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Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others.

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.

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.

Nineteen bacteriophages against five main capsular types of multidrug-resistant

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.

Effective, sustainable management of urban water systems, including drinking water, stormwater, wastewater, and natural water systems, is critical to the health and well-being of people in urban areas and the ecosystems that encompass them. The demands of human population growth, aging infrastructure, and changing climate will increase pressure on these systems and require future innovations in water management. Planning for urban water systems will increasingly require collaborations between water professionals and researchers to imagine, design and model the response of novel urban water systems to future conditions. We highlight benefits and challenges of transdisciplinary projects for integrated urban water management; organized broadly into: (1) engagement of water managers and planners; (2) transdisciplinary design of innovative systems, and once designed; (3) modeling and evaluation of urban water system response to various innovations. We describe the development of a multi-scale approach to design and evaluation of innovative urban water systems, and illustrate its application using examples from the Willamette River Basin and Portland, Oregon. The scenario-based approach described here offers several key contributions to the design and modeling of innovation. First, this process provides the opportunity to convene professionals and researchers, who do not typically collaborate, as participants in a collaborative process. Second, it engages participants in thinking together across land and water management sectors to develop plausible futures at multiple spatial extents and multidecadal time horizons. Third, it helps to identify critical gaps in extant water modeling capabilities, and thus helps define the near-term research agenda for modelers.

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.

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.

© 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group. 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.

Space heating is the single greatest source of building-related greenhouse gas emissions in the industrialized world, giving urgency to the development of strategies for carbon-free heating. Recent advances have shown that the direct capture, storage, and deployment of solar energy, without conversion to electricity, has considerable potential to address space-heating needs even in cold and cloudy climates. However, the solar energy available for direct heating at climatic and metropolitan scales is both unquantified and widely assumed to be negligible, impeding further investigation, development, and policy responses. To estimate the magnitude and distribution of solar resources concurrent with space-heating needs, we spatially integrate datasets characterizing solar radiation, outdoor temperature, and heating energy use across U.S. climates. Results show that the median resource incident upon collectors of residential scale (10 m2) and distribution is much greater than previously realized, equaling 7 MWh per household annually; by comparison, the median household heating need is currently 10.3 MWh. Unexpectedly, cloud-diffused solar radiation accounts for over one-quarter of this resource in all but semi-arid climates. Metropolitan residential resources exceed 5 TWh in areas including Detroit and Boston (cold continental), Washington D.C. (humid subtropical), Seattle and San Francisco (Mediterranean), and Denver (semi-arid), and national resources exceed 750 TWh annually, compared to approximately 1200 TWh of annual heating need. Current technology is able to capture and retain over half of a direct solar heating resource, revealing that the untapped U.S. solar heating potential is comparable to one-third of the national residential space-heating need and implying that analogous resources exist in analogous climates worldwide.

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.

Pseudomonas aeruginosa is a ubiquitous environmental microorganism that is also a common cause of nosocomial infections that vary in severity from chronic wound infections to pneumonia, bloodstream infections and sepsis. Its ability to survive in many different environments and persistently colonize human tissue is linked to its presence within biofilms that form on indwelling device surfaces such as plastics and stainless steel. Biofilm promotes bacterial adhesion and survival on surfaces, reduces susceptibility to desiccation, and the actions of antibiotics and disinfectants. Recent genome sequencing studies demonstrate that P. aeruginosa is a highly diverse species with a very large pan-genome consistent with its adaptability to differing environments. However, most MDR infections are caused by a small number of “high-risk” clones or lineages that recently emerged and spread globally.In our 2017 study of the resistome of P. aeruginosa we confirmed the power of genome-wide association (GWAS) techniques to explore the genetic basis of several antibiotic resistance phenotypes and discovered 46 novel putative resistance polymorphisms. In this study we sought to examine genetic associations within a subset of these isolates with simple biofilm phenotypes. We examined the genetic basis for biofilm production on polystyrene at room temperature (22°C) and body temperature (37°C) within a total of 280 isolates. 69% of isolates (n=193) produced more biofilm mass at 22°C, whilst those producing more biofilm at 37°C had reduced optical density 540 variation. We found statistically significant associations with IpxO and other genes associated with arsenic resistance to be significantly associated with this trait. IpxO which encodes a lipid A hydroxylase and arsenic reduction genes have previously been found to be associated with biofilm production in this species. We analyzed 260 ST111 and ST235 genomes and found considerable genetic variation between isolates in their content of genes previously found associated with biofilm production. This is indicative of a highly variable and flexible population within these clades with frequent emergence of successful sub-lineages. Analysis of 48 of these isolates’ ability to form biofilm on stainless steel surfaces showed that a ‘good’ biofilm-forming phenotype had significant intra-clone variation, independent of core genome phylogeny with pan-genome analysis, suggesting a possible association and involvement of components of the type IV secretion system. However, GWAS and pan-GWAS analyses yielded weaker statistical significance. This study confirms GWAS and pan-GWAS trait associations can be performed for biofilm phenotype and produce data in agreement with each other. This panel of 280 study isolates, matched to genomic data has potential for the investigation of other phenotypes in P. aeruginosa perhaps as part of a growing database / collection. A representative, curated, genome sequenced collection should increase in usefulness as it grows offering increasing statistical power.ImportanceP. aeruginosa is a major cause of multiply antibiotic infections worldwide but it is also found in many hospital and natural environments, especially aquatic ones. In this study we examined genetic polymorphism associated with biofilm production at room temperature and at body temperature, the biofilm associated gene repertoire of two major MDR clones and also genetic polymorphisms associated with biofilm production on stainless steel. Using these genome-wide and pan-genome wide association methods we identified / confirmed potential key genes involved in biofilm production and survival of P. aeruginosa. The study demonstrates the potential usefulness of large, genome sequenced isolate collections such as ours, to better understand the genetics underlying phenotypic diversity in this species.

Bacteriophage therapy is a promising new treatment that may help overcome the threat posed by antibiotic-resistant pathogenic bacteria, which are increasingly identified in hospitalized patients. The development of biocompatible and sustainable vehicles for incorporation of viable bacterial viruses into a wound dressing is a promising alternative. This article evaluates the antimicrobial efficacy of Bacteriophage K against Staphylococcus aureus over time, when stabilized and delivered via an oil-in-water nano-emulsion. Nano-emulsions were formulated via thermal phase inversion emulsification, and then bacterial growth was challenged with either native emulsion, or emulsion combined with Bacteriophage K. Bacteriophage infectivity, and the influence of storage time of the preparation, were assessed by turbidity measurements of bacterial samples. Newly prepared Bacteriophage K/nano-emulsion formulations have greater antimicrobial activity than freely suspended bacteriophage. The phage-loaded emulsions caused rapid and complete bacterial death of three different strains of S. aureus. The same effect was observed for preparations that were either stored at room temperature (18-20°C), or chilled at 4°C, for up to 10 days of storage. A response surface design of experiments was used to gain insight on the relative effects of the emulsion formulation on bacterial growth and phage lytic activity. More diluted emulsions had a less significant effect on bacterial growth, and diluted bacteriophage-emulsion preparations yielded greater antibacterial activity. The enhancement of bacteriophage activity when delivered via nano-emulsions is yet to be reported. This prompts further investigation into the use of these formulations for the development of novel anti-microbial wound management strategies.

Objective.We sought to identify hospital characteristics associated with community-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) carriage among inpatients.Design.Prospective cohort study.Setting.Orange County, California.Participants.Thirty hospitals in a single county.Methods.We collected clinical MRSA isolates from inpatients in 30 of 31 hospitals in Orange County, California, from October 2008 through April 2010. We characterized isolates by spa typing to identify CA-MRSA strains. Using California's mandatory hospitalization data set, we identified hospital-level predictors of CA-MRSA isolation.Results.CA-MRSA strains represented 1,033 (46%) of 2,246 of MRSA isolates. By hospital, the median percentage of CA-MRSA isolates was 46% (range, 14%–81%). In multivariate models, CA-MRSA isolation was associated with smaller hospitals (odds ratio [OR], 0.97, or 3% decreased odds of CA-MRSA isolation per 1,000 annual admissions;PP= .002), and hospitals with more patients with low comorbidity scores (OR, 1.3;P< .001). Results were similar when restricted to isolates from patients with hospital-onset infection.Conclusions.Among 30 hospitals, CA-MRSA comprised nearly half of MRSA isolates. There was substantial variability in CA-MRSA penetration across hospitals, with more CA-MRSA in smaller hospitals with healthier but socially disadvantaged patient populations. Additional research is needed to determine whether infection control strategies can be successful in targeting CA-MRSA influx.

We assessed characteristics associated with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) carriage among residents of 22 nursing homes. Of MRSA-positive swabs, 25% (208/824) were positive for CA-MRSA. Median facility CAMRSA percentage was 22% (range, 0%-44%). In multivariate models, carriage was associated with age less than 65 years (odds ratio, 1.2; P

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 β-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 use of bacteriophages (phages) to treat bacterial infections, known as phage therapy, has a history substantially longer than that of antibiotics, yet these drugs have been the treatment of choice in the West for over 60 years owing to efficacy, low toxicity and ease of production. Bacteria are becoming increasingly resistant to antibiotics while efforts to discover new agents have drastically reduced. Phages have co-evolved with their hosts over billions of years and have acquired mechanisms to counter bacterial defences such as extracellular biofilm production, which severely reduces the effectiveness of conventional antibiotics. Recent animal and human trials show phages to be safe, well-tolerated agents with a bright future as an alternative to chemical agents.

Bacteriophages were first identified in 1915 and were used as antimicrobial agents from 1919 onwards. Despite apparent successes and widespread application, early users did not understand the nature of these agents and their efficacy remained controversial. As a result, they were replaced in the west by chemical antibiotics once these became available. However, bacteriophages remained a common therapeutic approach in parts of Eastern Europe where they are still in use. Increasing levels of antibiotic-resistant bacterial infections are now driving demand for novel therapeutic approaches. In cases where antibiotic options are limited or nonexistent, the pressure for new agents is greatest. One of the most prominent areas of concern is multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa is a prominent member of this class and is the cause of damaging infections that can be resistant to successful treatment with conventional antibiotics. At the same time, it exhibits a number of properties that make it a suitable target for bacteriophage-based approaches, including growth in biofilms that can hydrolyse following phage infection. Pseudomonas aeruginosa provides a striking example of an infection where clinical need and the availability of a practical therapy coincide.

It is hypothesized that perennial freshwater tidal wetland habitat exports inorganic and organic material needed to support the estuarine food web and to create favorable habitat for aquatic organisms in San Francisco Estuary. It is also hypothesized that most of the material flux in this river-dominated region is controlled by river flow. The production and export of material by Liberty Island were measured and com- pared using discrete monthly and continuous (15 min) measurements of a suite of inorganic and organic materials and flow between 2004 and 2005. Seasonal material flux was estimated from monthly discrete data for inorganic nutrients, suspended solids and salts, organic carbon and nitrogen and phytoplankton and zooplankton group carbon and chlorophyll a and pheophytin pigment. Estimates of material flux from monthly values were compared with measured daily material flux values for chlorophyll a concentration, salt and suspended solids obtained from continuous measurements (15 min) using YSI water quality sondes. Phytoplankton carbon produced within the wetland was estimated by in situ primary productivity. Most inorganic and organic materials were exported from the wetland on an annual basis, but the magnitude and direction varied seasonally. Dissolved inorganic nutrients such as nitrate, soluble phosphorus, total phosphorus and silica as well as total suspended solids were exported in the summer while total and dissolved organic carbon were exported in the winter. Salts like chloride and bromide were exported in the fall. Chlorophyll a and pheophytin were exported in the fall and associated with diatom and cyanobacteria carbon. Mesozooplankton carbon was dominated by calanoid copepods and exported most of the year except summer. Continuous sampling revealed high hourly and daily variation in chlorophyll a, salt and total suspended solids flux due to high frequency changes in concentration and tidal flow. In fact, tidal flow rather than river discharge was responsible for 90% or more of the material flux of the wetland. These studies indicate that freshwater tidal wetlands can be a source of inorganic and organic material but the export of material is highly variable spatially and temporally, varies most closely with tidal flow and requires high frequency measurements of both tidal flow and material concentration for accurate estimates.

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 ( ) and hospital-onset ( ) USA300 compared to non-USA300 strains. P p .008 P p .03 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.

In the present study we give some direction on the selection of the most appropriate typing method(s) to be used for the characterization of Staphylococcus epidermidis , in view of the most recent findings on the evolution, population structure, and epidemiology of this species. In order to achieve this aim, quantitative assessment of the correlation of the results of three typing methods—pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCC mec ) typing, which target different regions of the chromosome that evolve at different rates—was performed. In order to evaluate the discriminatory ability and the strength and direction of the correlation of the different typing methods, Simpson's index of diversity (SID), the adjusted Rand coefficient (AR), and the Wallace coefficient ( W ) were calculated. PFGE was the most discriminatory method (SID = 99%), followed by MLST (SID = 90%) and SCC mec typing (SID = 75%). The values of AR and W (0.10 < AR < 0.30; 0.50 < W < 0.75) indicated that the partition of the same isolate collection by PFGE, MLST, and SCC mec typing provided results that had only a poor correlation with each other. However, the information provided by the combination of PFGE and SCC mec enabled the prediction of the results obtained by MLST at the level of the clonal complex with a high degree of precision ( W > 0.90). We propose that clones of S. epidermidis be defined by the combination of the PFGE type followed by the SCC mec type, which provides reliable information on the short-term epidemiology and the ability to predict with consistency long-term clonal evolution.

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCC mec ) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCC mec was acquired at least 56 times by S. epidermidis . Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 ( P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCC mec .

We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.

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.

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2 h post-infection (treatment) or 2 h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24 h for quantification of bacteria and phages. Survival rates at 24 h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI = 10; 47% vs. 20%, MOI = 1; and 33% vs. 7%, MOI = 0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI = 10; 53% vs. 13%, MOI = 1; 67% vs. 7%, MOI = 0.1). By 24 h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses.

Since their discovery near the beginning of the 20th century lytic viruses of bacteria known as bacteriophages (phages), have been widely used as antimicrobial agents even prior to their characterization as viruses. Early studies demonstrated their potential to treat infections in large but poorly described trials in cholera and dysentery patients. Phage therapy was commercialized by several large companies in the 1920s and 1930s but preparations were heterogeneous in quality and were recommended for the treatment of diseases that did not have a bacterial aetiology. The introduction of antibiotics into human and veterinary medicine side-lined the use of phage to pockets of use in countries in Eastern Europe and the countries of the former USSR where they are still used today. Phages were central to early discoveries in molecular biology and this and studies in animal models of infection in the 1980s led to a revival in interest as therapeutic agents. More recently, several early-stage clinical trials have been performed to demonstrate their safety in topical applications and a small phase 2/2a study showed efficacy in the treatment of antibiotic-resistant otitis. Phage genomics is still at an early stage and most phage genes are of unknown function, however expanding antibiotic resistance and a paucity of novel antibiotics has generated recent, further interest in their therapeutic development built upon genetically characterized bacteriophages.

Staphylococcal chromosome cassette mec (SCCmec) elements within major lineages of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) clones were characterised using intra-SCCmec multilocus sequencing. A strong correlation was observed between sequence- and PCR-based typing methods (p

The control of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection is of concern worldwide. Given the evidence that several pathogenic species replicate within amoebae and emerge more virulent and more resistant and the abundance of amoebae in healthcare settings, we investigated interactions of Acanthamoeba polyphaga with epidemic MRSA isolates. MRSA proliferated in the presence of amoebae, attributable partly to intracellular replication. Following 24 h of co-culture, confocal microscopy revealed that c. 50% amoebae had viable MRSA within phago-lysosomes and 2% of amoebae were heavily infected with viable cocci throughout the cytoplasm. Infection control strategies should recognize the contribution of protozoa.

Two hundred nonduplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates causing bacteremia in patients in four major Hong Kong hospitals during the period 2000 to 2001 were characterized by antibiogram, pulsed-field gel electrophoresis (PFGE) using SmaI restriction enzymes, and determination of staphylococcal cassette chromosome mec (SCC mec ) types. Nine PFGE types, A to I, were obtained. PFGE type A constituted 50% (99/200) of all isolates and was present in isolates from all four hospitals. PFGE types A to E, had previously been identified as the major types at one of the hospitals from 1988 to 2000. The majority had a resistance profile to tetracycline (T), erythromycin (E), clindamycin (D), gentamicin (G), tobramycin (To), and ciprofloxacin (Ci), and belonged to SCC mec type III; and representatives belonged to clonal complex 239 (CC239) (MRSA with SCC mec type III and sequence type 239, designated ST239-MRSA-III). PFGE types F to I were new patterns that had not been previously identified in isolates from Hong Kong. PFGE type F constituted 18% (35/200) of MRSAs, had resistance profile TEGToCi, and belonged to CC5 (ST5-MRSA-II). PFGE type G included 13% (26/200) of MRSAs, had resistance profile TECi, and belonged to CC45 with SCC mec type I or II. PFGE type H had characteristics similar to those of CC239, while PFGE type I included three isolates, two of which expressed resistance to oxacillin and fusidic acid only. Two of these strains had SCC mec IVa and carried sequence type 389, with a multilocus sequence typing allelic profile of 3-35-19-2-20-26-39. Contemporary MRSAs causing bacteremia in Hong Kong hospitals belong to three clonal complexes (CC5, CC45, and CC239). The most prevalent MRSA clone in Hong Kong belongs to CC239, with PFGE types A to E and H, SCC mec type III, ST239, and a resistance profile of TEDGToCi.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in Irish hospitals between 1971 and 2002 were characterized using multilocus sequence typing (MLST) ( n = 130) and SCC mec typing ( n = 172). Where atypical SCC mec typing results were obtained, PCR amplification of entire SCC mec elements, analysis of amplimer mobility, and nucleotide sequencing were undertaken. MLST revealed that 129/130 isolates had the same genotypes as internationally spread MRSA clones, including ST239, ST247, ST250, ST5, ST22, ST36, and ST8. A novel genotype, ST496, was identified in one isolate. Half of the isolates (86/172) had SCC mec type I, IA, II, III, or IV. The remaining 86 isolates harbored novel SCC mec variants in three distinct genetic backgrounds: (i) 74/86 had genotype ST8 and either one of five novel SCC mec II (IIA, IIB, IIC, IID, and IIE) or one of two novel SCC mec IV (IVE and IVF) variants; (ii) 3/86 had genotype ST239 and a novel SCC mec III variant; (iii) 9/86 had a novel SCC mec I variant associated with ST250. SCC mec IVE and IVF were similar to SCC mec IVc and IVb, respectively, but differed in the region downstream of mecA . The five SCC mec II variants were similar to SCC mec IVb in the region upstream of the ccr complex but otherwise were similar to SCC mec II, except for the following regions: SCC mec IIA and IID had a novel mec complex, A.4 (Δ mecI -IS 1182- Δ mecI-mecR1-mecA- IS 431mec ); SCC mec IIC and IIE had a novel mec complex, A.3 (IS 1182 -Δ mecI - mecR1-mecA- IS 431mec ); SCC mec IID and IIE lacked pUB110; SCC mec IIC and IIE lacked a region of DNA between Tn 554 and the mec complex; and SCC mec IIB lacked Tn 554 . This study has demonstrated a hitherto-undescribed degree of diversity within SCC mec .

Community methicillin-resistant Staphylococcus aureus (CMRSA) strains are being isolated with increasing frequency around the world. In Western Australia CMRSA are endemic in geographically remote communities and have been found to belong to five different contour-clamped homogeneous electric field (CHEF) electrophoretic patterns. Representatives of each of these CHEF patterns have been compared to CMRSA representative of CHEF patterns from other Australian states and New Zealand. With one exception, all of the isolates were nonmultiresistant and were not resistant to many antimicrobial agents other than the β-lactams. With one exception, which is not believed to be a CMRSA, all of the isolates harbored a β-lactamase plasmid. Erythromycin resistance was associated with a 2-kb plasmid. One of the β-lactamase plasmids was found to be able to acquire additional resistance determinants to become a multiple resistance plasmid. There were 10 multilocus sequence types belonging to eight distantly related clonal complexes of S. aureus . One new sequence type was found. Although most of the CMRSA harbored the type IVa SCC mec , a type IV structural variant was found and two new SCC mec types were identified. Protein A gene ( spa ) typing revealed two new spa types and, with two exceptions, corresponded to multilocus sequence typing. In contrast to other reports on CMRSA, most of the CMRSA strains studied here did not contain the Panton-Valentine leukocidin genes. The results also demonstrate that nonmultiresistant hospital strains such as UK EMRSA-15 may be able to circulate in the community and could be mistaken for CMRSA based on their resistance profiles.

Staphylococcus aureus is an important nosocomial and community-acquired pathogen. Its genetic plasticity has facilitated the evolution of many virulent and drug-resistant strains, presenting a major and constantly changing clinical challenge. We sequenced the ≈2.8-Mbp genomes of two disease-causing S. aureus strains isolated from distinct clinical settings: a recent hospital-acquired representative of the epidemic methicillin-resistant S. aureus EMRSA-16 clone (MRSA252), a clinically important and globally prevalent lineage; and a representative of an invasive community-acquired methicillin-susceptible S. aureus clone (MSSA476). A comparative-genomics approach was used to explore the mechanisms of evolution of clinically important S. aureus genomes and to identify regions affecting virulence and drug resistance. The genome sequences of MRSA252 and MSSA476 have a well conserved core region but differ markedly in their accessory genetic elements. MRSA252 is the most genetically diverse S. aureus strain sequenced to date: ≈6% of the genome is novel compared with other published genomes, and it contains several unique genetic elements. MSSA476 is methicillin-susceptible, but it contains a novel Staphylococcal chromosomal cassette (SCC) mec -like element (designated SCC 476 ), which is integrated at the same site on the chromosome as SCC mec elements in MRSA strains but encodes a putative fusidic acid resistance protein. The crucial role that accessory elements play in the rapid evolution of S. aureus is clearly illustrated by comparing the MSSA476 genome with that of an extremely closely related MRSA community-acquired strain; the differential distribution of large mobile elements carrying virulence and drug-resistance determinants may be responsible for the clinically important phenotypic differences in these strains.

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.

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in many countries is increasing and, in hospitals in some areas, more than half of all S. aureus disease isolates are MRSA. MRSA strains are becoming increasingly multiresistant, and have recently developed resistance to vancomycin, used successfully to treat MRSA for more than 30 years. This review summarises recent studies that have elucidated the evolutionary history of MRSA. The first MRSA isolate evolved from a sensitive, epidemic strain prevalent in Europe, and its progeny—the first MRSA clone—quickly spread to other continents. Analyses of epidemic MRSA isolates from hospitals in different countries by molecular methods, including multilocus sequence typing (MLST) and DNA microarray analysis, reveal that MRSA strains have evolved separately within five distinct epidemic, sensitive lineages. However, resistance has been transferred to S. aureus on many more than five occasions, as some lineages have acquired different structural types of the element carrying the methicillin resistance gene. The emergence of MRSA as a community pathogen has been noted in several countries, and MLST and SCCmec typing have been used to demonstrate that community-acquired MRSA strains are typically related only distantly to hospital MRSA strains, and thus represent novel acquisitions of SCCmec.

To analyse the mutations and epidemiology associated with fluoroquinolone-resistant pneumococci collected as part of the PROTEKT global surveillance programme during 1999-2000.Sixty-nine centres in 25 countries submitted a total of 3362 Streptococcus pneumoniae isolates, for which the MICs of antimicrobial agents were determined using NCCLS methodology.Levofloxacin resistance was low overall (1% worldwide), with higher rates in: Hong Kong (14.3%), South Korea (2.9%), USA (1.8%), Mexico (1.5%), Canada (1.4%) and Japan (1.3%). Levofloxacin resistance was very low or absent in European countries, and absent in Australia. Worldwide, there was a total of 35 levofloxacin-resistant isolates, of which 22 (63%) were resistant and 10 (29%) were intermediate to moxifloxacin. All levofloxacin-resistant isolates were susceptible to telithromycin (or =0.5 mg/L), linezolid (or =2 mg/L) and quinupristin/dalfopristin (or =1 mg/L). One or more mutations in the topoisomerase genes were identified in all levofloxacin-resistant isolates; most of these isolates (33/35) had a mutation in one of the DNA gyrase encoding genes (gyrA, gyrB) and one of the topoisomerase IV encoding genes (parC, parE). Eighteen (51%) isolates carried the same combination of amino acid substitutions: Ser-81--Phe in GyrA and Ser-79--Phe in ParC. Isolates displaying a levofloxacin MIC of 2-4 mg/L generally had no mutation or one mutation in either a DNA gyrase or a topoisomerase IV gene, although most mutations were in parC.Most levofloxacin-resistant isolates possess two mutations (one in DNA gyrase and one in topoisomerase IV). Although multilocus sequence typing demonstrated that most of these isolates were unrelated, 12 (34%) were the Spain23F-1 clone: 10 from Hong Kong and one each from Saskatchewan, Canada and Sao Paulo, Brazil.

SCC mec is a mobile genetic element that carries the gene ( mecA ) mediating methicillin resistance in staphylococci. For Staphylococcus aureus , four SCC mec types have been described, one (type IV) of which has been associated with newly identified community-acquired methicillin-resistant S. aureus . However, the distribution of SCC mec types among S. epidermidis is not known. SCC mec typing of a collection of 44 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered between 1973 and 1983 from the blood of patients with prosthetic valve endocarditis (PVE) was performed by PCR amplification of key genetic elements ( mecA , mecI , IS 1272 , and ccrAB ). Of the 44 isolates, 1 (2%) harbored SCC mec type I, 15 (34%) harbored type II, 12 (28%) harbored type III, and 16 (36%) harbored type IV. The complete nucleotide sequence of SCC mec type IV was determined for 16 isolates and found to be identical in size (24 kb) and 98% homologous to DNA sequences published for S. aureus . Type IV SCC mec was also common (5 of 10 isolates) among a geographically dispersed collection of 10 recent (1998 to 2001) S. epidermidis bloodstream isolates. Multilocus sequence typing (MLST) (using the same seven genes presently employed for S. aureus MLST) of these MRSE isolates and of 10 additional recent geographically dispersed methicillin-susceptible isolates demonstrated that all 16 PVE isolates and 2 of 5 recent isolates harboring type IV SCC mec were in three related clonal groups. All three MSSE PVE isolates recovered from patients between 1976 and 1979 were in the same clonal groups as type IV SCC mec MRSE isolates. These data support the hypothesis of intra- and interspecies transfer of type IV SCC mec and suggest that there are clonal associations in S. epidermidis that correlate with SCC mec type.

A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes. The evolutionary origins of MRSA are poorly understood, no rational nomenclature exists, and there is no consensus on the number of major MRSA clones or the relatedness of clones described from different countries. We resolve all of these issues and provide a more thorough and precise analysis of the evolution of MRSA clones than has previously been possible. Using multilocus sequence typing and an algorithm, burst , we analyzed an international collection of 912 MRSA and methicillin-susceptible S. aureus (MSSA) isolates. We identified 11 major MRSA clones within five groups of related genotypes. The putative ancestral genotype of each group and the most parsimonious patterns of descent of isolates from each ancestor were inferred by using burst , which, together with analysis of the methicillin resistance genes, established the likely evolutionary origins of each major MRSA clone, the genotype of the original MRSA clone and its MSSA progenitor, and the extent of acquisition and horizontal movement of the methicillin resistance genes. Major MRSA clones have arisen repeatedly from successful epidemic MSSA strains, and isolates with decreased susceptibility to vancomycin, the antibiotic of last resort, are arising from some of these major MRSA clones, highlighting a depressing progression of increasing drug resistance within a small number of ecologically successful S. aureus genotypes.

Bacteria have unusual and variable sex lives, ranging from near-celibacy to evident promiscuity. “Sex” in bacteria involves a recipient bacterium replacing, by homologous recombination, small regions of its genome with corresponding regions from other strains ( 1 ). Studies over the past 20 years have demonstrated that in relatively promiscuous bacterial species, frequent recombination can drive rapid diversification of strains. In more celibate species, diversification is much slower and depends on the slow accumulation of point mutations ( 1 , 2 ). Sorting out the roles that these processes play in the pace and pattern of bacterial evolution, however, has proven problematic. On page 430 of this issue, Croucher et al. ( 3 ) demonstrate a powerful new approach to the problem. By comparing the genomes of many isolates of a single strain of a bacterium that causes pneumonia, they were able to rapidly obtain a comprehensive evaluation of the role that recombination, point mutation and other genetic processes played in its diversification. The approach offers insight into how this pathogenic strain can rapidly evolve resistance to antibiotics and evade future vaccines, and promises to help researchers better understand how this resistant strain may have spread globally.