Allelic variation in genes encoding Panton–Valentine leukocidin from community-associated Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus isolates characteristically contain the genes for Panton-Valentine leukocidin (PVL), which is a proposed virulence factor. To determine whether different alleles of the PVL genes lukS-PV and lukF-PV occur, and whether they are associated with specific genetic lineages of S. aureus, sequences from 28 S. aureus isolates, representing four different multilocus sequence types, and bacteriophages SLT and PVL were compared. Seven nucleotide polymorphisms were identified, which defined three groups of the lukS-PV and lukF-PV sequence. Only one polymorphism resulted in an amino-acid change. Bacteriophage SLT and isolates of bacteriophage type 80/81 contained the prototypic (founder) lukS-PV and lukF-PV sequence. The alleles were not lineage-specific.     

Prevalence of Staphylococcus aureus carrying Panton–Valentine leukocidin genes among isolates from hospitalised patients in China

Between January 2005 and January 2006, 25 (12.8%) of 195 Staphylococcus aureus isolates were positive for Panton–Valentine leukocidin (PVL) genes in a teaching hospital in Wenzhou, China. Nineteen (11.9%) of 160 hospital-acquired isolates, and six (17.1%) of 35 community-acquired isolates, harboured lukS / F-PV . Six sequence types (ST88, ST239, ST398, ST25, ST30 and ST59) were found among 18 PVL-positive methicillin-resistant isolates with SCC mec types I, III, IIIA or IV. Only ST88 was found among seven PVL-positive methicillin-susceptible S. aureus isolates. The PVL-positive isolates were associated with lung infection, bloodstream infection and soft-tissue pyogenic infection. Overall, there was a high prevalence of PVL genes in genetically diverse S. aureus isolates.     

Serum antibodies against Panton–Valentine leukocidin in a normal population and during Staphylococcus aureus infection

To determine whether Staphylococcus aureus Panton–Valentine leukocidin (PVL) is expressed during human infection, anti-PVL antibody titres were compared in patients with PVL-positive and PVL-negative staphylococcal infections, and in patients with no evidence of S. aureus infection. Patients with PVL-positive strains had higher levels of anti-PVL antibodies than individuals of both control groups. The median anti-PVL titre increased 8.6-fold during the course of PVL-positive infection and 1.4-fold during PVL-negative infection. These results indicate that only PVL-positive S. aureus strains elicit significant anti-PVL antibody production in humans, and demonstrate the production of PVL during PVL-positive S. aureus infection. The protective role of this immune response remains to be established.     

Methicillin-resistant Staphylococcus aureus producing Panton–Valentine leukocidin as a cause of acute osteomyelitis in children

Staphylococcus aureus was identified as the cause of acute childhood osteomyelitis in 19 patients. A single clone of community-acquired methicillin-resistant S. aureus (MRSA) carrying the type IV mecA staphylococcal cassette chromosome and the Panton-Valentine leukocidin (PVL) genes was isolated from five patients. Among the remaining 14 patients, two methicillin-sensitive S. aureus (MSSA) isolates were PVL-positive. The maximal erythrocyte sedimentation rate and C-reactive protein values, and the time required for normalisation, were significantly different in patients with PVL-positive strains (MRSA and MSSA), suggesting that the production of PVL is an important factor that contributes to the course of the disease.     

The Panton–Valentine leukocidin vaccine protects mice against lung and skin infections caused by Staphylococcus aureus USA300

Methicillin-resistant Staphylococcus aureus is increasingly responsible for staphylococcal infections in the community. A large percentage of the community-acquired methicillin-resistant (CA-MRSA) strains in the USA produce Panton–Valentine leukocidin (PVL), which is associated with severe infections. The virulence of the clinical CA-MRSA strain USA300 was compared to that of its isogenic pvl- deleted mutant, and it was shown that PVL contributes to lung and muscle tissue destruction, respectively, in murine necrotizing pneumonia and skin infection models. Mice infected with the USA300 strain developed a dominant anti-PVL response. The PVL subunits were therefore tested as vaccinogens against this isolate, and their vaccine efficacy correlated with both the route of vaccination and infection. These data suggest that PVL is a virulence factor in murine CA-MRSA infections.     

Detection of Panton–Valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects

The prevalence of the Panton-Valentine leukocidin (PVL) gene in Staphylococcus aureus was investigated with a simple, reproducible and rapid real-time LightCycler SYBR Green I PCR assay. The PVL gene was detected in one isolate from 65 patients with S. aureus bacteraemia, in four isolates from 55 patients with respiratory tract infections, and in two isolates from 91 patients with cutaneous infections. In contrast, 15 of 25 cutaneous isolates of methicillin-resistant S. aureus (MRSA) were positive. All PVL-positive cutaneous MRSA isolates were community-acquired and comprised three different clones as determined by pulsed-field gel electrophoresis. The PVL gene was detected in isolates from patients with recurrent primary skin infections and S. aureus bacteraemia, but PVL did not seem to be an important virulence factor in the pathogenesis of staphylococcal bacteraemia.     

Methicillin-resistant Staphylococcus aureus producing Panton–Valentine leukocidin in a retrospective case series from 12 French hospital laboratories, 2000–2003

A retrospective analysis of hospital laboratory databases for 2000-2003 found that 0.4-1.0% of methicillin-resistant Staphylococcus aureus isolates had an antibiotic susceptibility pattern associated previously with the production of Panton-Valentine leukocidin (PVL). Of 81 isolates of this type, 35 were available for molecular testing. Each of the 35 available isolates carried the PVL genes, and 33 of these 35 isolates had an identical SmaI pulsed-field gel electrophoresis pattern.     

Microarray-based characterisation of a Panton–Valentine leukocidin-positive community-acquired strain of methicillin-resistant Staphylococcus aureus

Recent years have witnessed the emergence of novel methicillin-resistant Staphylococcus aureus (MRSA) strains that produce the potent toxin Panton-Valentine leukocidin (PVL). PVL-positive strains can cause complicated skin infections or necrotising pneumonia with high mortality, and these strains have the potential for epidemic spread in the community. In 2004-2005, two case clusters and two isolated cases were observed in eastern Saxony and southern Brandenburg. These were the first known infections with PVL-positive community-acquired MRSA (caMRSA) in this part of Germany. The isolates belonged to agr type III, spa type 44 or spa type 131, and showed a SmaI macrorestriction pattern that corresponded to caMRSA of clonal group ST80. The isolates were susceptible to levofloxacin, macrolides, clindamycin, gentamicin and vancomycin. Most isolates showed resistance to tetracycline and fusidic acid because of the presence of the tetK and far1 genes. A novel plasmid (designated pUB102) harbouring far1, tetK and blaZ was characterised and partially sequenced. Microarray analysis revealed that the caMRSA isolates harboured genes encoding several bi-component toxins (lukF/S-PVL, lukD/E, lukS/F plus hlgA, and another putative leukocidin homologue). Neither tst1 nor genes for enterotoxins A-Y were detected, but the isolates harboured several staphylococcal enterotoxin-like toxin genes (set genes), as well as genes encoding an epidermal cell differentiation inhibitor (edinB) and exfoliative toxin D (etD). Comparative analysis of other isolates from Australia, Germany, Switzerland and the UK showed that these isolates were representative of a widespread clone of caMRSA.     

Effect of antibiotics, alone and in combination, on Panton–Valentine leukocidin production by a Staphylococcus aureus reference strain

The capacity of Staphylococcus aureus strain LUG855 to release Panton–Valentine leukocidin (PVL) in the presence of sub-inhibitory concentrations of anti-staphylococcal drugs was examined. Oxacillin enhanced PVL release 2.5-fold, while clindamycin, linezolid, fusidic acid and rifampicin were inhibitory, and vancomycin, pristinamycin, tetracycline, ofloxacin and co-trimoxazole had no effect. In combination with oxacillin, sub-inhibitory concentrations of clindamycin or rifampicin inhibited PVL induction significantly, linezolid was less inhibitory, and fusidic acid did not inhibit PVL induction by oxacillin. These data support the use of oxacillin in combination with clindamycin, rifampicin or linezolid for the treatment of PVL-positive S. aureus infections.     

High diversity of Panton–Valentine leukocidin-positive, methicillin-susceptible isolates of Staphylococcus aureus and implications for the evolution of community-associated methicillin-resistant S. aureus

In total, 100 Staphylococcus aureus isolates from diverse cases of skin and soft-tissue infection at a university hospital in Saxony, Germany, were characterised using diagnostic microarrays. Virulence factors, including Panton-Valentine leukocidin (PVL), were detected and the isolates were assigned to clonal groups. Thirty isolates were positive for the genes encoding PVL. Only three PVL-positive methicillin-resistant S. aureus (MRSA) isolates were found, two of which belonged to European clone ST80-MRSA IV and one to USA300 strain ST8-MRSA IV. The remaining methicillin-susceptible PVL-positive isolates belonged to a variety of different multilocus sequence types. The predominant strains were agrI/ST22, agrII/CC5, agrIII/CC30 and agrIV/ST121. In order to check for possible bias caused by regional or local outbreak strains, an additional 18 methicillin-susceptible, PVL-positive isolates from the UK were tested. Approximately two-thirds of the UK isolates belonged to types that also comprised approximately two-thirds of the isolates from Saxony. Some methicillin-susceptible PVL-positive isolates (agrI/ST152, agrIII/ST80 and agrIII/ST96) closely resembled known epidemic community-acquired MRSA (CaMRSA) strains. These findings indicate that the current rise in PVL-positive CaMRSA could be caused by the dissemination of novel SCCmec elements among pre-existing PVL-positive strains, rather than by the spread of PVL phages among MRSA strains.     

Panton–Valentine leukocidin-positive Staphylococcus aureus skin and soft tissue infections among children in an emergency department in Madrid, Spain

Fifty-three children who attended the emergency department with community-associated (CA) Staphylococcus aureus skin and soft tissue infections (SSTIs) were enrolled in the study. Seven cases of infection (13.2%) were due to methicillin-resistant S. aureus (MRSA). Twelve of 46 available isolates (26.1%) were Panton–Valentine leukocidin (PVL)-positive. PVL-positive S. aureus SSTIs were more frequently associated with abscesses and cellulitis (75% vs. 38%, p 0.028), and more commonly required incision and drainage (75% vs. 21%, p 0.001). Most PVL-positive CA-MRSA isolates belonged to a single multilocus sequence type (ST8). In contrast, PVL-positive methicillin-susceptible S. aureus isolates belonged to four different sequence types (ST8, ST30, ST80, ST120).     

Distribution of the exfoliative toxin D gene in clinical Staphylococcus aureus isolates in France

Exfoliative toxin D (ETD) was identified recently as a new exfoliative toxin serotype. Like other exfoliative toxins, ETD induces intra-epidermal cleavage through the granular layer of the epidermis of neonatal mice. The distribution of ETD production was investigated in Staphylococcus aureus isolates from infected and colonised patients in France. The etd gene was found in 55 (10.5%) of 522 isolates tested. Isolates responsible for bullous impetigo and generalised staphylococcal scalded skin syndrome did not harbour etd, but etd was significantly more frequent in isolates causing cutaneous abscesses and furuncles. Most etd- and Panton-Valentine leukocidin-positive strains belonged to the clone of community-acquired methicillin-resistant S. aureus spreading currently throughout France.     

The molecular epidemiology and evolution of the Panton–Valentine leukocidin-positive, methicillin-resistant Staphylococcus aureus strain USA300 in Western Australia

Between 2003 and 2008, 76 clinical isolates of the Panton–Valentine leukocidin-positive Staphylococcus aureus strain 'West Australian methicillin-resistant Staphylococcus aureus (MRSA)-12' (WA MRSA-12) were recovered from 72 patients living in the Perth area in Western Australia. These isolates were found to belong to multilocus sequence type 8, and had a USA300-like pulsed-field gel electrophoresis pulsotype. All isolates were genotyped using diagnostic DNA arrays covering species markers, resistance factors, virulence-associated, as well as MSCRAMM (microbial surface components recognizing adhesive matrix molecules) genes to prove the identity between WA MRSA-12 and the pandemic strain USA300, as well as to detect possible genetic variability. In general, WA MRSA-12 isolates were similar to USA300, and the most common variant was identical to USA300- TC1516 . From this clone, most of the other variants may have evolved by a limited number of gene losses or acquisitions. Variations in carriage of virulence and resistance-associated genes allow distinction of variants or sub-clones. Altogether, 16 variants could be distinguished. They differed in the carriage of resistance genes ( blaZ/I/R , ermC , msrA + mpbBM , aadD + mupR , aphA3 + sat , tetK , qacC , merA/B/R/T ) of β-haemolysin-converting phages and of enterotoxins ( sek  +  seq , which were deleted in four isolates). Notably, the arginine catabolic mobile element (ACME) was absent in 12 isolates (15.8%). The mercury resistance ( mer ) operon, which is usually associated with SCC mec type III elements, was found in several ACME-negative isolates. The present study emphasises the importance of genotyping in detecting the introduction and evolution of significant MRSA strains within a community.     

Genetic diversity of community-associated methicillin-resistant Staphylococcus aureus in southern Stockholm, 2000–2005

This study investigated the molecular epidemiology of 104 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates from southern Stockholm during the period 2000–2005. The isolates were analysed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, staphylococcal chromosomal cassette (SCC) mec typing and detection of genes encoding Panton–Valentine leukocidin (PVL). Overall, 28 distinct PFGE patterns and 13 sequence types (STs) were identified. ST80, ST8, ST88 and ST150 were the major CA-MRSA clones in the area, and these accounted for 75% (78/104) of all CA-MRSA isolates. ST150 isolates, which have, to date, been found only in Sweden, were isolated exclusively from a group of homeless individuals. Eighty-six (83%) of the 104 isolates in the study possessed SCC mec IV, found in ten different STs, while 16 isolates possessed SCC mec V. The PVL genes were detected in 56% (58/104) of the isolates. Strain ST80-MRSA-IV carrying PVL genes predominated over the 6-year period and accounted for 38% of all isolates. However, a polyclonal tendency was observed among the CA-MRSA isolates recovered in recent years.     

Nasal carriage of Staphylococcus aureus, including community-associated methicillin-resistant strains, in Queensland adults

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are emerging in southeast Queensland, Australia, but the incidence of carriage of CA-MRSA strains is unknown. The aim of this study was to assess the nasal carriage rate of S. aureus , including CA-MRSA strains, in the general adult population of southeast Queensland. 396 patients presenting to general practices in two Brisbane suburbs and 303 volunteers randomly selected from the electoral rolls in the same suburbs completed a medical questionnaire and had nasal swabs performed for S. aureus . All isolates of S. aureus underwent antibiotic susceptibility testing and single-nucleotide polymorphism (SNP) and binary typing, including determination of Panton–Valentine leukocidin (PVL). The nasal carriage rate of methicillin-susceptible S. aureus (MSSA) was 202/699 (28%), a rate similar to that found in other community-based nasal carriage studies. According to multivariate analysis, nasal carriage of S. aureus was associated with male sex, young adult age group and Caucasian ethnicity. Only two study isolates (one MSSA and one CA-MRSA) carried PVL. The nasal carriage rate of MRSA was low, at 5/699 (0.7%), and only two study participants (0.3%) had CA-MRSA strains. CA-MRSA is an emerging cause of infection in southeast Queensland, but as yet the incidence of carriage of CA-MRSA in the general community is low.     

Community-acquired methicillin-resistant Staphylococcus aureus infections: an emerging threat in Spain

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has not been recognised previously as a cause of MRSA infections in Spain. Nineteen patients carrying Panton–Valentine leukocidin (PVL)-positive MRSA were identified in a Barcelona hospital, of whom 15 were immigrants, mostly from South America. Twelve developed skin and soft-tissue infections. The associated isolates carried the PVL gene and staphylococcal chromosomal cassette (SCC) mec IV. A dominant clone belonging to sequence type (ST)8 and related to the USA300 clone was identified by pulsed-field gel electrophoresis. This clone is emerging in Spain, primarily among immigrants from South America, but dissemination to the native Spanish population could increase.     

Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes

The epidemiology of methicillin-susceptible Staphylococcus aureus (MSSA) in Africa is poorly documented. From January 2007 to March 2008, 555 S. aureus isolates were collected from five African towns in Cameroon, Madagascar, Morocco, Niger, and Senegal; among these, 456 unique isolates were susceptible to methicillin. Approximately 50% of the MSSA isolates from each different participating centre were randomly selected for further molecular analysis. Of the 228 isolates investigated, 132 (58%) belonged to five major multilocus sequence typing (MLST) clonal complexes (CCs) (CC1, CC15, CC30, CC121 and CC152) that were not related to any successful methicillin-resistant S. aureus (MRSA) clones previously identified in the same study population. The luk-PV genes encoding Panton-Valentine leukocidin (PVL), present in 130 isolates overall (57%), were highly prevalent in isolates from Cameroon, Niger, and Senegal (West and Central Africa). This finding is of major concern, with regard to both a source of severe infections and a potential reservoir for PVL genes. This overrepresentation of PVL in MSSA could lead to the emergence and spread of successful, highly virulent PVL-positive MRSA clones, a phenomenon that has already started in Africa.     

多重PCR对金黄色葡萄球菌杀白细胞素基因的检测

目的应用多重PcR检测含Panton-Valentine杀白细胞素（PVL）基因的金黄色葡萄球菌。方法收集我院2005年1月至2006年1月从临床多种标本中分离的金黄色葡萄球菌，用多重PCR同时检测葡萄球菌16SrRNA基因、mecA基因和lukS/F-PV基因。多重PCR检测MRSA的SCCmec基因型及亚型。结果195株金黄色葡萄球菌经多重PCR检测，121株为耐甲氧西林金黄色葡萄球菌（MRSA），74株为甲氧西林敏感金黄色葡萄球菌（MSSA），共检测到26株金黄色葡萄球菌lukS/F-PV基因阳性，阳性率为13．3％（26，195）。其中19株为MRSA，阳性率为15．7％（19／121）；7株为MSSA，阳性率为12．2％（7／74）。19株lukS/F-PV基因阳性的MRSA的SCCmec基因型分别为SCCmec Ⅲ型10株、SCCmecⅢA型4株、SCCmecⅣ型4株及SCCmecⅠ型1株。26株lukS/F-PV基因阳性的分离株有11株分离自脓液或创面分泌物，10株分离自痰标本，3株分离自血液标本，2株分离自尿液。结论在温州地区分离的MRSA和MSSA中都能检测到Panton-Valentine杀白细胞素基因，含Panton-Valentine杀白细胞素基因MRSA的SCCmec基因型主要为SCCmec Ⅲ，含PVL基因的金黄色葡萄球菌主要引起化脓性感染和肺部感染。     

Comparative genomics and DNA array-based genotyping of pandemic Staphylococcus aureus strains encoding Panton-Valentine leukocidin

Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.