Staphylococcal Virulence Research Articles

Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Optimization and Evaluation of Triplex Real-time PCR Assay for Detection of Genes Encoding Staphylococcal Virulence and Methicillin Resistance Using Two Different Multi-channel Emission Instruments

Molecular characterization of staphylococcal cassette chromosome mec and virulence encoding genes in methicillin-resistant staphylococci at a medical center in Lebanon

Background: Methicillin-resistant staphylococci (MRS) are major human pathogens accounting for most hospital-acquired (HA) and community acquired (CA) infections worldwide. The recent increase in MRS in a medical center in Lebanon elicited the determination of SCCmec types, genotypes, and prevalence of Panton-Valentine leucociden (PVL) and toxic shock syndrome toxin-1 (TSST-1) among the MRS isolates. Methods: Thirty-six MRS isolates collected between October 2010 and September 2011 at a medical center, Lebanon were typed using phenotypic and genotypic methods. Antimicrobial susceptibility was determined using the disk diffusion agar method. SCCmec typing was performed by multiplex PCR and sequence analysis. The prevalence of the genes encoding PVL and TSST-1 virulence factors and their transcription levels, were determined respectively by PCR and semi-quantitative real-time PCR. The genomic relatedness of the isolates was assessed by random amplified polymorphic DNA (RAPD) analysis.Results: Antimicrobial susceptibility revealed three distinct antibiotypes. The predominant SCCmec type found among the MRS isolates was type IVa (51%). Twenty-nine percent harbored SCCmec type III and 14% harbored SCCmec type II. One isolate harbored SCCmec type IVc, and another harbored SCCmec type I. All methicillin-resistant Staphylococcus aureus (MRSA) isolates were negative for the gene encoding for PVL, and two were positive for the gene encoding for TSST-1. RAPD analysis demonstrated high genomic diversity among the MRS isolates.Conclusion: This study demonstrated the SCCmec types and the clonality of the MRS strains, allowing the differentiation between HA and CA-MRS strains. CA-MRS have increased in the hospital environment and rendered highly resistant to erythromycin and clindamycin.

Proteomics in studies of Staphylococcus aureus virulence.

Staphylococcus aureus is a widespread, opportunistic pathogen that causes community and hospital acquired infections. Its high pathogenicity is driven by multifactorial and complex mechanisms determined by the ability of the bacterium to express a wide variety of virulence factors. The proteome secreted into extracellular milieu is a rich reservoir of such factors which include mainly nonenzymatic toxins and enzymes. Simultaneously, membrane proteins, membrane-cell wall interface proteins and cell wall-associated proteins also strongly influence staphylococcal virulence. Proteomics shows a great potential in exploring the role of the extracellular proteome in cell physiology, including the pathogenic potential of particular strains of staphylococci. In turn, understanding the bacterial physiology including the interconnections of particular factors within the extracellular proteomes is a key to the development of the ever needed, novel antibacterial strategies. Here, we briefly overview the latest applications of gel-based and gel-free proteomic techniques in the identification of the virulence factors within S. aureus secretome and surfacome. Such studies are of utmost importance in understanding the host-pathogen interactions, analysis of the role of staphylococcal regulatory systems and also the detection of posttranslational modifications emerging as important modifiers of the infection process.

A study of Staphylococcus aureusnasal carriage, antibacterial resistance and virulence factor encoding genes in a tertiary care hospital, Kayseri, Turkey.

This study was to determine the virulence encoding genes, and the antibiotic resistance patterns of the Staphylococcus aureus isolates, which were isolated from the nasal samples of chest clinic patients. The nasal samples of the in-patients (431) and out-patients (1857) in Kayseri Training and Research Hospital's Chest Clinic, Kayseri, Turkey, were cultured on CHROMagar (Biolife, Italiana) S. aureus, and subcultured on sheep blood agar for the isolation of S. aureus. Disc diffusion method was used for antimicrobial susceptibility testing. The occurrence of the staphylococcal virulence encoding genes (enterotoksins [sea, seb, sec, see, seg, seh, sei, sej], fibronectin-binding proteins A, B [fnbA, fnbB], toxic shock syndrome toxin-1 [tst]) were detected by polymerase chain reaction. Forty-five of the 55 (81.8%) S. aureus isolates from inpatients, and 319 (90.6%) isolates from tested 352 out-patient's isolates were suspected to all the antibiotics tested. methicillin-resistant S. aureus (MRSA) was detected in 1.2% of S. aureus isolates. Rifampin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, gentamicin resistance rates were 1.2%, 1.7%, 2.0%, 8.8%, and 1.2%, respectively. The isolates were susceptible to teicoplanin and vancomycin. The genes most frequently found were tst (92.7%), seg (85.8%), sea (83.6%), fnbA (70.9%). There was no statistical significance detected between MRSA and mecA-negative S. aureus isolates in encoding genes distribution (P > 0.05). Our results show that virulence factor encoding genes were prevalent in patients with S. aureus carriage, whereas antibiotic resistance was low. These virulence determinants may increase the risk for subsequent invasive infections in carriers.

Changes in Caenorhabditis elegans immunity and Staphylococcal virulence factors during their interactions

The nematode Caenorhabditis elegans is used as a model system for the study of host-pathogen interactions. Lipoteichoic acid (LTA) is one of the major virulent and immunostimulatory components found in gram positive bacteria. The current study used LTA isolated from Staphylococcus aureus and pathogenic and non-pathogenic Staphylococcus epidermidis. The overall physiological assays revealed that LTA exposed C. elegans show a significant reduction in the life span, production of eggs and progenies. To understand the involvement of innate immune specific players at the mRNA level, the regulation of few candidate antimicrobial genes was studied during Staphylococcal LTA exposures. qPCR analysis indicated an upregulation of antimicrobial peptides during LTA exposures. To understand the involvement of LTA and other virulent genes during infection, the regulation of LTA synthase and a few virulence genes was monitored during host exposure. The qPCR analyses indicated the upregulation of ltaS and other virulence genes (atoxin, sak, ssaA and fbe) during infection. Ability of the pathogens to modify their internal machinery during host presence was monitored by Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltametric analyses. The FTIR results indicated distinct alterations of peaks from Staphylococcal LTA composition between control and the host exposed. Further, EIS and CV data displayed clear differences between the host exposed Staphylococcal samples compared to their respective unexposed controls. The pathogenic and non-pathogenic strains showed different types of regulations and interactions during host exposures. The observed modifications clearly suggest that the Gram positive pathogen changes its LTA production and possibly the structure to cause a severe pathogenic effect on an interacting host.

Flipping the switch: tools for detecting small molecule inhibitors of staphylococcal virulence.

Through the expression of the accessory gene regulator quorum sensing cascade, Staphylococcus aureus is able to produce an extensive array of enzymes, hemolysins and immunomodulators essential to its ability to spread through the host tissues and cause disease. Many have argued for the discovery and development of quorum sensing inhibitors (QSIs) to augment existing antibiotics as adjuvant therapies. Here, we discuss the state-of-the-art tools that can be used to conduct screens for the identification of such QSIs. Examples include fluorescent reporters, MS-detection of autoinducing peptide production, agar plate methods for detection of hemolysins and lipase, High performance liquid chromatography-detection of hemolysins from supernatants, and cell-toxicity assays for detecting damage (or relief thereof) against human keratinocyte cells. In addition to providing a description of these various approaches, we also discuss their amenability to low-, medium-, and high-throughput screening efforts for the identification of novel QSIs.

Correlation Between Serum 25-Hydroxyvitamin D and Virulence Genes of Staphylococcus aureus Isolates Colonizing Children with Atopic Dermatitis.

The skin of children with atopic dermatitis (AD) is colonized with Staphylococcus aureus more frequently than that of their peers. We investigated the prevalence of skin and nares colonization by S.aureus in children with AD, the virulence genes of the isolates, and their association with allergy, AD severity, and serum vitamin D (25(OH)D). This was an observational, cross-sectional study in a sample of children diagnosed with AD in two settings in Spain. The samples were collected in 2012. Swabs from affected skin and nares were taken for microbiologic culture. The prevalence of S.aureus and presence of 17 staphylococcal virulence genes were studied using polymerase chain reaction. A total of 114 patients with a mean age of 5.7±4.1 (range 3mos to 14yrs) were included in the study. Swabs were taken from the skin of 113 individuals with AD and from the nares of 85; 28.3% had S.aureus on the skin, which was significantly associated with positive allergen-specific immunoglobulin E antibodies and higher Scoring Atopic Dermatitis (SCORAD) scores in the multivariate analysis. The presence of virulence factors tsst-1, eta, cna, aur, and sec in cutaneous S.aureus isolates was associated with lower serum levels of 25(OH)D. S.aureus on nasal swabs correlated with its presence on the skin and was associated with lower 25(OH)D levels. In conclusion, S.aureus colonization is associated with allergy and severity in AD, whereas certain virulence genes are associated with lower serum 25(OH)D levels.

Methicillin-susceptible Staphylococcus aureus clonal complex 398: high prevalence and geographical heterogeneity in bone and joint infection and nasal carriage

The prevalence of clonal complex (CC) 398 methicillin-susceptible Staphylococcus aureus (MSSA) was unexpectedly high among bone and joint infections (BJIs) and nasal-colonizing isolates in France, with surprising geographical heterogeneity. With none of the major, most-known staphylococcal virulence genes, MSSA CC398 BJI was associated with lower biological inflammatory syndrome and lower treatment failure rates.

Contribution of Lactococcus lactis reducing properties to the downregulation of a major virulence regulator in Staphylococcus aureus, the agr system.

Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.

Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins

The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1–3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)–liquid ordered (lo) phase. Our results show that the PSMβ class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure–function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells.

The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell.

Inhibition of staphylococcal biofilm-related gene transcription by rhodomyrtone, a new antibacterial agent

Biofilms are one of the most important staphylococcal virulence factors, which are often associated with antibiotic resistance in the organisms. Our previous studies have demonstrated that rhodomyrtone from Rhodomyrtus tomentosa leaves exerts the activity against biofilm-forming staphylococci. In this study, we further evaluated the efficacy of the compound on transcription of biofilm-related genes including atl and ica, as well as their products in representative biofilm-forming strains Staphylococcus aureus SA113 and Staphylococcus epidermidis O47. Minimal inhibitory concentration of rhodomyrtone against S. aureus and S. epidermidis was 0.5 μg/mL. The transcription of biofilm-related genes was investigated using Northern blot analysis. Reduction in atl and ica transcription signals following rhodomyrtone treatment was observed. A different autolysin profile was detected in the rhodomyrtone-treated cells with respect to the untreated ones. In contrast, no differences in the production of polysaccharide intercellular adhesin, encoded by ica, of rhodomyrtone-treated and the untreated cells were observed.

The psmα locus regulates production of Staphylococcus aureus alpha-toxin during infection.

Staphylococcus aureus is a leading cause of human bacterial infection, causing a wide spectrum of disease ranging from skin and soft tissue infections to life-threatening pneumonia and sepsis. S. aureus toxins play an essential role in disease pathogenesis, contributing to both immunomodulation and host tissue injury. Prominent among these toxins are the membrane-active pore-forming cytolysin alpha-toxin (Hla) and the amphipathic α-helical phenol-soluble modulin (PSM) peptides. As deletion of either the hla or psm locus leads to a phenotypically similar virulence defect in skin and soft tissue infection, we sought to determine the relative contribution of each locus to disease pathogenesis. Here we show that production of Hla can be modulated by PSM expression. An S. aureus mutant lacking PSM expression exhibits a transcriptional delay in hla mRNA production and therefore fails to secrete normal levels of Hla at early phases of growth. This leads to attenuation of virulence in vitro and in murine skin and lung models of infection, correlating with reduced recovery of Hla from host tissues. Production of Hla and restoration of staphylococcal virulence can be achieved in the psm mutant by plasmid-driven overexpression of hla. Our study suggests the coordinated action of Hla and PSMs in host tissue during early pathogenesis, confirming a major role for Hla in epithelial injury during S. aureus infection. These findings highlight the possibility that therapeutics targeting PSM production may simultaneously prevent Hla-mediated tissue injury.

Quorum sensing-mediated regulation of staphylococcal virulence and antibiotic resistance.

Accessory gene regulator (agr)-mediated quorum sensing plays a central role in staphylococcal pathogenesis. It primarily upregulates secreted virulence factors and downregulates cell surface proteins, thereby governing invasiveness of staphylococci and cell dispersal from biofilms. Except for α- and β-PSMs, which are directly controlled by AgrA, the effector functions of agr are primarily mediated by RNAIII, a regulatory RNA encoded by this operon. agr phenotype and expression considerably influence the chronicity of an infection. It has also been linked with altered susceptibility of Staphylococcus aureus against antibiotics. Four classes of S. aureus and Staphylococcus epidermidis AIPs exist based on sequence variation, and lead to inter-strain and species cross-inhibition. Certain agr classes have been associated with specific clonal complexes, disease syndromes and intermediate-susceptibility to glycopeptides. It is also being investigated as a prophylactic and therapeutic target. This article describes the presently available literature regarding the role of agr in S. aureus and S. epidermidis infections.

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli.

Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the histidine kinase SaeS recognizes specific host stimuli is unknown. After mutagenizing the predicted extracellular loop of SaeS, we discovered one methionine residue (M31) was essential for the ability of S. aureus to transcribe sae target genes, including hla, lukAB/lukGH, and hlgA. This single M31A mutation also significantly reduced cytotoxicity in human neutrophils to levels observed in cells following interaction with ΔsaeS. Another important discovery was that mutation of two aromatic anchor residues (W32A and F33A) disrupted the normal basal signaling of SaeS in the absence of inducing signals, yet both mutant kinases had appropriate activation of effector genes following exposure to neutrophils. Although the transcriptional profile of aromatic mutation W32A was consistent with that of WT in response to human α-defensin 1, mutant kinase F33A did not properly transcribe the γ-toxin genes in response to this stimulus. Taken together, our results provide molecular evidence for how SaeS recognizes host signals and triggers activation of select virulence factors to facilitate evasion of innate immunity. These findings have important implications for signal transduction in prokaryotes and eukaryotes due to conservation of aromatic anchor residues across both of these domains and the important role they play in sensor protein structure and function.

Cutaneous Immune Defenses Against Staphylococcus aureus Infections.

Staphylococcus aureus (S. aureus) is a virulent bacterium that abundantly colonizes inflammatory skin diseases. Since S. aureus infections occur in an impaired skin barrier, it is important to understand the protective mechanism through cutaneous immune responses against S. aureus infections and the interaction with Staphylococcal virulence factors. In this review, we summarize not only the pathogenesis and key elements of S. aureus skin infections, but also the cutaneous immune system against its infections and colonization. The information obtained from this area may provide the groundwork for further immunomodulatory therapies or vaccination strategies to prevent S. aureus infections.

Molecular Analysis of the Interaction between Staphylococcal Virulence Factor Sbi-IV and Complement C3d

Staphylococcus aureus expresses numerous virulence factors that aid in immune evasion. The four-domain staphylococcal immunoglobulin binding (Sbi) protein interacts with complement component 3 (C3) and its thioester domain (C3d)-containing fragments. Recent structural data suggested two possible modes of binding of Sbi domain IV (Sbi-IV) to C3d, but the physiological binding mode remains unclear. We used a computational approach to provide insight into the C3d-Sbi-IV interaction. Molecular dynamics (MD) simulations showed that the first binding mode (PDB code 2WY8) is more robust than the second (PDB code 2WY7), with more persistent polar and nonpolar interactions, as well as conserved interfacial solvent accessible surface area. Brownian dynamics and steered MD simulations revealed that the first binding mode has faster association kinetics and maintains more stable intermolecular interactions compared to the second binding mode. In light of available experimental and structural data, our data confirm that the first binding mode represents Sbi-IV interaction with C3d (and C3) in a physiological context. Although the second binding mode is inherently less stable, we suggest a possible physiological role. Both binding sites may serve as a template for structure-based design of novel complement therapeutics.

Hyperexpression of α-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus

BackgroundThe community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined.ResultsHere, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus.ConclusionsThese data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

Children with Invasive Staphylococcus aureus Disease Exhibit a Potently Neutralizing Antibody Response to the Cytotoxin LukAB

Despite the importance of Staphylococcus aureus as a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity of S. aureus toward human neutrophils. Children with culture-proven S. aureus infection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers against S. aureus exotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently described S. aureus bicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicity in vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies to S. aureus antigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is produced in vivo and that it elicits a functional humoral response.

Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Ireland from 2002 to 2011: 21 Clones, Frequent Importation of Clones, Temporal Shifts of Predominant Methicillin-Resistant S. aureus Clones, and Increasing Multiresistance

There has been a worldwide increase in community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) infections. CA-MRSA isolates commonly produce the Panton-Valentine leukocidin toxin encoded by the pvl genes lukF-PV and lukS-PV. This study investigated the clinical and molecular epidemiologies of pvl-positive MRSA and methicillin-susceptible S. aureus (MSSA) isolates identified by the Irish National MRSA Reference Laboratory (NMRSARL) between 2002 and 2011. All pvl-positive MRSA (n=190) and MSSA (n=39) isolates underwent antibiogram-resistogram typing, spa typing, and DNA microarray profiling for multilocus sequence type, clonal complex (CC) and/or sequence type (ST), staphylococcal cassette chromosome mec type assignment, and virulence and resistance gene detection. Where available, patient demographics and clinical data were analyzed. The prevalence of pvl-positive MRSA increased from 0.2% to 8.8%, and that of pvl-positive MSSA decreased from 20% to 2.5% during the study period. The pvl-positive MRSA and MSSA isolates belonged to 16 and 5 genotypes, respectively, with CC/ST8-MRSA-IV, CC/ST30-MRSA-IV, CC/ST80-MRSA-IV, CC1/ST772-MRSA-V, CC30-MSSA, CC22-MSSA, and CC121-MSSA predominating. Temporal shifts in the predominant pvl-positive MRSA genotypes and a 6-fold increase in multiresistant pvl-positive MRSA genotypes occurred during the study period. An analysis of patient data indicated that pvl-positive S. aureus strains, especially MRSA strains, had been imported into Ireland several times. Two hospital and six family clusters of pvl-positive MRSA were identified, and 70% of the patient isolates for which information was available were from patients in the community. This study highlights the increased burden and changing molecular epidemiology of pvl-positive S. aureus in Ireland over the last decade and the contribution of international travel to the influx of genetically diverse pvl-positive S. aureus isolates into Ireland.