Biofilm associated genotypes of multiple antibiotic resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous environmental microorganism and also a common cause of infection. Its ability to survive in many different environments and persistently colonize humans is linked to its presence in biofilms formed on indwelling device surfaces. Biofilm promotes adhesion to, and survival on surfaces, protects from desiccation and the actions of antibiotics and disinfectants. RESULTS: We examined the genetic basis for biofilm production on polystyrene at room (22 °C) and body temperature (37 °C) within 280 P. aeruginosa. 193 isolates (69 %) produced more biofilm at 22 °C than at 37 °C. Using GWAS and pan-GWAS, we found a number of accessory genes significantly associated with greater biofilm production at 22 °C. Many of these are present on a 165 kb region containing genes for heavy metal resistance (arsenic, copper, mercury and cadmium), transcriptional regulators and methytransferases. We also discovered multiple core genome SNPs in the A-type flagellin gene and Type II secretion system gene xpsD. Analysis of biofilm production of isolates of the MDR ST111 and ST235 lineages on stainless-steel revealed several accessory genes associated with enhanced biofilm production. These include a putative translocase with homology to a Helicobacter pylori type IV secretion system protein, a TA system II toxin gene and the alginate biosynthesis gene algA, several transcriptional regulators and methytransferases as well as core SNPs in genes involved in quorum sensing and protein translocation. CONCLUSIONS: Using genetic association approaches we discovered a number of accessory genes and core-genome SNPs that were associated with enhanced early biofilm formation at 22 °C compared to 37 °C. These included a 165 kb genomic island containing multiple heavy metal resistance genes, transcriptional regulators and methyltransferases. We hypothesize that this genomic island may be associated with overall genotypes that are environmentally adapted to survive at lower temperatures. Further work to examine their importance in, for example gene-knockout studies, are required to confirm their relevance. GWAS and pan-GWAS approaches have great potential as a first step in examining the genetic basis of novel bacterial phenotypes.

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak.

BACKGROUND: Isolates of methicillin-resistant Staphylococcus aureus (MRSA) belonging to a single lineage are often indistinguishable by means of current typing techniques. Whole-genome sequencing may provide improved resolution to define transmission pathways and characterize outbreaks. METHODS: We investigated a putative MRSA outbreak in a neonatal intensive care unit. By using rapid high-throughput sequencing technology with a clinically relevant turnaround time, we retrospectively sequenced the DNA from seven isolates associated with the outbreak and another seven MRSA isolates associated with carriage of MRSA or bacteremia in the same hospital. RESULTS: We constructed a phylogenetic tree by comparing single-nucleotide polymorphisms (SNPs) in the core genome to a reference genome (an epidemic MRSA clone, EMRSA-15 [sequence type 22]). This revealed a distinct cluster of outbreak isolates and clear separation between these and the nonoutbreak isolates. A previously missed transmission event was detected between two patients with bacteremia who were not part of the outbreak. We created an artificial "resistome" of antibiotic-resistance genes and demonstrated concordance between it and the results of phenotypic susceptibility testing; we also created a "toxome" consisting of toxin genes. One outbreak isolate had a hypermutator phenotype with a higher number of SNPs than the other outbreak isolates, highlighting the difficulty of imposing a simple threshold for the number of SNPs between isolates to decide whether they are part of a recent transmission chain. CONCLUSIONS: Whole-genome sequencing can provide clinically relevant data within a time frame that can influence patient care. The need for automated data interpretation and the provision of clinically meaningful reports represent hurdles to clinical implementation. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).

Molecular tracing of the emergence, adaptation, and transmission of hospital-associated methicillin-resistant Staphylococcus aureus.

Hospital-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a global health burden dominated by a small number of bacterial clones. The pandemic EMRSA-16 clone (ST36-II) has been widespread in UK hospitals for 20 y, but its evolutionary origin and the molecular basis for its hospital association are unclear. We carried out a Bayesian phylogenetic reconstruction on the basis of the genome sequences of 87 S. aureus isolates including 60 EMRSA-16 and 27 additional clonal complex 30 (CC30) isolates, collected from patients in three continents over a 53-y period. The three major pandemic clones to originate from the CC30 lineage, including phage type 80/81, Southwest Pacific, and EMRSA-16, shared a most recent common ancestor that existed over 100 y ago, whereas the hospital-associated EMRSA-16 clone is estimated to have emerged about 35 y ago. Our CC30 genome-wide analysis revealed striking molecular correlates of hospital- or community-associated pandemics represented by mobile genetic elements and nonsynonymous mutations affecting antibiotic resistance and virulence. Importantly, phylogeographic analysis indicates that EMRSA-16 spread within the United Kingdom by transmission from hospitals in large population centers in London and Glasgow to regional health-care settings, implicating patient referrals as an important cause of nationwide transmission. Taken together, the high-resolution phylogenomic approach used resulted in a unique understanding of the emergence and transmission of a major MRSA clone and provided molecular correlates of its hospital adaptation. Similar approaches for hospital-associated clones of other bacterial pathogens may inform appropriate measures for controlling their intra- and interhospital spread.

Diversity of methicillin-resistant Staphylococcus aureus strains isolated from residents of 26 nursing homes in Orange County, California.

Nursing homes represent a unique and important methicillin-resistant Staphylococcus aureus (MRSA) reservoir. Not only are strains imported from hospitals and the community, strains can be transported back into these settings from nursing homes. Since MRSA bacteria are prevalent in nursing homes and yet relatively poorly studied in this setting, a multicenter, regional assessment of the frequency and diversity of MRSA in the nursing home reservoir was carried out and compared to that of the MRSA from hospitals in the same region. The prospective study collected MRSA from nasal swabbing of residents of 26 nursing homes in Orange County, California, and characterized each isolate by spa typing. A total of 837 MRSA isolates were collected from the nursing homes. Estimates of admission prevalence and point prevalence of MRSA were 16% and 26%, respectively. The spa type genetic diversity was heterogeneous between nursing homes and significantly higher overall (77%) than the diversity in Orange County hospitals (72%). MRSA burden in nursing homes appears largely due to importation from hospitals. As seen in Orange County hospitals, USA300 (sequence type 8 [ST8]/t008), USA100 (ST5/t002), and a USA100 variant (ST5/t242) were the dominant MRSA clones in Orange County nursing homes, representing 83% of all isolates, although the USA100 variant was predominant in nursing homes, whereas USA300 was predominant in hospitals. Control strategies tailored to the complex problem of MRSA transmission and infection in nursing homes are needed in order to minimize the impact of this unique reservoir on the overall regional MRSA burden.

Differences in methicillin-resistant Staphylococcus aureus strains isolated from pediatric and adult patients from hospitals in a large county in California.

Studies of U.S. epidemics of community- and health care-associated methicillin-resistant Staphylococcus aureus (MRSA) suggested differences in MRSA strains in adults and those in children. Comprehensive population-based studies exploring these differences are lacking. We conducted a prospective cohort study of inpatients in Orange County, CA, collecting clinical MRSA isolates from 30 of 31 Orange County hospitals, to characterize differences in MRSA strains isolated from children compared to those isolated from adults. All isolates were characterized by spa typing. We collected 1,124 MRSA isolates from adults and 159 from children. Annual Orange County population estimates of MRSA inpatient clinical cultures were 119/100,000 adults and 22/100,000 children. spa types t008, t242, and t002 accounted for 83% of all isolates. The distribution of these three spa types among adults was significantly different from that among children (χ(2) = 52.29; P < 0.001). Forty-one percent of adult isolates were of t008 (USA300), compared to 69% of pediatric isolates. In multivariate analyses, specimens from pediatric patients, wounds, non-intensive care unit (ICU) wards, and hospitals with a high proportion of Medicaid-insured patients were significantly associated with the detection of t008 strains. While community- and health care-associated MRSA reservoirs have begun to merge, significant differences remain in pediatric and adult patient populations. Community-associated MRSA spa type t008 is significantly more common in pediatric patients.

Isolation and Characterisation of Bacteriophage Selective for Key Acinetobacter baumannii Capsule Chemotypes.

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (-20 to 60 °C) and pH (5.0-9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38-39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections.

Phenotypic and Genotypic Characterization of Novel Polyvalent Bacteriophages With Potent In Vitro Activity Against an International Collection of Genetically Diverse Staphylococcus aureus.

Phage therapy recently passed a key milestone with success of the first regulated clinical trial using systemic administration. In this single-arm non-comparative safety study, phages were administered intravenously to patients with invasive Staphylococcus aureus infections with no adverse reactions reported. Here, we examined features of 78 lytic S. aureus phages, most of which were propagated using a S. carnosus host modified to be broadly susceptible to staphylococcal phage infection. Use of this host eliminates the threat of contamination with staphylococcal prophage - the main vector of S. aureus horizontal gene transfer. We determined the host range of these phages against an international collection of 185 S. aureus isolates with 56 different multilocus sequence types that included multiple representatives of all epidemic MRSA and MSSA clonal complexes. Forty of our 78 phages were able to infect > 90% of study isolates, 15 were able to infect > 95%, and two could infect all 184 clinical isolates, but not a phage-resistant mutant generated in a previous study. We selected the 10 phages with the widest host range for in vitro characterization by planktonic culture time-kill analysis against four isolates:- modified S. carnosus strain TM300H, methicillin-sensitive isolates D329 and 15981, and MRSA isolate 252. Six of these 10 phages were able to rapidly kill, reducing cell numbers of at least three isolates. The four best-performing phages, in this assay, were further shown to be highly effective in reducing 48 h biofilms on polystyrene formed by eight ST22 and eight ST36 MRSA isolates. Genomes of 22 of the widest host-range phages showed they belonged to the Twortvirinae subfamily of the order Caudovirales in three main groups corresponding to Silviavirus, and two distinct groups of Kayvirus. These genomes assembled as single-linear dsDNAs with an average length of 140 kb and a GC content of c. 30%. Phages that could infect > 96% of S. aureus isolates were found in all three groups, and these have great potential as therapeutic candidates if, in future studies, they can be formulated to maximize their efficacy and eliminate emergence of phage resistance by using appropriate combinations.

Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

Further understanding of Pseudomonas aeruginosa's ability to horizontally acquire virulence: possible intervention strategies.

Introduction: Pseudomonas aeruginosa is a common, ubiquitous bacterium that is found in natural environments but is also a successful opportunistic pathogen of humans and plants. The reasons for this flexibility and evolutionary success can be attributed to its ability to readily acquire new genes to ensure its survival enabling it to survive desiccation, the action of antimicrobial compounds and invade new territories such as modern hospitals with high levels of antibiotic usage.Areas covered: Literature was searched using PubMed and Web of science (05/19 to 05/20). Identified studies paint a picture of a dynamic, highly variable population shaped by frequent intra- and inter-species horizontal gene transfer resulting in a species able to resist the action of antibiotics and deploy multiple virulence strategies controlled by complex quorum-sensing systems. We investigate possible control measures including anti-virulence and environmental control measures.Expert opinion: P.aeruginosa is a resilient, richly diverse species but also a global health threat due to the emergence and global dissemination of successful multiresistant clones that resist all antibiotics. Genomics offers the potential for rapid identification of 'high-risk' clones to guide chemotherapy, but novel control measures are also required to slow the species progression to pan-resistance.

A population-based study of the incidence and molecular epidemiology of methicillin-resistant Staphylococcus aureus disease in San Francisco, 2004-2005.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections have become a major public health problem in both the community and hospitals. Few studies have characterized the incidence and clonal composition of disease-causing strains in an entire population. Our objective was to perform a population-based survey of the clinical and molecular epidemiology of MRSA disease in San Francisco, California. METHODS: We prospectively collected 3985 MRSA isolates and associated clinical and demographic information over a 12-month period (2004-2005) at 9 San Francisco-area medical centers. A random sample of 801 isolates was selected for molecular analysis. RESULTS: The annual incidence of community-onset MRSA disease among San Francisco residents was 316 cases per 100,000 population, compared with 31 cases per 100,000 population for hospital-onset disease. Persons who were aged 35-44 years, were men, and were black had the highest incidence of community-onset disease. The USA300 MRSA clone accounted for 234 cases of community-onset disease and 15 cases of hospital-onset disease per 100,000 population, constituting an estimated 78.5% and 43.4% of all cases of MRSA disease, respectively. Patients with community-onset USA300 MRSA versus non-USA300 MRSA disease were more likely to be male, be of younger age, and have skin and soft-tissue infections. USA300 strains were generally more susceptible to multiple antibiotics, although decreased susceptibility to tetracycline was observed in both community-onset (P = .008) and hospital-onset (P = .03) USA300 compared to non-USA300 strains. CONCLUSIONS: The annual incidence of community-onset MRSA disease in San Francisco is substantial, surpassing that of hospital-onset disease. USA300 is the predominant clone in both the community and hospitals. The dissemination of USA300 from the community into the hospital setting has blurred its distinction as a community-associated pathogen.

Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa.

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

Re-emergence of early pandemic Staphylococcus aureus as a community-acquired meticillin-resistant clone.

During the 1950s, the notorious penicillin-resistant clone of Staphylococcus aureus known as phage type 80/81 emerged and caused serious hospital-acquired and community-acquired infections worldwide. This clone was largely eliminated in the 1960s, concurrent with the widespread use of penicillinase-resistant beta lactams. We investigated whether early 80/81 isolates had the genes for Panton-Valentine leucocidin, a toxin associated with virulence in healthy young people. Multilocus sequence analysis suggested that descendants of 80/81 have acquired meticillin resistance, are re-emerging as a community-acquired meticillin-resistant S aureus (MRSA) clone, and represent a sister lineage to pandemic hospital-acquired MRSA.

Analyses of clonality and the evolution of bacterial pathogens.

The existence of bacterial clones was evident in early phenotypic studies that recognised high levels of similarity in geographically and temporally separated isolates. Multilocus sequence typing (MLST) has become the most common method for genetically characterizing clones of several bacterial pathogens, allowing the tracking of hypervirulent/antibiotic-resistant lineages. MLST has also been used to examine the way that bacterial populations, and in particular, bacterial clones evolve. Visualisation of MLST datasets has required the development of novel tools, such as 'eBURST', a key program in constructing evolutionary models that detail how methicillin resistant Staphylococcus aureus (MRSA) and other clones emerge and spread.

A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions.

Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred.

An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

Despite the importance of phages in driving horizontal gene transfer (HGT) among pathogenic bacteria, the underlying molecular mechanisms mediating phage adsorption to S. aureus are still unclear. Phage ϕ11 is a siphovirus with a high transducing efficiency. Here, we show that the tail protein Gp45 localized within the ϕ11 baseplate. Phage ϕ11 was efficiently neutralized by anti-Gp45 serum, and its adsorption to host cells was inhibited by recombinant Gp45 in a dose-dependent manner. Flow cytometry analysis demonstrated that biotin-labelled Gp45 efficiently stained the wild-type S. aureus cell but not the double knockout mutant ΔtarM/S, which lacks both α- and ß-O-GlcNAc residues on its wall teichoic acids (WTAs). Additionally, adsorption assays indicate that GlcNAc residues on WTAs and O-acetyl groups at the 6-position of muramic acid residues in peptidoglycan are essential components of the ϕ11 receptor. The elucidation of Gp45-involved molecular interactions not only broadens our understanding of siphovirus-mediated HGT, but also lays the groundwork for the development of sensitive affinity-based diagnostics and therapeutics for S. aureus infection.

The evolution of a resistant pathogen--the case of MRSA.

Recent advances in molecular epidemiological methods have elucidated the evolutionary pathways by which modern methicillin-resistant Staphylococcus aureus have evolved. Only five major lineages of hospital methicillin-resistant Staphylococcus aureus exist, but these are globally distributed and evolving resistance to even the newest antibiotics.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE: P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Genetic analysis of Staphylococcus aureus from intravenous drug user lesions.

Multilocus sequence typing (MLST) of 48 methicillin-sensitive Staphylococcus aureus isolates from intravenous drug user abscesses/soft-tissue infections revealed 12 sequence types (STs) belonging to eight genetically distinct lineages. Only two novel STs were recovered (one isolate of each), indicating that isolates in this study were similar to those from previous studies of disease and carriage. However, ST59, the most common genotype recovered (from six individuals), may be adept at causing subcutaneous lesions in this patient population, as it is rare in carriage and disease. PCR detection of 22 toxin genes revealed a high prevalence of the gene for staphylococcal enterotoxin B compared with previous studies, indicating that this toxin may promote infections in this patient group.