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Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

ABSTRACT: Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates. This assay facilitates the rapid identification of PVL-positive isolates of MRSA.

SUBMITTER: McDonald RR

PROVIDER: S-EPMC1317231 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Development of a triplex real-time PCR assay for detection of Panton-Valentine leukocidin toxin genes in clinical isolates of methicillin-resistant Staphylococcus aureus.

McDonald Ryan R RR Antonishyn Nick A NA Hansen Toni T Snook Laelie A LA Nagle Evelyn E Mulvey Michael R MR Levett Paul N PN Horsman Greg B GB

Journal of clinical microbiology 20051201 12

Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates. This assay facilitates the rapid identification of PVL-positive isolates of MRSA. ...[more]

PMID: 16333116

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Global distribution of Panton-Valentine leukocidin--positive methicillin-resistant Staphylococcus aureus, 2006.

Project description:We determined the agr type, multilocus sequence type, protein A gene type (spa typing), toxin gene profile, and antimicrobial drug resistance profile of 469 isolates of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus isolates (PVL-positive CA-MRSA). The isolates had been collected from around the world from 1999 through 2005 by the French National Reference Center for Staphylococci. We found that some continent-specific clones described in 2003, such as clone ST8, have now spread all over the world. Likewise, some PVL-positive CA-MRSA have spread to several countries on various continents. New clones have emerged (e.g., ST377) on new genetic backgrounds. PVL-positive CA-MRSA that were usually susceptible to most antistaphylococcal antimicrobial agents have acquired new resistance determinants (e.g., to gentamicin) in certain countries. The major trait shared by all these clones is a short staphylococcal chromosomal cassette mec element of type IV or V.

| S-EPMC2725977 | biostudies-literature

Panton-Valentine leukocidin-positive Staphylococcus aureus, Singapore.

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Contribution of Panton-Valentine leukocidin in community-associated methicillin-resistant Staphylococcus aureus pathogenesis.

Project description: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 (Delta pvl) 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.

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Molecular Detection of Panton Valentine Leukocidin Toxin in Clinical Isolates of Staphylococcus aureus from Kiambu County, Kenya.

Project description:Panton-Valentine leukocidin gene is produced by Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus isolates as a pore-forming toxin is largely responsible for skin and soft tissue illnesses. MRSA produces PVL toxins through lukS and lukF proteins causing tissue necrosis by damaging membrane of the defense cells. Presence of PVL toxin was tested from the 54 S. aureus clinical isolates obtained from Thika and Kiambu Level 5 Hospitals, in Kiambu County, Kenya, by Geno Type® MRSA assay (Hain Life Science, Nehren, Germany). DNA was isolated from freshly harvested bacterial cultures by spin column using Geno Type DNA isolation kit. The detection of PVL toxins was performed by amplification of genomic DNA and by reverse hybridization that identifies PVL genes using Geno Type MRSA kit. Out of 138 samples that were collected from patients in Kiambu County, 54 S. aureus isolates were obtained, of which 14 (25.9%; 95% CI?=?11.9-38.9) samples had PVL toxins. The isolates that were obtained from the female patients had a higher PVL toxin prevalence of 35.7%, while the isolates collected from the male patients had a lower prevalence of 15.4% (P = 0.09). The pediatrics department had the highest PVL gene prevalence compared to outpatient department and surgical units (P = 0.08). However, the age groups of patients and the hospital attended by patients showed no significant difference in terms of PVL gene prevalence (P = 0.26). Therefore, the patients' gender and hospital units were not significantly associated with PVL gene prevalence (P = 0.08). This study shows that PVL positive isolates occur in the sampled hospitals in the county and female as well as children must be taken into consideration among patients with wound infections when isolating S. aureus.

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Monomicrobial Fournier's Gangrene Caused by Panton-Valentine Leukocidin-negative Methicillin-susceptible Staphylococcus aureus ST8 in Japan.

Project description:Methicillin-resistant Staphylococcus aureus USA300, belonging to sequence type (ST) 8, is a rare cause of necrotizing fasciitis in the USA. We herein report a case of monomicrobial Fournier's gangrene caused by an ST8, methicillin-susceptible Staphylococcus aureus (designated ksw1). Whole-genome sequencing and analyses for virulence determinants revealed that, unlike USA300, ksw1 lacked virulence genes, such as Panton-Valentine leukocidin and SCCmec, while harboring the toxic shock syndrome toxin-1 gene. These genomic features correlate with ST8 CA-MRSA/J, which is the major genotype of ST8 in Japan.

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Distinct bacteriophages encoding Panton-Valentine leukocidin (PVL) among international methicillin-resistant Staphylococcus aureus clones harboring PVL.

Project description:Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (?PVL, ?108PVL, ?SLT, ?Sa2MW, ?Sa2USA, and ?Sa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ?Sa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains.

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Basis of virulence in a Panton-Valentine leukocidin-negative community-associated methicillin-resistant Staphylococcus aureus strain.

Project description:Community-associated (CA) infections with methicillin-resistant Staphylococcus aureus (MRSA) are on a global rise. However, analysis of virulence characteristics has been limited almost exclusively to the US endemic strain USA300. CA-MRSA strains that do not produce Panton-Valentine leukocidin (PVL) have not been investigated on a molecular level. Therefore, we analyzed virulence determinants in a PVL-negative CA-MRSA strain, ST72, from Korea. Genome-wide analysis identified 3 loci that are unique to that strain, but did not affect virulence. In contrast, phenol-soluble modulins (PSMs) and the global virulence regulator Agr strongly affected lysis of neutrophils and erythrocytes, while ?-toxin and Agr had a major impact on in vivo virulence. Our findings substantiate the general key roles these factors play in CA-MRSA virulence. However, our analyses also showed noticeable differences to strain USA300, inasmuch as ?-toxin emerged as a much more important factor than PSMs in experimental skin infection caused by ST72.

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Prevalence of Panton-Valentine leukocidin in methicillin-resistant Staphylococcus aureus clinical isolates at a university hospital in Northern Cyprus: a pilot study.

Project description:OBJECTIVE:Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) is a healthcare problem worldwide. There are no reports on the virulence characteristics of MRSA in Northern Cyprus (NC). This study aimed to determine the presence of pvl among MRSA isolates from patients admitted to a university hospital in NC using molecular methods. Fifty S. aureus strains were included in this study. BD Phoenix automated identification system was used for bacterial identification and antibiotic susceptibility testing. Methicillin resistance was confirmed by disc diffusion assay. Presence of nuc and mecA genes was tested by multiplex PCR. Detection of pvl gene was performed by single-target PCR. RESULTS:Out of 50 S. aureus isolates identified as MRSA by BD Phoenix system, 3 were susceptible to cefoxitin with disc diffusion assay and were confirmed as methicillin-sensitive S. aureus (MSSA). All isolates (n?=?50, 100%) tested positive for the presence nuc gene and 68% (n?=?34/50) were mecA positive. pvl was detected in 27.7% (n?=?13/47) of the MRSA isolates. Among PVL-positive MRSA isolates, 69.2% (9/13) were inpatients. PVL-MRSA was more common in isolates from deep tracheal aspirate (30.8%, 4/13) and abscess/wound (23.1%, 3/13). This represents the first study of PVL presence among MRSA in hospital setting in NC.

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Detection of Panton-valentine Leukocidin Gene Isoforms of Staphylococcus aureus Isolates in Al-Zahra Hospital, Isfahan-Iran.

Project description:Panton-Valentine leukocidin (PVL) is a gamma-toxin produced by Staphylococcus aureus encoded by genes lukS/lukF-PV with several single-nucleotide polymorphisms. A mutation at nucleotide position 527 results in substitution of histidine (H) to arginine (R) at amino acid 176. The groups defined based on the amino acid change, the "R isoform" group and the "H isoform" group. The purpose of this study was to determine the frequency of PVL gene isoforms in S. aureus strains isolated from patients at Al-Zahra Hospital Isfahan and molecular characterization of PVL-positive methicillin-resistant S. aureus (MRSA) strains including the detection of mecA gene and staphylococcal chromosomal cassette mec (SCCmec) typing.In this study, 130 isolates of S. aureus were collected from Al-Zahra Hospital. The PVL gene identified using polymerase chain reaction (PCR); PCR products were sequenced to identify the type of isoform. The molecular characterization of isolates of PVL-positive MRSA including detection of mecA gene by PCR and also SCCmec typing was performed by multiplex PCR.Out of 130 isolates, 23% were positive for the presence of PVL genes. The PVL positive isolates were comprised 37% (11/30) of methicillin-resistant isolates and 63% (19/30) of methicillin-susceptible S. aureus (MSSA) isolates. The results showed that 17 isolated carrying isoform H and 13 isolated carrying the R isoform.The PVL gene was predominantly found in MSSA isolates. There was no relation between PVL isoforms and the presence of mecA and SCCmec types.

| S-EPMC5549547 | biostudies-other

Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.

Project description:Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

| S-EPMC2711303 | biostudies-literature

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