USA300 Lineage Research Articles

Fibronectin-binding proteins are required for biofilm formation by community-associated methicillin-resistantStaphylococcus aureusstrain LAC

Community-associated methicillin-resistant Staphylococcus aureus of the USA300 lineage is emerging as an important cause of medical device-related infection. However, few factors required for biofilm accumulation by USA300 strains have been identified, and the processes involved are poorly understood. Here, we identify S. aureus proteins required for the USA300 isolate LAC to form biofilm. A mutant with a deletion of the fnbA and fnbB genes did not express the fibronectin-binding proteins FnBPA and FnBPB and lacked the ability to adhere to fibronectin or to form biofilm. Biofilm formation by the mutant LAC∆fnbAfnbB could be restored by expression of FnBPA or FnBPB from a plasmid demonstrating that both of these proteins can mediate biofilm formation when expressed by LAC. Expression of FnBPA and FnBPB increased bacterial aggregation suggesting that fibronectin-binding proteins can promote the accumulation phase of biofilm. Loss of fibronectin-binding proteins reduced the initial adherence of bacteria, indicating that these proteins are also involved in primary attachment. In summary, these findings improve our understanding of biofilm formation by the USA300 strain LAC by demonstrating that the fibronectin-binding proteins are required.

Transduction of Staphylococcal Cassette Chromosome mec Elements between Strains of Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process.

Rapid and high-resolution distinction of community-acquired and nosocomial Staphylococcus aureus isolates with identical pulsed-field gel electrophoresis patterns and spa types

Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the ‘classical’ community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control.

Genetic structure of community acquired methicillin-resistant Staphylococcus aureus USA300

BackgroundCommunity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a significant bacterial pathogen that poses considerable clinical and public health challenges. The majority of the CA-MRSA disease burden consists of skin and soft tissue infections (SSTI) not associated with significant morbidity; however, CA-MRSA also causes severe, invasive infections resulting in significant morbidity and mortality. The broad range of disease severity may be influenced by bacterial genetic variation.ResultsWe sequenced the complete genomes of 36 CA-MRSA clinical isolates from the predominant North American community acquired clonal type USA300 (18 SSTI and 18 severe infection-associated isolates). While all 36 isolates shared remarkable genetic similarity, we found greater overall time-dependent sequence diversity among SSTI isolates. In addition, pathway analysis of non-synonymous variations revealed increased sequence diversity in the putative virulence genes of SSTI isolates.ConclusionsHere we report the first whole genome survey of diverse clinical isolates of the USA300 lineage and describe the evolution of the pathogen over time within a defined geographic area. The results demonstrate the close relatedness of clinically independent CA-MRSA isolates, which carry implications for understanding CA-MRSA epidemiology and combating its spread.

Virulence strategies of the dominant USA300 lineage of community-associated methicillin-resistantStaphylococcus aureus(CA-MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to worldwide health. Historically, MRSA clones have strictly been associated with hospital settings, and most hospital-associated MRSA (HA-MRSA) disease resulted from a limited number of virulent clones. Recently, MRSA has spread into the community causing disease in otherwise healthy people with no discernible contact with healthcare environments. These community-associated MRSA clones (CA-MRSA) are phylogenetically distinct from traditional HA-MRSA clones, and CA-MRSA strains seem to exhibit hypervirulence and more efficient host : host transmission. Consequently, CA-MRSA clones belonging to the USA300 lineage have become dominant sources of MRSA infections in North America. The rise of this successful USA300 lineage represents an important step in the evolution of emerging pathogens and a great deal of effort has been exerted to understand how these clones evolved. Here, we review much of the recent literature aimed at illuminating the source of USA300 success and broadly categorize these findings into three main categories: newly acquired virulence genes, altered expression of common virulence determinants and alterations in protein sequence that increase fitness. We argue that none of these evolutionary events alone account for the success of USA300, but rather their combination may be responsible for the rise and spread of CA-MRSA.

Nuclease Modulates Biofilm Formation in Community-Associated Methicillin-Resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.

Molecular Distinctions Exist Between Community-associated Methicillin-resistant Staphylococcus aureus Colonization and Disease-associated Isolates in Children

To define the molecular epidemiology of colonization and disease-associated isolates of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Laboratory-based comparative study of clinical staphylococcal isolates. We analyzed 255 pediatric CA-MRSA isolates for molecular characteristics associated with colonization and disease. We used polymerase chain reaction to determine the presence of Panton-Valentine Leukocidin and the lantibiotic element, bsaB, and to characterize the staphylococcal cassette chromosome mec type and accessory gene regulator locus. Pulsed-field gel electrophoresis was used to determine genetic relatedness between strains. A total of 150 isolates were obtained from patients with clinical disease (37 invasive infections, 113 noninvasive infections) and 105 from subjects with nasal colonization alone. Of 150 disease-associated isolates, 123 (82%) belonged to pulsed-field gel electrophoresis group USA300, whereas only 19 (18%) of 105 colonization isolates were of the USA300 lineage. Colonization isolates were less likely to possess staphylococcal cassette chromosome mec type IV, Panton-Valentine Leukocidin, or agr type 1 (P < 0.001). Colonization strains of CA-MRSA in children differ significantly from those strains recovered from patients with staphylococcal infections. This suggests that only colonization with specific strain types, rather than methicillin-resistant Staphylococcus aureus colonization in general, increases the risk for CA-MRSA disease.

Antimicrobial Activity of Community-associated Methicillin-resistant Staphylococcus aureus Is Caused by Phenol-soluble Modulin Derivatives

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are causing an ongoing pandemic of mostly skin and soft tissue infections. The success of CA-MRSA as pathogens is due to a combination of antibiotic resistance with high virulence. In addition, it has been speculated that CA-MRSA strains such as the epidemic U.S. clone USA300 have increased capacity to colonize human epithelia, owing to bacteriocin-based bacterial interference. We here analyzed the molecular basis of antimicrobial activity detected in S. aureus strains, including those of the USA300 lineage. In contrast to a previous hypothesis, we found that this activity is not due to expression of a lantibiotic-type bacteriocin, but proteolytically processed derivatives of the phenol-soluble modulin (PSM) peptides PSMα1 and PSMα2. Notably, processed PSMα1 and PSMα2 exhibited considerable activity against Streptococcus pyogenes, indicating a role of PSMs in the interference of S. aureus strains with the competing colonizing pathogen. Furthermore, by offering a competitive advantage during colonization of the human body, the characteristically high production of PSMs in USA300 and other CA-MRSA strains may thus contribute not only to virulence but also the exceptional capacity of those strains to sustainably spread in the population, which so far has remained poorly understood.

Distribution of the ACME-arcA gene among methicillin-resistant Staphylococcus aureus from England and Wales

The ST8-SCCmecIVa (USA300) methicillin-resistant Staphylococcus aureus (MRSA) clone can harbour the arginine catabolic mobile element (ACME). The arc gene cluster within the ACME may function as a virulence or strain survival factor. We determined the distribution of the ACME-associated arcA gene among genetically diverse MRSA from around England and Wales. MRSA isolates (n = 203) of diverse genetic types, referred to the England and Wales Staphylococcus reference laboratory, were tested for the presence of the ACME-arcA gene. ACME-arcA-positive isolates were characterized by toxin gene profiling, PFGE and spa sequence typing. MICs of a range of antimicrobials were also determined. The ACME-arcA gene was detected in 17 isolates. Twelve were related to known ST8-MRSA-SCCmecIVa isolates of the USA300 lineage by pulsotype and were resistant to oxacillin, with variable ciprofloxacin and erythromycin resistance. Outside the USA300 lineage, four of the remaining five ACME-arcA isolates were closely related ST97-MRSA-SCCmecV, Panton-Valentine leucocidin (PVL)-negative, resistant to oxacillin and variously resistant to erythromycin, ciprofloxacin, clindamycin, gentamicin, tetracycline and fusidic acid. The remaining isolate was ST1, PVL-positive and resistant to fusidic acid as well as oxacillin. Thirteen out of the 17 isolates were associated with skin and soft tissue infections. The detection of ACME-arcA in diverse MRSA types highlights the mobility of the elements encoding ACME-arcA genes. The diversity of strain types and resistance profiles among ACME-arcA-encoding MRSA is a cause for public-health concern and demands continued surveillance and close monitoring.

Complicated adult epiglottitis due to methicillin-resistant Staphylococcus aureus

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is emerging as an important pathogen. However, MRSA rarely causes epiglottitis. We report an adult with epiglottitis due to a CA-MRSA isolate of the USA300 lineage with Panton-Valentine leukocidin. Maintaining vigilance for CA-MRSA as a cause of an expanding spectrum of severe infections is warranted.

Characteristics ofStaphylococcus aureusInfections, Chicago Pediatric Hospital

Invasive and skin community-associated (CA)–methicillin-resistant Staphylococcus aureus (MRSA) isolates from children were matched with invasive CA–methicillin-sensitive S. aureus strains during 2000–2004. Isolates were analyzed for presence of Panton-Valentine leukocidin. A USA400 lineage clone (n = 6) and the predominant USA300 lineage clone emerged.