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Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation.

SUBMITTER: Sung K

PROVIDER: S-EPMC8800443 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

Sung Kidon K Gudeta Dereje D DD Park Miseon M Chon Jungwhan J Kim Sungguk S Nawaz Mohamed S MS Foley Steven L SL Khan Saeed A SA

Microbiology resource announcements 20220127 2

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation. ...[more]

PMID: 35084222

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WGS of Panton-Valentine leucocidin methicillin-resistant Staphylococcus aureus

Project description:WGS of Panton-Valentine leucocidin methicillin-resistant Staphylococcus aureus

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Differential distribution and expression of Panton-Valentine leucocidin among community-acquired methicillin-resistant Staphylococcus aureus strains.

Project description:Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging threat worldwide. CA-MRSA strains differ from hospital-acquired MRSA strains in their antibiotic susceptibilities and genetic backgrounds. Using several genotyping methods, we clearly define CA-MRSA at the genetic level and demonstrate that the prototypic CA-MRSA strain, MW2, has spread as a homogeneous clonal strain family that is distinct from other CA-MRSA strains. The Panton-Valentine leucocidin (PVL)-encoding genes, lukF and lukS, are prevalent among CA-MRSA strains and have previously been associated with CA-MRSA infections. To better elucidate the role of PVL in the pathogenesis of CA-MRSA, we first analyzed the distribution and expression of PVL among different CA-MRSA strains. Our data demonstrate that PVL genes are differentially distributed among CA-MRSA strains and, when they are present, are always transcribed, albeit with strain-to-strain variability of transcript levels. To directly test whether PVL is critical for the pathogenesis of CA-MRSA, we evaluated the lysis of human polymorphonuclear leukocytes (PMNs) during phagocytic interaction with PVL-positive and PVL-negative CA-MRSA strains. Unexpectedly, there was no correlation between PVL expression and PMN lysis, suggesting that additional virulence factors underlie leukotoxicity and, thus, the pathogenesis of CA-MRSA.

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A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin.

Project description:BackgroundStaphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern.ResultsThe present study focused on the development of a pentaplex PCR assay for the rapid detection of MRSA. The assay simultaneously detected five genes, namely 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, lukS that encodes production of Panton-Valentine leukocidin (PVL), a necrotizing cytotoxin, and one internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity and specificity of the pentaplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the pentaplex PCR at the DNA level was found to be 10 ng DNA. The analytical specificity was evaluated with 34 reference staphylococci and non-staphylococcal strains and was found to be 100%. The diagnostic evaluation of MRSA carried out using 230 clinical isolates, showed 97.6% of sensitivity, 99.3% of specificity, 98.8% of positive predictive value and 98.6% of negative predictive value compared to the conventional method. The presence of an internal control in the pentaplex PCR assay is important to exclude false-negative cases.ConclusionThe pentaplex PCR assay developed was rapid and gave results within 4 h, which is essential for the identification of Staphylococcus spp., virulence and their resistance to methicillin. Our PCR assay may be used as an effective surveillance tool to survey the prevalence of MRSA and PVL-producing strains in hospitals and the community.

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Nasal carriage of multi-drug resistant Panton-Valentine leucocidin-positive methicillin-resistant Staphylococcus aureus in children in Tripoli-Libya.

Project description:Methicillin-resistant Staphylococcus aureus (MRSA) colonized children are at an increased risk of developing infections than methicillin-sensitive S. aureus colonized children. Nasal specimens from inpatient children, mothers of inpatient children, healthcare workers, and outpatient children at Tripoli Children Hospital (TCH) were examined for MRSA by chromogenic MRSA ID medium. Susceptibility of MRSA isolates to antibiotics was determined by the disc diffusion method. The nasal carriage rate of MRSA among inpatient children (8.3%, 24 of 289), their mothers (11%, 22 of 200), and healthcare workers (12.4%, 22 of 178) was significantly higher than among outpatient children (2.2%, 2 of 91) (P < 0.05, P < 0.02, and P < 0.006, respectively). Of the examined MRSA isolates (N = 35) 10 (28.6%) were positive for Panton-Valentine leucocidin genes by polymerase chain reaction. Multidrug resistance was found in 24.3% (17 of 70) of MRSA isolates. Nasal carriage of multidrug-resistant Panton-Valentine leucocidin-positive MRSA is not uncommon among inpatient children and their mothers in Tripoli.

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Molecular characterization and panton-valentine leucocidin typing of community-acquired methicillin-sensitive Staphylococcus aureus clinical isolates.

Project description:Limited comprehensive molecular typing data exist currently for Panton-Valentine leucocidin (PVL)-positive, methicillin-sensitive Staphylococcus aureus (PVL-MSSA) clinical isolates. Characterization of PVL-MSSA isolates by multilocus sequence typing (MLST) and spa typing in this study showed a genetic similarity to PVL-positive, methicillin-resistant S. aureus (PVL-MRSA) strains, although three novel spa types and a novel MLST (ST1518) were detected. Furthermore, the detection of PVL phages and haplotypes in PVL-MSSA identical to those previously found in PVL-MRSA isolates highlights the role these strains may play as precursors of emerging lineages of clinical significance.

| S-EPMC3421788 | biostudies-literature

Bioguided Isolation of Active Compounds from Rhamnus alaternus against Methicillin-Resistant Staphylococcus aureus (MRSA) and Panton-Valentine Leucocidin Positive Strains (MSSA-PVL).

Project description:Despite intensified efforts to develop an effective antibiotic, S. aureus is still a major cause of mortality and morbidity worldwide. The multidrug resistance of bacteria has considerably increased the difficulties of scientific research and the concomitant emergence of resistance is to be expected. In this study we have investigated the in vitro activity of 15 ethanol extracts prepared from Moroccan medicinal plants traditionally used for treatment of skin infections. Among the tested species I. viscosa, C. oxyacantha, R. tinctorum, A. herba alba, and B. hispanica showed moderate anti-staphylococcal activity. However, R. alaternus showed promising growth-inhibitory effects against specific pathogenic bacteria especially methicillin-susceptible Staphylococcus aureus Panton-Valentine leucocidin positive (MSSA-PVL) and methicillin-resistant S. aureus (MRSA). The bioguided fractionation of this plant using successive chromatographic separations followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS) including EIMS and HREIMS analysis yielded the emodin (1) and kaempferol (2). Emodin being the most active with MICs ranging between 15.62 and 1.95 µg/mL and showing higher activity against the tested strains in comparison with the crude extract, its mechanism of action and the structure-activity relationship were interestingly discussed. The active compound has not displayed toxicity toward murine macrophage cells. The results obtained in the current study support the traditional uses of R. alaternus and suggest that this species could be a good source for the development of new anti-staphylococcal agents.

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Flow cytometric determination of Panton-Valentine leucocidin S component binding.

Project description:The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (Kd = 0.07 +/- 0.02 nM, n = 5) and monocytes (Kd = 0.020 +/- 0.003 nM, n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

| S-EPMC98170 | biostudies-literature

Panton-Valentine leucocidin expression by Staphylococcus aureus exposed to common antibiotics.

Project description:ObjectivesWe set out to investigate the impact of common antibiotics on Panton-Valentine Leucocidin (PVL) expression by methicillin-sensitive Staphylococcus aureus (MSSA). PVL expression by methicillin-resistant S. aureus (MRSA) is reportedly enhanced by β-lactams, but inhibited by protein-synthesis inhibitors, a fact that has influenced management of infections associated with PVL. Although PVL is more frequently associated with MSSA than MRSA in the UK, the effect of antibiotics on PVL expression by MSSA has not been fully addressed.MethodsMSSA was cultured in vitro with varying concentrations of flucloxacillin, clindamycin or linezolid and PVL expression measured by qRT-PCR and Western blotting. A murine MSSA abscess model was developed to measure leucocidin expression in vivo following antibiotic treatment.Results9% (27/314) of MSSA isolates from patients with uncomplicated community skin/soft tissue infections were positive for PVL genes (lukFS-PV). PVL expression by MSSA in broth was unaffected by varying concentrations of flucloxacillin, clindamycin or linezolid. In a murine abscess model, treatment with flucloxacillin did, however, enhance in vivo MSSA lukF-PV transcription and this was sustained even when flucloxacillin was combined with clindamycin, or clindamycin plus linezolid. Notwithstanding increased leucocidin transcription, functional leucotoxin activity was not enhanced. Treatment with flucloxacillin plus clindamycin significantly decreased leucotoxin activity, but the addition of a second protein synthesis inhibitor, linezolid, did not confer benefit.ConclusionsOur results suggest flucloxacillin in combination with a single protein-synthesis inhibitor such as clindamycin would give the best treatment outcome.

| S-EPMC4535317 | biostudies-literature

Genomic characterization of a unique Panton-Valentine leucocidin-positive community-associated methicillin-resistant <i>Staphylococcus aureus</i> lineage increasingly impacting on Australian indigenous communities.

Project description:In 2010 a single isolate of a trimethoprim-resistant multilocus sequence type 5, Panton-Valentine leucocidin-positive, community-associated methicillin-resistant Staphylococcus aureus (PVL-positive ST5 CA-MRSA), colloquially named WA121, was identified in northern Western Australia (WA). WA121 now accounts for ~14 % of all WA MRSA infections. To gain an understanding of the genetic composition and phylogenomic structure of WA121 isolates we sequenced the genomes of 155 WA121 isolates collected 2010-2021 and present a detailed genomic description. WA121 was revealed to be a single clonally expanding lineage clearly distinct from sequenced ST5 strains reported outside Australia. WA121 strains were typified by the presence of the distinct PVL phage φSa2wa-st5, the recently described methicillin resistance element SCCmecIVo carrying the trimethoprim resistance (dfrG) transposon Tn4791, the novel β-lactamase transposon Tn7702 and the epidermal cell differentiation inhibitor (EDIN-A) plasmid p2010-15611-2. We present evidence that SCCmecIVo together with Tn4791 has horizontally transferred to Staphylococcus argenteus and evidence of intragenomic movement of both Tn4791 and Tn7702. We experimentally demonstrate that p2010-15611-2 is capable of horizontal transfer by conjugative mobilization from one of several WA121 isolates also harbouring a pWBG749-like conjugative plasmid. In summary, WA121 is a distinct and clonally expanding Australian PVL-positive CA-MRSA lineage that is increasingly responsible for infections in indigenous communities in northern and western Australia. WA121 harbours a unique complement of mobile genetic elements and is capable of transferring antimicrobial resistance and virulence determinants to other staphylococci.

| S-EPMC10763498 | biostudies-literature

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

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