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Background: Members of the bacterial genus Salmonella cause salmonellosis, a disease with a spectrum of clinical presentations from a self-limiting gastroenteritis to more severe bacteremia, organ failure and sepsis. The genus consists of over 2,600 serological variants (serovars). Important differences in the pathogenesis of Salmonella serovars have been noted. Objective: The purpose of this study was to determine which Salmonella serovars were more likely to be associated with bacteremia in Canada. Methods: Information on the total number of Salmonella infections and blood isolations reported to the National Enteric Surveillance Program (NESP) from 2006 to 2019 was extracted for each serovar. The risk (proportion) and likelihood (odds) of bacteremia were calculated for all serovars. Results: Of the 96,082 Salmonella cases reported to the NESP during the 14-year study period, 4.4% (95% CI: 4.3%–4.6%) were bacteremic. Twenty nontyphoidal Salmonella (NTS) serovars were associated with lower rates of bacteremia compared to all NTS serovars, and 19 NTS serovars were identified as having higher rates. Heidelberg, Oranienburg, Schwarzengrund, Virchow, Panama and Poona among the top 25 most commonly reported serovars in Canada during the study period. Conclusion: The identification of serovars associated with Salmonella bacteremia in Canada is a first step towards understanding differences in pathogenesis and disease presentation.

Bacterial carbapenem resistance is a major public health concern since these antimicrobials are often the last resort to treat serious human infections. To evaluate methodologies for detection of carbapenem resistance, carbapenem-tolerant bacteria were isolated from wastewater treatment plants in Toronto, Ottawa, and Arnprior, Ontario. A total of 135 carbapenem-tolerant bacteria were recovered. Polymerase chain reaction (PCR) indicated the presence of carbapenem hydrolysing enzymes KPC ( n = 10), GES ( n = 5), VIM ( n = 7), and IMP ( n = 1), and β-lactamases TEM ( n = 7), PER ( n = 1), and OXA-variants ( n = 16). A subset of 46 isolates were sequenced and analysed using ResFinder and CARD-RGI. Both programs detected carbapenem resistance genes in 35 sequenced isolates and antimicrobial resistance genes (ARGs) conferring resistance to multiple class of other antibiotics. Where β-lactamase resistance genes were not initially identified, lowering the thresholds for ARG detection enabled identification of closely related β-lactamases. However, no known carbapenem resistance genes were found in seven sequenced Pseudomonas spp. isolates. Also of note was a multi-drug-resistant Klebsiella pneumoniae isolate from Ottawa, which harboured resistance to seven antimicrobial classes including β-lactams. These results highlight the diversity of genes encoding carbapenem resistance in Ontario and the utility of whole genome sequencing over PCR for ARG detection where resistance may result from an assortment of genes.

Over the past 15 years, multiple foodborne outbreaks have occurred in Canada due to the presence of Salmonella enterica in frozen breaded chicken products. These chicken products were raw and required cooking in conventional household ovens to inactivate any pathogens that they may have been harboring. During the course of food safety investigations associated with these outbreaks, many consumers reported using alternative household appliances such as air fryers to cook these products. The effectiveness of these appliances for the inactivation of pathogens in food is not known. Here, we compare the ability of a toaster oven, air fryer, deep fryer, and conventional oven to inactivate a cocktail of Salmonella Enteritidis in frozen breaded chicken strips. Deep frying was the most effective cooking method, demonstrating a median 7-log reduction; the conventional oven was next with a median 6-log reduction. Both the air fryer and toaster oven performed poorly, with respective median 4- and 3-log reductions. Overall, the results of this study suggest the revision of cooking instructions is required for the safe household use of toaster ovens and air fryers. HIGHLIGHTS

Background Salmonella enterica is a leading cause of foodborne illness worldwide resulting in considerable public health and economic costs. Testing for the presence of this pathogen in food is often hampered by the presence of background microflora that may present as Salmonella (false positives). False positive isolates belonging to the genus Citrobacter can be difficult to distinguish from Salmonella due to similarities in their genetics, cell surface antigens, and other phenotypes. In order to understand the genetic basis of these similarities, a comparative genomic approach was used to define the pan-, core, accessory, and unique coding sequences of a representative population of Salmonella and Citrobacter strains. Results Analysis of the genomic content of 58 S. enterica strains and 37 Citrobacter strains revealed the presence of 31,130 and 1540 coding sequences within the pan- and core genome of this population. Amino acid sequences unique to either Salmonella (n = 1112) or Citrobacter (n = 195) were identified and revealed potential niche-specific adaptations. Phylogenetic network analysis of the protein families encoded by the pan-genome indicated that genetic exchange between Salmonella and Citrobacter may have led to the acquisition of similar traits and also diversification within the genera. Conclusions Core genome analysis suggests that the Salmonella enterica and Citrobacter populations investigated here share a common evolutionary history. Comparative analysis of the core and pan-genomes was able to define the genetic features that distinguish Salmonella from Citrobacter and highlight niche specific adaptations.

The incidence of Salmonella enterica infection resulting from consumption of chicken products has historically been elevated in British Columbia compared with the rest of Canada. Raw frozen breaded chicken products are often implicated as the source of infection as there is a potential for consumers to not cook these products adequately. This occurs because the production process for these foods involves par-frying, a step which lends a cooked appearance to the product surface without reaching the internal temperatures required to fully inactivate potential pathogens. A survey of frozen chicken products from 10 retail stores of various sizes was conducted in order to determine the type and source of frozen chicken products that are available for purchase in British Columbia. Information on 391 individual products was collected and 50 were sampled for microbiological testing. Raw frozen breaded chicken products represented 59% of the frozen chicken products available to consumers at retail; 34% of these raw products were made by a single processor. The same processor was also found to have the highest proportion (33%) of samples testing positive for Salmonella. Whole genome sequencing of isolates obtained during this study revealed that majority of these isolates were phylogenetically related to clinical isolates of Salmonella. A substantial reduction of risk and increased consumer protection may be achieved by implementing a kill step (e.g., cook process that has been validated to achieve a 7-log reduction) during production of these products.

Since 2015, 11 recalls of live oyster shellstock have been issued in Canada due to the presence of Salmonella enterica. Six of those recalls took place in 2018. To understand this increase, fundamental information is needed on the relationship between S. enterica and oysters. The aims of this study were to address important data gaps concerning the levels of Salmonella in naturally contaminated oysters and the ability of this pathogen to survive in live oyster shellstock. Enumeration data were evaluated for five oyster and clam samples collected from the east coast of Canada from 2015 to 2018. The reported levels were

Salmonella is a major foodborne pathogen and is responsible for a range of diseases. Not all Salmonella contributes to severe health outcomes as there is a large degree of genetic heterogeneity among the 2,600 serovars within the genus. This variability across Salmonella serovars is linked to numerous genetic elements that dictate virulence. While several genetic elements encode virulence factors with well-documented contributions to pathogenesis, many genetic elements implicated in Salmonella virulence remain uncharacterized. Many pathogens encode a family of E3 ubiquitin ligases that are delivered into the cells that they infect using a Type 3 Secretion System (T3SS). These effectors, known as NEL-domain E3s, were first characterized in Salmonella. Most Salmonella encodes the NEL-effectors sspH2 and slrP, whereas only a subset of Salmonella encodes sspH1. SspH1 has been shown to ubiquitinate the mammalian protein kinase PKN1, which has been reported to negatively regulate the pro-survival program Akt. We discovered that SspH1 mediates the degradation of PKN1 during infection of a macrophage cell line but that this degradation does not impact Akt signaling. Genomic analysis of a large collection of Salmonella genomes identified a putative new gene, sspH3, with homology to sspH1. SspH3 is a novel NEL-domain effector.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

The incidence of

Since 2015, 11 recalls of live oyster shellstock have been issued in Canada due to the presence of

Between November 2018 and May 2019, Canada experienced a nationwide salmonellosis outbreak linked to the presence of Salmonella enterica ser. Enteritidis in frozen profiteroles. Analysis of the implicated food products revealed low levels of Salmonella ranging from 0.2 to 0.7 MPN/100g. Water activity and pH of the food samples ranged from 0.9479 to 0.9867 and 4.6-6.8 respectively indicating conditions conducive to bacterial growth. Higher levels of the hygiene indicators Enterobacteriaceae and coliforms were associated with Salmonella positive samples compared to Salmonella negative samples. Investigation of the relationship between storage conditions, temperature, and pathogen levels during thawing revealed that the profiteroles reached temperatures permissive to pathogen growth (≥5 °C) much sooner than pathogen growth was observed and that the composition of the food matrix can influence bacterial levels upon thawing. Collectively these data can be used to inform guidance to minimize the risk of infection from the consumption of contaminated cream-filled frozen desserts.

Various methods have been described to isolate third generation cephalosporin (3GC) resistant Enterobacteriaceae from foods, but it is not known how comparable they are between studies. Here, the performance of five enrichment broths and two selective agars are compared for their ability to isolate 3GC resistant Enterobacteriaceae from retail chicken, beef, pork, and veal samples. The results showed equivalence between Enterobacteriaceae enrichment broth (EE), lauryl sulfate broth (LST), and modified typtone soy broth (mTSB). Lower isolation rates were observed when LST and mTSB were supplemented with the 3GC antibiotic cefotaxime . The overall performance of MacConkey agar supplemented with cefotaxime and a proprietary selective agar (ESBL CHROMagar) was equivalent, although differences linked to the microbiota of specific meat commodities were noted. Regardless of the isolation method, further screening was required to confirm the taxonomy and resistance of the presumptive positive strains. Approximately 40% of confirmed 3GC resistant foodborne Enterobacteriaceae strains tested positive for extended spectrum beta-lactamase (ESBL) activity. Strains that were resistant to ceftriaxone and susceptible to cefoxitin were more likely to test positive for ESBL activity, as were strains that possessed either of two ESBL genes ( blaSHV or blaTEM). Based on our results, we recommend using an antibiotic-free enrichment broth, two selective agars, and an isolate screening strategy to isolate 3GC resistant Enterobacteriaceae from retail meats. Antibiotic susceptibility testing and/or PCR screening for blaSHV or blaTEM can then be used to identify ESBL producing strains among the 3GC resistant meat isolates. The adoption of this approach by the research community will enable more effective monitoring of antibiotic resistance rates and trends among foodborne Enterobacteriaceae over time and across jurisdictions.

A significant data gap exists with respect to the levels of pathogens in foods implicated in foodborne outbreaks. These data are essential for the quantification of pathogen exposure via the ingestion of contaminated food. Here we report the levels of the foodborne pathogen Salmonella in comminuted raw chicken products that had been breaded and then frozen. The products investigated were collected during four food safety investigations of foodborne outbreaks that occurred in Canada from 2014 to 2016. Most-probable-number (MPN) distribution analysis of the food samples revealed Salmonella levels of 0.0018 to 3 MPN/g, which is equivalent to 1 MPN per 0.33 to 556 g of product. These data suggest low levels of Salmonella may be associated with foodborne outbreaks.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing

Despite the increasing popularity of sprouted chia and flax seed powders, no data have been reported on their intrinsic physicochemical properties and background microflora. Here, we report the moisture content, water activity, pH, and fatty acid methyl ester and bacteriological profiles of 19 sprouted chia and flax seed samples, 10 of which were associated with an outbreak of salmonellosis in Canada and the United States. The physicochemical parameters of the Salmonella-positive samples did not differ significantly from those of the negative samples. However, the higher Enterobacteriaceae and coliform levels on the contaminated powders were associated with the presence of Salmonella. Enumeration of Salmonella by the most probable number (MPN) method revealed concentrations ranging from 1 MPN per 3 g of powder to 1 MPN per 556 g of powder. The results of this study demonstrate that low numbers of Salmonella may be linked to foodborne outbreaks.

Salmonella enterica is one of the pathogens that is frequently identified as the cause of fresh produce-related outbreaks. Biofilm formation is a factor that can contribute to pathogen survival on produce surface. The goal of our current research was to investigate the survival of five S. enterica strains representing different serotypes (i.e., Typhimurium, Enteritidis, Daytona, Poona, and Newport) on whole mini cucumbers stored at refrigeration (4°C) and room temperature (22°C). We also determined the strains survival on glass slides and in phosphate-buffered saline at 4 and 22°C, as well as the ability to form biofilms on a solid-liquid interphase. A rapid decrease in cell density (>4-log reduction over 8 days) of all five tested strains was observed on glass slides, while a slower die-off (

We evaluated the relative response of generic Escherichia coli (GEC), Shiga toxin-producing E. coli (STEC) and Salmonella enterica to heat, alkaline or acid treatments. GEC included strains from carcasses (n = 24) and trim (n = 25) at a small beef plant where no decontamination interventions are used and at a large plant where multiple decontamination interventions are used (carcass n = 25 and trim n = 25). STEC strains belonging to nine serogroups, included isolates from cattle (n = 53), beef (n = 16) and humans (n = 44). S. enterica strains belonging to 29 serotypes, included isolates from humans (n = 30), poultry (n = 26), pork (n = 10) and beef (n = 33). Strains were grown in Brain Heart Infusion (BHI) broth and subjected to the following treatments: 60 °C for 2 min, 5% lactic acid (pH 2.9) for 1 h at 4 °C, or NaOH (pH 11.0) for 2 h at 4 °C. Median log reductions of the GEC populations after heat, alkaline and acid treatment ranged from 2.3 to 3.8, 0.7 to 2.2 and 0.7 to 1.2 log CFU/mL, respectively. No statistically significant difference in reductions was observed between carcass GEC or trim GEC from the large or small plant, except for a greater reduction in trim GEC from the small plant. Median reductions of the STEC populations ranged from 3.3 to 3.5, 0.0 to 0.6, and 0.3 to 0.5 log CFU/mL after heat, alkaline and acid treatment, respectively. The median reductions were not dependent upon isolation source, except between STEC cattle and human isolates after alkaline treatment, where the reduction of the former was higher by 0.6 log unit. For the Salmonella populations, median log reductions ranged from 3.5 to 4.0, 1.7 to 2.4 and 3.7 to 4.1 log CFU/mL after heat, alkaline and acid treatment, respectively. The reductions were not isolation source related. The median log reductions were in the order GEC STEC Salmonella after heat treatment and STEC GEC Salmonella after alkaline or acid treatment. Overall, the relative response of GEC, STEC and Salmonella in the model system suggests that exposure to heat or pH-based decontamination interventions in meat plants is not associated with increased resistance among E. coli strains in these environments, and total E. coli counts on beef can be indicative of treatment efficacy for the control of Salmonella by heat, lactic acid and alkaline treatment and for the control of STEC subjected to heat.

High-pressure processing is a nonthermal method of food preservation that uses pressure to inactivate microorganisms. To ensure the effective validation of process parameters, it is important that the design of challenge protocols consider the potential for resistance in a particular species. Herein, the responses of 99 diverse Salmonella enterica strains to high pressure are reported. Members of this population belonged to 24 serovars and were isolated from various Canadian sources over a period of 26 years. When cells were exposed to 600 MPa for 3 min, the average reduction in cell numbers for this population was 5.6 log 10 CFU/ml, with a range of 0.9 log 10 CFU/ml to 6 log 10 CFU/ml. Eleven strains, from 5 serovars, with variable levels of pressure resistance were selected for further study. The membrane characteristics (propidium iodide uptake during and after pressure treatment, sensitivity to membrane-active agents, and membrane fatty acid composition) and responses to stressors (heat, nutrient deprivation, desiccation, and acid) for this panel suggested potential roles for the cell membrane and the RpoS regulon in mediating pressure resistance in S. enterica . The data indicate heterogeneous and multifactorial responses to high pressure that cannot be predicted for individual S. enterica strains. IMPORTANCE The responses of foodborne pathogens to increasingly popular minimal food decontamination methods are not understood and therefore are difficult to predict. This report shows that the responses of Salmonella enterica strains to high-pressure processing are diverse. The magnitude of inactivation does not depend on how closely related the strains are or where they were isolated. Moreover, strains that are resistant to high pressure do not behave similarly to other stresses, suggesting that more than one mechanism might be responsible for resistance to high pressure and the mechanisms used may vary from one strain to another.

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23 different integrase genes within the food-associated isolates, but only identified four different phages and integrase genes within clonal isolates of S. Enteritidis and S. Heidelberg. These results demonstrate the potential usefulness of PCR based detection of prophage integrase genes as a rapid indicator of genome diversity in S. enterica.

A 2014 foodborne salmonellosis outbreak in Canada and the United States implicated, for the first time, sprouted chia seed powder as the vehicle of transmission. Here, we report the draft whole genome sequences of two Salmonella enterica strains isolated from sprouted powders related to the aforementioned outbreak.

The diversity of the genus Salmonella is reflected in the physiological adaptations used by its members in response to stressors such as high pressure. Here we report the draft whole genome sequences of 11 Salmonella enterica strains, five sensitive strains and six demonstrating high levels of pressure resistance.

Probiotic organisms have shown promise in treating diseases. Previously, we have reported on the efficacy of microencapsulated Lactobacillus reuteri NCIMB 30242 in a yogurt formulation at lowering serum cholesterol levels in otherwise healthy hypercholesterolemic adults. This study investigates the safety and toxicology of oral ingestion of microencapsulated L. reuteri NCIMB 30242 in a yogurt formulation. A randomized group of 120 subjects received a dose of 5 × 10(10) CFU microencapsulated L. reuteri NCIMB 30242 in yogurt (n=59) or placebo yogurt (n=61) twice/day for 6 weeks. Clinical chemistry and hematological parameters of safety were analyzed. Fecal samples were collected at these time points for the analysis of deconjugated bile acids. The frequency, duration and intensity of adverse events (AEs) and clinical significance of safety parameters were recorded for both groups. No clinically significant differences between the probiotic yogurt and placebo yogurt treated groups were detected in either the blood clinical chemistry or hematology results and there was no significant increase in fecal deconjugated bile acids (P>0.05) between treated and control groups. The frequency and intensity of AEs was similar in the two groups. These results demonstrate the safe use of this formulation in food.

The expression of virulence factors in Staphylococcus aureus is tightly coordinated by a vast network of regulatory molecules. In this report, we characterize a genetic locus unique to staphylococci called rsr that has a role in repressing two key virulence regulators, sarR and agr . Using strain SH1000, we showed that the transcription of virulence effectors, such as hla , sspA , and spa , is altered in an rsr mutant in a way consistent with agr upregulation. Analysis of RNAIII expression of the agr locus in rsr and rsr-sarR mutants indicated that rsr likely contributes to agr expression independently of SarR. We also provide evidence using a murine model of S. aureu s skin infection that the effects mediated by rsr reduce disease progression.

The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1 ) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to β-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro , virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.

Staphylococcus aureus is a remarkably adaptable organism capable of multiple modes of growth in the human host, as a part of the normal flora, as a pathogen, or as a biofilm. Many of the regulatory pathways governing these modes of growth are centered on the activities of two regulatory molecules, the DNA binding protein SarA and the regulatory RNAIII effector molecule of the agr system. Here, we describe the modulation of these regulators and their downstream target genes by SarZ, a member of the SarA/MarR family of transcriptional regulators. Transcriptional and phenotypic analyses of a sarZ mutant demonstrated that the decreased transcription of mgrA and the agr RNAIII molecule was accompanied by increased transcription of spa (protein A) and downregulation of hla (alpha-hemolysin) and sspA (V8 protease) transcripts when compared to its isogenic parent. The decrease in protease activity was also associated with an increase in SarA expression. Consistent with an increase in SarA levels, the sarZ mutant displayed an enhanced ability to form biofilms. Together, our results indicate that SarZ may be an important regulator governing the dissemination phase of S. aureus infections, as it promotes toxin expression while repressing factors required for biofilm formation.

MgrA is a pleiotropic regulator that controls autolysis, virulence, and efflux pump activity in Staphylococcus aureus . We recently found that mgrA mutants of strains RN6390, SH1000, and MW2 also displayed enhanced biofilm formation compared with their respective parents. The biofilms formed by mgrA mutants of RN6390 and MW2 are independent of sigB and ica loci, two genetic elements that have been previously associated with biofilm formation in S. aureus . Biofilms formed by mgrA mutants are dependent on the expression of surface proteins mediated by the sortase gene srtA . Extracellular DNA was also a crucial component of the early biofilm of mgrA mutants. Genetic analysis indicated that biofilm formation in mgrA mutants is mediated in part by agr RNAIII, a genetic locus regulated by mgrA . Additionally, SarA is important to biofilm formation in mgrA mutants since the double sarA mgrA mutants failed to form biofilms compared to single mgrA mutants of RN6390 and MW2. However, the SarA-mediated effect is independent of agr and proteases such as V8 protease and aureolysin. Collectively, our data showed MgrA to be a repressor of biofilm formation, and biofilms formed by mgrA mutants have features that are distinct from other reported biofilm types in S. aureus .

Staphylococcus aureus is widely appreciated as an opportunistic pathogen, primarily in hospital-related infections. However, recent reports indicate that S. aureus infections can now occur in other wise healthy individuals in the community setting. The success of this organism can be attributed to the large array of regulatory proteins, including the SarA protein family, used to respond to changing microenvironments. Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: 1) single domain proteins; 2) double domain proteins and 3) MarR homologs. Structural studies have also demonstrated that SarA, SarR, SarS, MgrA, and thus possibly all members of this protein family are winged helix proteins with minor variations. Mutagenesis studies of SarA disclose that the winged helix motifs are important for DNA binding and function. Recent progress concerning the functions and plausible mechanisms of regulation of SarA and its homologs are discussed.

Pseudomonas species have over 300 known and putative nutrient uptake systems enabling them to metabolize a large number of organic compounds, and thus inhabit many diverse ecological niches. The outer membrane of these organisms acts as a semi-permeable barrier, excluding many classes of potentially toxic molecules from the cell. Nutrients use specialized water-filled channels called porins to traverse this barrier. Entry into the cell is mediated by one of four classes of cytoplasmic membrane transporters: glycerol facilitators, phosphotransferase systems, primary active transporters, and secondary active transporters. The class of transporter used is dependent on the environmental conditions, as well as the type and concentration of solute. The recent advances in elucidating the structures and functional mechanisms of these uptake systems will be discussed in this review.

Deoxyribonucleic acid (DNA) electrophoresis and blotting are techniques commonly used to visualise DNA. Both techniques use simple, inexpensive protocols that rely on fundamental properties of DNA, and these protocols have changed little since they were introduced. Electrophoresis relies on the negative electrical charge of DNA to draw these molecules through a gel, separating DNA molecules on the basis of size. Southern blotting makes use of sequence complementarity to identify specific DNA fragments on the basis of their sequences. Despite the introduction of more powerful methods such as polymerase chain reaction and DNA sequencing, electrophoresis and blotting techniques are still widely used to identify DNA fragments of interest, including the identification of unusual structures within DNA and the analysis of larger fragments which are not easily analysed by other methods. Key Concepts: DNA electrophoresis and blotting are fundamental methods of molecular biology. Basic physical and chemical properties of DNA are used to analyse unknown sequences. Gel electrophoresis uses an electrical current to separate DNA by size. Southern blotting locates a specific DNA sequence on a gel using a probe sequence that is its chemical match. Minor variations on these procedures can improve results for specific experiments. These techniques are still used to analyse unknown DNA molecules and identify DNA fragments of interest. The principles behind electrophoresis and blotting can also be seen in newer methods. Keywords: DNA detection; DNA analysis; gel electrophoresis; alkaline blotting; southern blotting; hybridisation

OprG of Pseudomonas aeruginosa is a member of the very large and widely distributed but poorly characterized OmpW (PF0392) family of outer membrane proteins. It was established here that OprG was highly transcribed in anaerobic environments rich in iron via the ANR regulator. In the absence of OprG, P. aeruginosa was significantly less cytotoxic toward human bronchial epithelial cells. Planar bilayer studies indicated that purified OprG formed cationic-selective channels with a conductance of 500 pS in 1 M KCl; however, contrary to previous reports, OprG did not appear to be involved in either iron or antibiotic uptake.