George R. Golding

Livestock-associated Methicillin- Resistant Staphylococcus aureus Sequence Type 398 in Humans, Canada

Recent emergence of infections resulting from this strain is of public health concern.

Comparative Genomics of Canadian Epidemic Lineages of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has disseminated throughout Canadian hospitals and communities. Pulsed-field gel electrophoresis of over 9,300 MRSA isolates obtained from the Canadian Nosocomial Infection Surveillance Program has identified 10 epidemic strain types in Canada (CMRSA1 to CMRSA10). In an attempt to determine specific genetic factors that have contributed to their high prevalence in community and/or hospital settings, the genomic content of representative isolates for each of the 10 Canadian epidemic types was compared using comparative genomic hybridizations. Comparison of the community-associated Canadian epidemic isolates (CMRSA7 and CMRSA10) with the hospital-associated Canadian epidemic isolates revealed one open reading frame (ORF) (SACOL0046) encoding a putative protein belonging to a metallo-beta-lactamase family, which was present only in the community-associated Canadian epidemic isolates. A more restricted comparison involving only the most common hospital-associated Canadian epidemic isolates (CMRSA1 and CMRSA2) with the community-associated Canadian epidemic isolates did reveal additional factors that might be contributing to their prevalence in the community and hospital settings, which included ORFs encoding potential virulence factors involved in capsular biosynthesis, serine proteases, epidermin, adhesion factors, regulatory functions, leukotoxins, and exotoxins.

A Preliminary Guideline for the Assignment of Methicillin-Resistant Staphylococcus aureus to a Canadian Pulsed-Field Gel Electrophoresis Epidemic Type Using spa Typing

BACKGROUND Increasing rates of methicillin-resistant Staphylococcus aureus (MRSA) infections on a global scale is a major health concern. In Canada, there are 10 known epidemic types of MRSA as determined by pulsed-field gel electrophoresis (PFGE). Despite the excellent discriminatory power of PFGE, there are several disadvantages of using this technique, such as high degree of labour intensity and the inability to easily develop an MRSA typing database due to the subjective interpretation of results. OBJECTIVES The purpose of the present study was to determine whether spa typing, an established DNA sequence-based typing method, could be used as an alternative to PFGE for the typing of Canadian MRSA (CMRSA) epidemic isolates. RESULTS spa types were determined for 1488 CMRSA isolates, and the method was analyzed for its ability to identify and cluster CMRSA1-10 strains. Minimal spanning tree analysis of 1452 spa types revealed individual clonal clusters for PFGE epidemic types CMRSA1, 2, 7 and 8, but spa typing could not distinguish CMRSA5 from CMRSA9 and CMRSA10, and CMRSA3 from CMRSA4 and CMRSA6. However, specific spa types were generally associated with only one PFGE epidemic type. Based on these results, a spa typing guideline for CMRSA isolates was developed and tested using the first 300 MRSA isolates received in 2007 through the Canadian Nosocomial Infection Surveillance Program. CONCLUSIONS The high concordance of spa types with PFGE epidemic types using this guideline demonstrated the feasibility of spa typing as a more rapid and less technically demanding alternative typing method for MRSA in Canada.

Secondary Chromosomal Attachment Site and Tandem Integration of the Mobilizable Salmonella Genomic Island 1

Background The Salmonella genomic island 1 is an integrative mobilizable element (IME) originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104. SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. Previous studies have shown that SGI1 integrates site-specifically into the S. enterica, Escherichia coli, or Proteus mirabilis chromosome at the 3′ end of thdF gene (attB site). Methodology/Principal Findings Here, we report the transfer of SGI1 to a ΔthdF mutant of S. Typhimurium LT2. In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude. Through DNA sequencing SGI1 was shown to integrate specifically into a secondary attachment site (2nd attB), which is located in the intergenic region between the chromosomal sodB and purR genes. At this 2nd attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of ΔthdF mutant) contained tandem SGI1 arrays. Moreover, in wild type S. Typhimurium LT2 transconjugants, SGI1 integrated into both attachment sites, i.e., thdF and sodB-purR. The formation of SGI1 tandem arrays occurred in both specific attB sites. There was heterogeneity in the size of the SGI1 tandem arrays detected in single transconjugant colonies. Some arrays consisted as far as six SGI1s arranged in tandem. These tandem arrays were shown to persist during serial passages with or without antibiotic selection pressure. Conclusions/Significance The ability of integration into two distinct chromosomal sites and tandem array formation of SGI1 could contribute to its spread and persistence. The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1st or 2nd specific SGI1 attB sites of Salmonella.

Changing epidemiology of methicillin-resistant Staphylococcus aureus in Canada

OBJECTIVES To compare the demographics, antimicrobial susceptibilities and molecular epidemiology of community-associated (CA) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus (MRSA) in Canada. METHODS Between 2007 and 2011, 1266 MRSA were collected from inpatients and outpatients attending tertiary-care medical centres across Canada. Susceptibility testing was performed using broth microdilution and isolates were characterized by spa typing and PCR to detect the Panton-Valentine leucocidin (PVL) gene. Detection of heterogeneous vancomycin-intermediate S. aureus (hVISA) was performed using the Etest macromethod and confirmed by population analysis profiling. RESULTS The annual proportion of S. aureus that were methicillin resistant decreased from 26.1% in 2007 to 19.3% in 2011 (P= 0.0002). Of 1266 MRSA isolated, 366 (28.9%) were CA-MRSA genotypes and 868 (68.6%) were HA-MRSA genotypes. The proportion of MRSA represented by CA-MRSA genotypes increased from 19.7% to 36.4% between 2007 and 2011 (P < 0.0001). CMRSA10 (USA300) was the predominant CA-MRSA genotype (22.1%); the most common HA-MRSA genotype was CMRSA2 (USA100/800) (58.1%). PVL was detected in 328/366 (89.6%) of CA-MRSA genotypes and 6/868 (0.7%) of HA-MRSA genotypes. The hVISA phenotype was detected in 7/27 (25.9%) of MRSA with a vancomycin MIC of 2 mg/L. CONCLUSIONS The most frequent CA-MRSA genotype was CMRSA10 (USA300), while CMRSA2 (USA100/800) was the predominant HA-MRSA genotype. Despite a decrease in the numbers of MRSA, the proportion of CMRSA10 (USA300) CA-MRSA has risen significantly between 2007 and 2011 in Canada.

Effect of pH on Intracellular Accumulation of Trace Concentrations of Hg(II) in Escherichia coli under Anaerobic Conditions, as Measured Using a mer-lux Bioreporter

ABSTRACT The effects of pH on the uptake and accumulation of Hg(II) by Escherichia coli were determined at trace, environmentally relevant, concentrations of Hg and under anaerobic conditions. Hg(II) accumulation was measured using inducible light production from E. coli HMS174 harboring a mer-lux bioreporter plasmid (pRB28). The effect of pH on the toxicity of higher concentrations of Hg(II) was measured using a constitutive lux plasmid (pRB27) in the same bacterial host. In this study, intracellular accumulation and toxicity of Hg(II) under anaerobic conditions were both significantly enhanced with decreasing pH over the pH range of 8 to 5. The pH effect on Hg(II) accumulation was most pronounced at pHs of <6, which substantially enhanced the Hg(II)-dependent light response. This enhanced response did not appear to be due to pH stress, as similar results were obtained whether cells were grown at the same pH as the assay or at a different pH. The enhanced accumulation of Hg(II) was also not related to differences in the chemical speciation of Hg(II) in the external medium resulting from the changes in pH. Experiments with Cd(II), also detectable by the mer-lux bioreporter system, showed that Cd(II) accumulation responded differently to pH changes than the net accumulation of Hg(II). Potential implications of these findings for our understanding of bacterial accumulation of Hg(II) under anaerobic conditions and for bacteria-mediated cycling of Hg(II) in aquatic ecosystems are discussed. Arguments are provided suggesting that this differential accumulation is due to changes in uptake of mercury.

A comparison of risk factors associated with community-associated methicillin-resistant and -susceptible Staphylococcus aureus infections in remote communities

In this case-control study, cases [community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), n=79] and controls [community-associated methicillin-susceptible S. aureus (CA-MSSA), n=36] were defined as a laboratory-confirmed infection in a patient with no previous hospital-associated factors. Skin and soft tissue were the predominant sites of infection, both for cases (67.1%) and controls (55.6%). Most of the cases (79.7%) and controls (77.8%) were aged <30 years. Investigations did not reveal any significant statistical differences in acquiring a CA-MRSA or CA-MSSA infection. The most common shared risk factors included overcrowding, previous antibiotic usage, existing skin conditions, household exposure to someone with a skin condition, scratches/insect bites, and exposure to healthcare workers. Similar risk factors, identified for both CA-MRSA and CA-MSSA infections, suggest standard hygienic measures and proper treatment guidelines would be beneficial in controlling both CA-MRSA and CA-MSSA in remote communities.

Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.

High rates of Staphylococcus aureus USA400 infection, Northern Canada.

Surveillance of Staphylococcus aureus infections in 3 northern remote communities of Saskatchewan was undertaken. Rates of methicillin-resistant infections were extremely high (146–482/10,000 population), and most (98.2%) were caused by USA400 strains. Although USA400 prevalence has diminished in the United States, this strain is continuing to predominate throughout many northern communities in Canada.

Changing epidemiology of methicillin-resistant Staphylococcus aureus in Alberta, Canada: population-based surveillance, 2005-2008.

SUMMARYIncreasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has been reported in Canada. We report the results of a prospective surveillance of MRSA infections in Alberta over a consecutive 3-year period. A total of 8910 unique clinical MRSA isolates was analysed from July 2005 to June 2008. The incidence of MRSA infection increased over the study period and was highest in males, age group ⩾85 years, and the Calgary Area. CMRSA10 (USA300) and CMRSA2 (USA100/800) were the most common PFGE strain types, representing 53·0% and 27·9% of all isolates, respectively. Significant differences were noted between MRSA strains in the source of infection and antimicrobial susceptibility. The incidence of MRSA infection in Alberta has nearly doubled in the last 3 years; this is attributed to the emergence of CMRSA10 as the predominant strain.