PVL-positive Community-associated Methicillin-resistant Staphylococcus Aureus Research Articles

Predominance of t355/ST152/SCCmec V clonal type among PVL-positive MRSA isolates in a tertiary care hospital in Belgrade, Serbia.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is continually changing. Frequency of genotypes typical for community-associated MRSA (CA-MRSA) is increasing in hospitals, as well as resistance to antimicrobial agents. Moreover, different clones predominate in different geographic regions, and temporal shifts occur in the predominant clonal type. The aim of this study was to estimate the prevalence of MRSA, CA-MRSA and PVL-positive MRSA isolates from patients hospitalised in the Military Medical Academy (MMA) and from outpatients, and to perform genotyping of PVL-positive MRSA isolates. MRSA isolates were obtained by standard microbiological techniques. PVL-positive MRSA were detected by single PCR. Determination of SCCmec types in MRSA isolates was done using multiplex PCR and genotyping of PVL-positive MRSA by PFGE, MLST and spa typing. The prevalence of MRSA among S. aureus isolates from different clinical specimens was 43.4%. In outpatients the prevalence of MRSA was 3.2%. SCCmec types specific for CA-MRSA were found in 26% of MRSA isolates from hospitalised patients. In groups, hospitalised patients and outpatients, the prevalence of PVL-positive MRSA isolates was 4%, and all of them harboured SCCmec type V genetic element. PFGE revealed minor differences between four groups of PVL-positive MRSA isolates, but all of them belonged to ST152, and all except one were of the t355 spa type. High prevalence of MRSA and CA-MRSA in MMA, especially the presence of PVL-positive CA-MRSA, represent a serious health threat for patients. Genotype t355/ST152/SCCmec V is the dominant MRSA clone among PVL-positive CA-MRSA.

Panton-Valentine Leukocidin Facilitates the Escape of Staphylococcus aureus From Human Keratinocyte Endosomes and Induces Apoptosis

Skin and soft-tissue infections (SSTIs) caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have emerged as major health problems throughout the world. Most SSTI CA-MRSA strains produce Panton-Valentine leukocidin (PVL), but its contribution to CA-MRSA pathogenesis is poorly defined. Here, we used an endemic PVL-positive SSTI-causing CA-MRSA strain from Taiwan, together with an isogenic PVL-knockout mutant (Δpvl) and complemented PVL-positive derivative, to evaluate the role of PVL in the pathogenesis of CA-MRSA in the RHEK-1 human keratinocyte cell line and a rabbit skin infection model. We found that both PVL-positive CA-MRSA and isogenic Δpvl strains attached and were engulfed into endosomes of RHEK-1 cells within 1 hour following infection. However, by 2 hours after infection PVL-positive CA-MRSA more effectively disrupted endosomes, escaped into the cytoplasm, and replicated intracellularly. By 6 hours after infection, the PVL-positive strain caused significantly more caspase-dependent keratinocyte apoptosis than the isogenic Δpvl mutant. In the rabbit infection model, 1 week following infection the wild-type strain produced significantly more widespread lesions and cell apoptosis than the isogenic Δpvl mutant. These findings indicate that PVL is an important virulence factor that enables CA-MRSA to produce necrotizing skin infections by allowing the bacteria to escape from endosomes, replicate intracellularly, and induce apoptosis.

Trial to control an outbreak of Panton-Valentine leukocidin–positive methicillin-resistant Staphylococcus aureus at a boarding school in Japan

Our retrospective investigation of methicillin-resistant Staphylococcus aureus (MRSA) infection at a hospital in Japan around 2007 suggested dissemination of community-associated MRSA (CA-MRSA) strains among healthy students in a Japanese boarding school, which frequently caused skin disease and exhibited the same antibiogram patterns. Active surveillance of skin diseases for 6 months after May 2008, examination of MRSA carriage in selected high-risk groups, and investigation of their life circumstances, including environmental cultures, were conducted in the school. Furthermore, we strengthened hygiene practices and improved recognized risk factors from November 2008 and observed the occurrence of skin diseases and MRSA carriage rate for the evaluation of infection controls. We identified 21 patients with skin diseases in whom MRSA strains were isolated. MRSA colonization rates in 3 selected groups ranged from 7.6% to 36.6%. The rates of both skin disease and MRSA carriage decreased significantly after infection controls were introduced. Genetic analysis revealed a main dissemination of a PVL-positive SCCmec IVc clone (41/47 isolates in total), presenting as a different pulsed-field type than USA300. This first report of a PVL-positive CA-MRSA outbreak in Japan demonstrates systematic management of dissemination by conducting surveillance in a closed community.

Panton–Valentine leukocidin-encoding bacteriophage and gene sequence variation in community-associated methicillin-resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) often produce Panton–Valentine leukocin (PVL), which is encoded by two co-transcribed genes located on lysogenized bacteriophages. Six PVL-encoding temperate phages have been described and single nucleotide polymorphisms (SNPs) in the PVL genes have been reported. In the present study, 22 PVL-positive CA-MRSA isolates were chosen to reflect the diversity of multilocus sequence type (MLST) clonal complexes (CC) identified in our hospital. Isolates were characterized by antimicrobial resistance profile, staphylococcal cassette chromosome mec (SCCmec) and spa type, pulsed-field gel electrophoresis profile and MLST. Primers were designed to sequence the lukSF-PV genes. PVL-encoding phages were characterized using a PCR-based assay. SNPs were identified at seven locations in the lukSF-PV genes, which varied with S. aureus MLST lineage. One SNP was nonsynonymous. All CC80 and some CC1 isolates carried FSa2mw; CC8, CC88 and CC154 isolates harboured PVL-encoding elongated head-type phages; and some CC59 isolates harboured a FSa2958-like phage. Novel or variant phages were present in CC5 and some CC1 and CC59 isolates. The PVL gene sequence and the PVL-encoding phage varied with lineage. Further work is required to determine whether PVL sequence and/or phage variations result in biological differences.

Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis

BackgroundExtensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.Methodology/Principal FindingsWe compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×105 and 4×108 CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.Conclusions/SignificancePVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.

Antimicrobial susceptibility ofStaphylococcus aureusisolated from children with impetigo in China from 2003 to 2007 shows community-associated methicillin-resistantStaphylococcus aureusto be uncommon and heterogeneous

The number of patients with impetigo caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been increasing. To investigate the antimicrobial susceptibility of S. aureus causing impetigo in children in China from 2003 to 2007 and further characterize isolates of CA-MRSA. We examined 984 S. aureus isolates for antimicrobial susceptibility to 11 antimicrobials using the agar dilution method. CA-MRSA isolates were analysed for Panton-Valentine leucocidin (PVL) genes, and staphylococcal cassette chromosome mec (SCCmec) typing was performed. The largest proportion (94.5%) of strains were resistant to penicillin, followed by erythromycin (86.2%) and clindamycin (69.6%). In total 772 of 984 (78.5%) S. aureus strains were multiresistant. The incidence of CA-MRSA was 1.1%, with a high rate of resistance to clindamycin (90.9%) and tetracycline (72.7%), but all were susceptible to ciprofloxacin. The susceptibility profiles of MRSA to other antimicrobial agents were similar to those of methicillin-sensitive S. aureus (MSSA). None of the S. aureus strains were resistant to vancomycin and fusidic acid; moreover, only one strain was resistant to mupirocin. Typing of the SCCmec showed that 54.5% were type IV, 18.2% were type V and 9.1% were type VI. All the PVL-positive CA-MRSA carried SCCmec type IV. CA-MRSA is still relatively uncommon and heterogeneous in children in China. Penicillin and erythromycin are no longer appropriate agents. Effective antibiotic agents for patients with impetigo are mupirocin and fusidic acid.

Community-associated Staphylococcus aureus infections and nasal carriage among children: molecular microbial data and clinical characteristics

An increasing number of infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying the Panton-Valentine leukocidin (PVL) genes was recently identified in Greece. In the present study, 170 patients with S. aureus infections and 123 uninfected children (<15 years old) who had been tested for nasal carriage were evaluated during a 2-year period. The MecA, PVL and superantigen family genes, and MRSA clones, were investigated by molecular methods. Sites of infection and laboratory findings for patients were recorded. The results were compared and statistically analysed. Among 123 uninfected children 73 (59%) carried S. aureus, including four MRSA strains. Of these, three MRSA and three methicillin-sensitive S. aureus (MSSA) strains were PVL-positive (p <0.0001). Ninety-six patients (96/170) exhibited skin and soft-tissue infections (SSTIs), and 74 exhibited invasive infections. The incidence of staphylococcal infections increased during July to September each year. In total, 110 S. aureus isolates were PVL-positive (81 from SSTIs and 29 from invasive infections, p <0.0001). Ninety-nine out of 106 MRSA (93%) isolates from 170 patients carried the PVL genes (p <0.0001); 97 belonged to the clonal complex CC80. Leukocyte and polymorphonuclear cell counts were higher among children with MRSA infections (p <0.005). MSSA predominated among patients with invasive infections (43/74), and carried mainly genes of the superantigen family. Children <5 years of age showed a higher risk of MRSA infection. The present study demonstrates that infections due to PVL-positive CA-MRSA spread easily among children, and SSTIs can lead to invasive infections. Nasal colonization may be an additional factor contributing to the emergence of CA-MRSA.

Contribution of Panton-Valentine Leukocidin in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Δpvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis.

Community-associated methicillin-resistant and methicillin-sensitive Staphylococcus aureus: skin and soft tissue infections in Hong Kong

This prospective study assessed the epidemiology of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among patients with purulent skin and soft tissue infections (SSTIs) in Hong Kong. Among 298 patients with SSTIs, 10.4% (13/125) of all S. aureus isolates and 5% (12/241) of all abscesses were attributed to pvl-positive CA-MRSA. Overall, 77% and 69.9% of CA-MRSA and methicillin-sensitive S. aureus (MSSA) were susceptible to erythromycin, 77% and 74.8% to clindamycin, 100% and 97.1% to minocycline, and 100% and 98.1% to rifampin, respectively. Filipino ethnicity was the only clinical and epidemiologic factor significantly associated with CA-MRSA infection (odds ratio, 14.8; 95% confidence interval, 3.3–70.0; P < 0.001). Pulsed-field gel electrophoresis analysis showed that 6 CA-MRSA isolates belonged to the ST30-HKU100 clone, 5 belonged to the ST59-HKU200 clone, and 1 was singleton. Features of HKU100 isolates include SCC mec type IV, agr3, spa t019, and pan-susceptibility to non–β-lactam antibiotics. In contrast, HKU200 isolates are characterized by having SCC mec type IV or V, agr4, spa t437, and variable non–β-lactam susceptibility profiles. The major CA-MRSA spa types were shared by a minority of the MSSA.