbored its own dominant STs. Pulsed-field gel electrophoresis showed diversity within the same epidemic S. aureus clones originating from the same wards.ConclusionThere is still a high prevalence of MRSA infections in the teaching hospital in Shanghai. There were also differences in the major infection types caused by MRSA and MSSA, and hospital-acquired S. aureus infections in the ICU of Huashan Hospital pose a greater threat to patient safety than in other wards. The high proportion of multiple antibiotic and chlorhexidine-based antiseptic-resistant clones in this hospital underscores the need for more effective infection control measures to help curtail dissemination of MRSA to hospitalized patients.

Community-acquired methicillin-resistant Staphylococcus aureus clones circulating in Belgium from 2005 to 2009: changing epidemiology

The present study reports the evolution of the demographic characteristics and the molecular epidemiology of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) in Belgium from 2005 to 2009. Four hundred and ten CA-MRSA isolates were prospectively collected and screened for the presence of Panton-Valentin leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) encoding genes, while clinical information were recorded. PVL- and TSST-1-positive isolates were genotyped by pulsed-field gel electrophoresis (PFGE). Staphylococcal cassette chromosome mec (SCCmec) type, spa type and multilocus sequence type (MLST) were determined on representative isolates. One hundred and fifty-nine (39 %) isolates were PVL-positive. PVL-positive isolates were significantly more frequently isolated from skin or soft tissue than PVL-negative isolates, causing mainly subcutaneous abscesses and furuncles. Patients with PVL-positive CA-MRSA were significantly younger than patients with PVL-negative CA-MRSA. Eighty-seven percent of the PVL-positive isolates belonged to a limited number (n = 7) of PFGE types belonging to sequence types (ST) ST80, ST8, ST30, ST5, ST152, ST338 and a new ST, a single-locus variant of ST1. A temporal evolution of the distribution of these PFGE types was observed, characterised by (1) the dissemination of the ST8-SCCmecIV arcA-positive (USA300) genotype and (2) a genetic diversification. Forty-seven (11 %) strains were TSST-1-positive, of which 65 % clustered into four PFGE types, all belonging to ST5. The epidemiology of CA-MRSA in Belgium is changing, as the rapid diffusion of the USA300 clone seems to occur, together with a clonal diversification.

Dissemination of Panton–Valentine leukocidin‐positive methicillin‐resistant Staphylococcus aureus in Okinawa, Japan

Panton-Valentine leukocidin (PVL) is a pore-forming cytotoxin that is produced by Staphylococcus aureus closely associated with skin and soft-tissue infections (SSTI). PVL-positive S.aureus strains have been identified worldwide, including in the USA; however, few studies have reported the presence of these strains in Japan. In this study, we prospectively investigated the prevalence of PVL in S.aureus strains from outpatients presenting with SSTI in Okinawa and characterized the PVL-positive S.aureus strains by polymerase chain reaction (PCR) and multilocus sequence typing (MLST). From 2008-2010, 499 clinical samples were obtained from 497 people. S.aureus was identified in 274 samples, and 36% (99 of 274) were methicillin-resistant S.aureus (MRSA). Seventeen (6.2%) PVL-positive S.aureus strains were detected by PCR, and 12 of the 17 PVL-positive strains were MRSA. Most PVL-positive S.aureus caused furuncles or carbuncles. Nine of the 17 PVL-positive isolates had an ST8 MRSA genotype and most harbored SCCmec type IVa and the arcA gene of the arginine catabolic mobile element, which is identical to the USA300 clone prevalent in the USA. PVL-positive S.aureus strains were more likely to be resistant to erythromycin (65%) and levofloxacin (53%). PVL-positive S.aureus strains have emerged and are spreading as a causative pathogen for SSTI in Okinawa.

The molecular epidemiology of the highly virulent ST93 Australian community Staphylococcus aureus strain.

In Australia the PVL - positive ST93-IV [2B], colloquially known as “Queensland CA-MRSA” has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.

Risk factors and molecular analysis of Panton-Valentine leukocidin-positive methicillin-susceptible Staphylococcus aureus colonization and infection in children

Limited information is available regarding the role Panton-Valentine leukocidin (PVL) plays in methicillin-susceptible Staphylococcus aureus (MSSA). In this study, we compared the frequency of the PVL gene between MSSA strains isolated from patients with MSSA infections and MSSA strains isolated from patients with evidence of MSSA nasal colonization. We also explored the role that the PVL toxin plays in the ability of MSSA to cause disease as well as the phylogenetic relationship between these S aureus strains. The presence of MSSA strains was screened among children aged <18 years during routine health maintenance visits and among children aged <7 years at daycare centers or kindergartens during the 2003 to 2008 period. At the same time, clinical MSSA isolates were recovered from patients with various types of bacterial infections. Polymerase chain reaction was applied to detect the presence of the PVL and SEB genes in these strains. The strains were also subjected to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) studies. Clinical features were compared between patients with PVL-positive and those with PVL-negative isolates. A total of 495 colonizing MSSA and 71 clinical MSSA isolates were used. The prevalence of PVL-positive S aureus was significantly higher among clinical isolates than among colonizing isolates (14/71, 19.7%; 5/495, 1.0%; p<0.05). In addition, we found that patients with PVL-positive MSSA infections had a significantly longer duration of fever and tended to have higher C-reactive protein levels than patients with PVL-negative MSSA infections. MLST typing of the 19 PVL-positive MSSA isolates revealed ST59, a strain that is similar to the MLST type of community-associated methicillin-resistant Staphylococcus aureus found in Taiwan. The PFGE typing of PVL-positive/ST59 MSSA isolates revealed multiple pulsotypes. The prevalence of the PVL gene was significantly higher among clinical strains of MSSA (19.7%) than among colonizing strains (1.0%). In addition, patients infected with PVL-positive MSSA strains had fever for a significantly longer duration and tended to have higher C-reactive protein levels than patients with PVL-negative MSSA infections. Our findings imply that PVL may play an important role in the pathogenesis of S aureus infection.

High clonal heterogeneity of Panton–Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus strains from skin and soft-tissue infections in the Province of Bolzano, Northern Italy

Panton–Valentine leukocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to characterise 10 PVL-positive MRSA isolates collected during February 2010 to January 2011 from skin and soft-tissue infections in the North Italian Province of Bolzano. Accessory gene regulator (agr) typing, staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene typing, multilocus sequence typing, toxin gene profiling, polymerase chain reaction for type I arginine catabolic mobile element (ACME) and antimicrobial resistance typing were applied to the isolates. Eight different CA-MRSA clones were identified, including ST30-IVc, ST772-V, ST80-IVc, ST5-IVc, ST88-IVa, ST93-IVa, ST8-IVc and the type I ACME-positive ST8-IVa. The high heterogeneity of PVL-positive MRSA probably reflects the introduction of different clones by international travellers or immigrants.

Molecular characterization of Staphylococcus aureus carrying the panton-valentine leucocidin genes in northern Spain

To study the prevalence of the Panton-Valentine leucocidin (PVL) gene in methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus obtained in Gipuzkoa, northeastern area of the Basque Country, north-central Spain, and perform the molecular characterization of PVL-positive isolates. Molecular studies comprised: PVL gene detection by PCR, staphylococcal chromosome cassette mec (SCCmec) typing, spa sequencing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and detection of the arginine catabolic mobile element (ACME). Between 1978 and 2006, only two (0.3%) of the 686 MRSA isolates studied were positive for the PVL gene. This percentage increased between 2007 and 2009, when the PVL gene was detected in 30 of the 679 MRSA (4.4%) and in nine of the 1227 MSSA (0.7%) isolates. The 41 PVL-positive isolates characterized had eight different sequence types (STs). Twenty-three MRSA PVL-positive isolates were ST8, spa type t008, seven of which were ACME positive, erythromycin-resistant and showed the PFGE pattern (90-100% similarity) of the USA300 clone. ST8 was also the most prevalent ST among the nine MSSA PVL-positive isolates. The current epidemiology of PVL-positive MRSA in our region more closely resembles that of the USA rather than that of other European countries, being USA300 or USA300-like isolates the most prevalent ones.