Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Injury of Macrophages Induced by Clostridium perfringens Type C Exotoxins.

Clostridium perfringens is a kind of anaerobic Gram-positive bacterium that widely exists in the intestinal tissue of humans and animals. And the main virulence factor in Clostridium perfringens is its exotoxins. Clostridium perfringens type C is the main strain of livestock disease, its exotoxins can induce necrotizing enteritis and enterotoxemia, which lead to the reduction in feed conversion, and a serious impact on breeding production performance. Our study found that treatment with exotoxins reduced cell viability and triggered intracellular reactive oxygen species (ROS) in human mononuclear leukemia cells (THP-1) cells. Through transcriptome sequencing analysis, we found that the levels of related proteins such as heme oxygenase 1 (HO-1) and ferroptosis signaling pathway increased significantly after treatment with exotoxins. To investigate whether ferroptosis occurred after exotoxin treatment in macrophages, we confirmed that the protein expression levels of antioxidant factors glutathione peroxidase 4/ferroptosis-suppressor-protein 1/the cystine/glutamate antiporter solute carrier family 7 member 11 (GPX4/FSP1/xCT), ferroptosis-related protein nuclear receptor coactivator 4/transferrin/transferrin receptor (NCOA4/TF/TFR)/ferritin and the level of lipid peroxidation were significantly changed. Based on the above results, our study suggested that Clostridium perfringens type C exotoxins can induce macrophage injury through oxidative stress and ferroptosis.

Toxin-triggered liposomes for the controlled release of antibiotics to treat infections associated with the gram-negative bacterium, Aggregatibacter actinomycetemcomitans.

Antibiotic resistance has become an urgent threat to health care in recent years. The use of drug delivery systems provides advantages over conventional administration of antibiotics and can slow the development of antibiotic resistance. In the current study, we developed a toxin-triggered liposomal antibiotic delivery system, in which the drug release is enabled by the leukotoxin (LtxA) produced by the Gram-negative pathogen, Aggregatibacter actinomycetemcomitans. LtxA has previously been shown to mediate membrane disruption by promoting a lipid phase change in nonlamellar lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE). In addition, LtxA has been observed to bind strongly and nearly irreversibly to membranes containing large amounts of cholesterol. Here, we designed a liposomal delivery system composed of N-methyl-DOPE and cholesterol to take advantage of these interactions. Specifically, we hypothesized that liposomes composed of N-methyl-DOPE and cholesterol, encapsulating antibiotics, would be sensitive to LtxA, enabling controlled antibiotic release. We observed that liposomes composed of N-methyl-DOPE were sensitive to the presence of low concentrations of LtxA, and cholesterol increased the extent and kinetics of content release. The liposomes were stable under various storage conditions for at least 7 days. Finally, we showed that antibiotic release occurs selectively in the presence of an LtxA-producing strain of A. actinomycetemcomitans but not in the presence of a non-LtxA-expressing strain. Together, these results demonstrate that the designed liposomal vehicle enables toxin-triggered delivery of antibiotics to LtxA-producing strains of A. actinomycetemcomitans.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Membrane Interaction Characteristics of the RTX Toxins and the Cholesterol-Dependence of Their Cytolytic/Cytotoxic Activity.

RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria. They are secreted as water-soluble proteins that are able to bind to the host cell membrane and insert hydrophobic segments into the lipid bilayer that ultimately contribute to the formation of transmembrane pores. Ion diffusion through these pores leads then to cytotoxic and cytolytic effects on the hosts. Several reports have evidenced that the binding of several RTX toxins to the target cell membrane may take place through a high-affinity interaction with integrins of the ß2 family that is highly expressed in immune cells of the myeloid lineage. However, at higher toxin doses, cytotoxicity by most RTX toxins has been observed also on ß2-deficient cells in which toxin binding to the cell membrane has been proposed to occur through interaction with glycans of glycosylated lipids or proteins present in the membrane. More recently, cumulative pieces of evidence show that membrane cholesterol is essential for the mechanism of action of several RTX toxins. Here, we summarize the most important aspects of the RTX toxin interaction with the target cell membrane, including the cholesterol dependence, the recent identification in the sequences of several RTX toxins of linear motifs coined as the Cholesterol Recognition/interaction Amino acid Consensus (CRAC), and the reverse or mirror CARC motif, which is involved in the toxin-cholesterol interaction.

Significant role of pyocyanin and exotoxin A in the pathogenesis of Pseudomonas aeruginosa isolated from hospitalized patients.

AIM: Due to the importance of exotoxin A and pyocyanin in the pathogenicity of this bacterium, we decided to evaluate the prevalence of genes encoding these virulence factors in clinical isolates of P.aeruginosa.

Antimicrobial Resistance and the Prevalence of the Panton-Valentine Leukocidin Gene among Clinical Isolates of Staphylococcus aureus in Lithuania.

This study aimed to determine resistance to antimicrobials of Staphylococcus aureus strains isolated from clinical specimens in Lithuanian hospitals and to identify the genes conferring resistance and virulence. The study was carried out from June 2019 to September 2021. S. aureus strains were isolated from skin, soft tissues, blood, lower respiratory tract, urine and other specimens. Antibiotic susceptibility testing was performed using the disc diffusion method according to EUCAST guidelines. All isolates were analyzed for detection of the ermA, ermC, mecA, mecC, tetK, tetM, and lukF-PV genes by multiplex real-time PCR. The 16S rRNA coding sequence was applied as an internal PCR control. Altogether, 745 S. aureus strains were analyzed. Antimicrobial susceptibility testing revealed that all isolates were susceptible to rifampin and vancomycin. Of the 745 strains, 94.8% were susceptible to tetracycline, 94.5% to clindamycin, and 88.3% to erythromycin. The lowest susceptibility rate was found for penicillin (25.8%). Six percent of the tested strains were methicillin-resistant S. aureus (MRSA). The majority of methicillin-resistant strains were isolated from skin and soft tissues (73.3%), with a smaller portion isolated from blood (17.8%) and respiratory tract (8.9%). The ermC gene was detected in 41.1% of erythromycin-resistant S. aureus strains, whereas ermA was detected in 32.2% of erythromycin-resistant S. aureus strains. 69.2% of tetracycline-resistant S. aureus strains had tetK gene, and 28.2% had tetM gene. 7.3% of S. aureus isolates harbored lukF-PV gene. The frequency of the pvl gene detection was significantly higher in MRSA isolates than in methicillin-susceptible S. aureus isolates (p < 0.0001).

Dandelion-derived vesicles-laden hydrogel dressings capable of neutralizing Staphylococcus aureus exotoxins for the care of invasive wounds.

Delayed wound healing caused by bacterial infection remains a major challenge in clinical treatment. Exotoxins incorporated in bacterial extracellular vesicles play a key role as the disease-causing virulence factors. Safe and specific antivirulence agents are expected to be developed as an effective anti-bacterial infection strategy, instead of single antibiotic therapy. Plant-derived extracellular vesicle-like nanoparticles have emerged as promising therapeutic agents for skin diseases, but the elucidations of specific mechanisms of action and clinical transformation still need to be advanced. Here, dandelion-derived extracellular vesicle-like nanoparticles (TH-EVNs) are isolated and exert antivirulence activity through specifically binding to Staphylococcus aureus (S. aureus) exotoxins, thereby protecting the host cell from attack. The neutralization of TH-EVNs against exotoxins has considerable binding force and stability, showing complete detoxification effect in vivo. Then gelatin methacryloyl hydrogel is developed as TH-EVNs-loaded dressing for S. aureus exotoxin-invasive wounds. Hydrogel dressings demonstrate good physical and mechanical properties, thus achieving wound retention and controlled release of TH-EVNs, in addition to promoting cell proliferation and migration. In vivo results show accelerated re-epithelialization, promotion of collagen maturity and reduction of inflammation after treatment. Collectively, the developed TH-EVNs-laden hydrogel dressings provide a potential therapeutic approach for S. aureus exotoxin- associated trauma.

Epidemiology and clinical features of Skin and Soft Tissue Infections Caused by PVL-Positive and PVL-Negative Methicillin-Resistant Staphylococcus aureus Isolates in inpatients in China: a single-center retrospective 7-year study.

Previous studies have mainly focused on outpatient cases of skin and soft tissue infections (SSTIs), with limited attention to inpatient occurrences. Thus, we aimed to compare the clinical parameters of inpatients with SSTIs, performed genomic characterization, and determined the subtypes of Panton-Valentine leucocidin (PVL) bacteriophages of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from these patients. We found that PVL-positive patients had shorter hospital stays (mean, 9 vs. 24 days; p < 0.001) and abscess resolution durations (mean, 8 vs. 13 days; p < 0.01). PVL-positive MRSA-induced SSTIs were more frequently associated with abscesses [36/55 (65.5%) vs. 15/124 (12.1%), p < 0.001], with 52.7% undergoing incision and drainage; over 80% of PVL-negative patients received incision, drainage, and antibiotics. In PVL-positive patients receiving empirical antibiotics, anti-staphylococcal agents such as vancomycin and linezolid were administered less frequently (32.7%, 18/55) than in PVL-negative patients (74.2%, 92/124), indicating that patients with PVL-positive SSTIs are more likely to require surgical drainage rather than antimicrobial treatment. We also found that the ST59 lineage was predominant, regardless of PVL status (41.3%, 74/179). Additionally, we investigated the linear structure of the lukSF-PV gene, revealing that major clusters were associated with specific STs, suggesting independent acquisition of PVL by different strain types and indicating that significant diversity was observed even within PVL-positive strains detected in the same facility. Overall, our study provides comprehensive insights into the clinical, genetic, and phage-related aspects of MRSA-induced SSTIs in hospitalized patients and contributes to a more profound understanding of the epidemiology and evolution of these pathogens in the Chinese population.

2B4: A potential target in Staphylococcus aureus associated allergic inflammation.

Staphylococcus aureus (SA) and its exotoxins activate eosinophils (Eos) and mast cells (MCs) via CD48, a GPI-anchored receptor belonging to the signaling lymphocytes activation molecules (SLAM) family. 2B4 (CD244), an immuno-regulatory transmembrane receptor also belonging to the SLAM family, is the high-affinity ligand for CD48. 2B4 is expressed on several leukocytes including NK cells, T cells, basophils, monocytes, dendritic cells (DCs), and Eos. In the Eos and MCs crosstalk carried out by physical and soluble interactions (named the 'allergic effector unit', AEU), 2B4-CD48 binding plays a central role. As CD48 and 2B4 share some structural characteristics and SA colonization accompanies most of the allergic diseases, we hypothesized that SA exotoxins (e.g. Staphylococcus enterotoxin B, SEB) can also bind and activate 2B4 and thereby possibly further aggravate inflammation. To check our hypothesis, we used in vitro, in silico, and in vivo methods. By enzyme-linked immunosorbent assay (ELISA), flow cytometry (FC), fluorescence microscopy, and microscale thermophoresis, we have shown that SEB can bind specifically to 2B4. By Eos short- and long-term activation assays, we confirmed the functionality of the SEB-2B4 interaction. Using computational modeling, we identified possible SEB-binding sites on human and mouse 2B4. Finally, in vivo, in an SEB-induced peritonitis model, 2B4-KO mice showed a significant reduction of inflammatory features compared with WT mice. Altogether, the results of this study confirm that 2B4 is an important receptor in SEB-mediated inflammation, and therefore a role is suggested for 2B4 in SA associated inflammatory conditions.

Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL + -Staphylococcus aureus infections.

Staphylococcus aureus strains that produce the toxin Panton-Valentine leukocidin (PVL-SA) frequently cause recurrent skin and soft tissue infections. PVL binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs), but the pathomechanism has not been extensively studied. Furthermore, it is unclear why some individuals colonized with PVL-SA experience recurring infections whereas others are asymptomatic. We thus aimed to (1) investigate how PVL exerts its pathogenicity on neutrophils and (2) identify factors that could help to explain the predisposition of patients with recurring infections. We provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH oxidase and reactive oxygen species production. Moreover, through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, and as such they were unable to kill S. aureus. Furthermore, we found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to be killed at a low PVL concentration than control neutrophils. Neutrophils from patients that experience recurring PVL-positive infections may thus be more sensitive to PVL-induced NET formation, which might impair their ability to combat the infection.

Interaction of zincite, alpha-terpineol, geranyl acetate, linalool, myrcenol, terpinolene, and thymol with virulence factors of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Staphylococcus aureus.

BACKGROUND: Based on gas chromatography - mass spectrometry (GC-MS) results of a previous study, six metabolites including alpha-terpineol, geranyl acetate, linalool, myrcenol, terpinolene, and thymol showed significantly higher amounts relative to other metabolites. METHODS: A continuation of the previous study, the interaction of these metabolites with the main virulence factors of P. aeruginosa (pseudomonas elastase and exotoxin A), Staphylococcus aureus (alpha-hemolysin and protein 2a), Mycobacterium tuberculosis (ESX-secreted protein B and the serine/threonine protein kinase), and Escherichia coli (heat-labile enterotoxin and Shiga toxin) were evaluated by molecular docking study and molecular simulation. RESULTS: In the case of Shiga toxin, higher and lower binding affinities were related to alpha-terpinolene and zincite with values of -5.8 and -2.6 kcal/mol, respectively. For alpha-hemolysin, terpinolene and alpha-terpinolene demonstrated higher binding affinities with similar energies of -5.9 kcal/mol. Thymol and geranyl acetate showed lower binding energy of -5.7 kcal/mol toward protein 2a. Furthermore, thymol had a higher binding affinity toward heat-labile enterotoxin and ESX-secreted protein B with values of -5.9 and -6.1 kcal/mol, respectively. CONCLUSIONS: It is concluded that the availability of secondary metabolites of A. haussknechtii surrounding zinc oxide (ZnO) NPs can hinder P. aeruginosa by inactivating Pseudomonas elastase and exotoxin.

Spatiotemporally controlled Pseudomonas exotoxin transgene system combined with multifunctional nanoparticles for breast cancer antimetastatic therapy.

The tumor microenvironment is a barrier to breast cancer therapy. Cancer-associated fibroblast cells (CAFs) can support tumor proliferation, metastasis, and drug resistance by secreting various cytokines and growth factors. Abnormal angiogenesis provides sufficient nutrients for tumor proliferation. Considering that CAFs express the sigma receptor (which recognizes anisamide, AA), we developed a CAFs and breast cancer cells dual-targeting nano drug delivery system to transport the LightOn gene express system, a spatiotemporal controlled gene expression consisting of a light-sensitive transcription factor and a specific minimal promoter. We adopted RGD (Arg-Gly-Asp) to selectively bind to the αvß3 integrin on activated vascular endothelial cells and tumor cells. After the LightOn system has reached the tumor site, LightOn gene express system can spatiotemporal controllably express toxic Pseudomonas exotoxin An under blue light irradiation. The LightOn gene express system, combined with multifunctional nanoparticles, achieved high targeting delivery efficiency both in vitro and in vivo. It also displayed strong tumor and CAFs inhibition, anti-angiogenesis ability and anti-metastasis ability, with good safety. Moreover, it improved survival rate, survival time, and lung metastasis rate in a mouse breast cancer model. This study proves the efficacy of combining the LightOn system with targeted multifunctional nanoparticles in tumor and anti-metastatic therapy and provides new insights into tumor microenvironment regulation.

[Genitoanal infections caused by Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus : Smear infection or sexually transmitted disease?] / Genitoanale Infektionen durch Panton-Valentine-Leukozidin(PVL)-positiven Staphylococcus aureus : Schmierinfektion oder sexuell übertragbare Erkrankung?

Panton-Valentine leukocidin (PVL) is a pore-forming exotoxin produced by certain Staphylococcus (S.) aureus strains, which is responsible for the increased virulence of the pathogen. Thus, infections caused by PVL-positive S. aureus tend to recur. Usually, the infection is a smear infection, which can cause folliculitis and purulent lid margin inflammation in addition to the classic mucocutaneous abscesses. Recently, recurrent genitoanal infections caused by PVL-positive S. aureus have also been described. In most cases, this is a sexually transmitted disease. Currently, it is assumed that most infections are imported from abroad. In addition to treatment of these infections, decolonization should be performed for prophylaxis of recurrence.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. "Toxin-based therapy" targets diseased cells using three strategies. Targeted cancer therapy, like antibody-toxin conjugates, fusion toxins, or "suicide gene therapy", can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection's critical role.

Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin.

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.

Detection of virulence genes of Staphylococcus aureus isolated from raw beef for retail sale in the markets of Ulaanbaatar city, Mongolia.

BACKGROUND: Staphylococcus aureus (S. aureus) is a highly virulent pathogen that causes food-borne illness, food poisoning, skin and soft tissue infections, abscesses, mastitis, and bacteremia. It is common for meat and meat products to become contaminated with S. aureus due to dirty hands, food storage conditions, food production processes, and unhygienic conditions, causing food poisoning. Therefore, we aimed to isolate S. aureus strain from the raw beef and reveal virulence genes and antibiotic resistance profile from isolated S. aureus strains. METHODS: In this study, 100 samples of raw beef were collected from 4 major market stalls in Ulaanbaatar city, Mongolia. S. aureus was detected according to the ISO 6888-1:2021 standard, and the nucA gene encoding the species-specific thermonuclease was amplified and confirmed by polymerase chain reaction (PCR). In the strains of S. aureus isolated from the samples, the genes encoding the virulence factors including sea, sed, tsst, eta, etb, and mecA were amplified by multiplex PCR. These genes are encoded staphylococcal enterotoxin A, enterotoxin D, toxic shock syndrome toxin, exotoxin A, exotoxin B and penicillin-binding protein PBP 2A, respectively. Antibiotic sensitivity test was performed by the Kirby-Bauer disc diffusion method. The Clinical and Laboratory Standard Institute guidelines as CLSI M100-S27 was used for analysis of the data. RESULTS: Thirty-five percent of our samples were detected contaminated with of the S. aureus strains. Subsequently, antibiotic resistance was observed in the S. aureus contaminated samples. Among our samples, the highest rates of resistance were determined against ampicillin (97.1%), oxacillin (88.6%), and penicillin (88.6%), respectively. Three genes including mecA, sea, and tsst from six virulence genes were detected in 17% of S. aureus strain-contaminated samples by multiplex PCR. The sed, etb and eta genes were detected in the 2.9%, 11.4% and 5.7% of our samples, respectively. CONCLUSION: The results show that S. aureus related contamination is high in the raw beef for retail sale and prevalent S. aureus strains are resistant to all antibiotics used. Also, our results have demonstrated that there is a high risk for food poisoning caused by antibiotic resistant S. aureus in the raw beef and it may establish public health issues. Genes encoding for both heat-resistant and nonresistant toxicity factors were detected in the antibiotic resistant S. aureus strains and shown the highly pathogenic. Finally, our study is ensuring to need proper hygienic conditions during beef's preparation and sale.

Significant increase in the prevalence of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus, particularly the USA300 variant ΨUSA300, in the Japanese community.

IMPORTANCE: USA300 is an MRSA clone producing PVL, a toxin associated with SSTIs. ΨUSA300 is a USA300 variant recently identified in Japan by Takadama et al. (15). Here, we found that the prevalence rate of PVL-positive MRSA in S. aureus was elevated in the Japanese community, and ΨUSA300 accounted for most of them. ΨUSA300 strains have been isolated from several areas in Japan and were associated with deep-seated SSTIs. This study highlighted the emerging threat posed by ΨUSA300 in Japan.