Phylogenomic Analysis of Salmonella enterica subsp. enterica Serovar Bovismorbificans from Clinical and Food Samples Using Whole Genome Wide Core Genes and kmer Binning Methods to Identify Two Distinct Polyphyletic Genome Pathotypes

Salmonella enterica subsp. enterica serovar Bovismorbificans has caused multiple outbreaks involving the consumption of produce, hummus, and processed meat products worldwide. To elucidate the intra-serovar genomic structure of S. Bovismorbificans, a core-genome analysis with 2690 loci (based on 150 complete genomes representing Salmonella enterica serovars developed as part of this study) and a k-mer-binning based strategy were carried out on 95 whole genome sequencing (WGS) assemblies from Swiss, Canadian, and USA collections of S. Bovismorbificans strains from foodborne infections. Data mining of a digital DNA tiling array of legacy SARA and SARB strains was conducted to identify near-neighbors of S. Bovismorbificans. The core genome analysis and the k-mer-binning methods identified two polyphyletic clusters, each with emerging evolutionary properties. Four STs (2640, 142, 1499, and 377), which constituted the majority of the publicly available WGS datasets from >260 strains analyzed by k-mer-binning based strategy, contained a conserved core genome backbone with a different evolutionary lineage as compared to strains comprising the other cluster (ST150). In addition, the assortment of genotypic features contributing to pathogenesis and persistence, such as antimicrobial resistance, prophage, plasmid, and virulence factor genes, were assessed to understand the emerging characteristics of this serovar that are relevant clinically and for food safety concerns. The phylogenomic profiling of polyphyletic S. Bovismorbificans in this study corresponds to intra-serovar variations observed in S. Napoli and S. Newport serovars using similar high-resolution genomic profiling approaches and contributes to the understanding of the evolution and sequence divergence of foodborne Salmonellae. These intra-serovar differences may have to be thoroughly understood for the accurate classification of foodborne Salmonella strains needed for the uniform development of future food safety mitigation strategies

Physiological contribution of the Pseudomonas aeruginosa OprD family of porins

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins to facilitate nutrient uptake. These channels have substrate-specific binding sites that selectively permit the passage of related classes of molecules. In this study, the identification of a novel 19 member family of porins is reported. The members of this family share a considerable degree of protein sequence conservation (46% to 57%) and fall into one of two phylogenetically distinct clusters; one bearing high similarity to the basic amino acid specific porin OprD and the other being most similar to the phenylacetic acid uptake porin, PhaK, of P. putida . The physiological contribution of this family was investigated by predicting substrates for of each homologue based on the genomic context of their genes. These predictions were then tested by performing growth curves with porin deficient mutants in minimal media containing the proposed substrate as the sole carbon source. The following substrates were identified for 7 of the 18 novel homologues: OpdB - proline, OpdC - histidine, OpdP - glycine-glutamate, OpdT - tyrosine, OpdH - cis-aconitate, OpdK--vanillate and OpdO--pyroglutamate. Functional overlap was observed between the basic amino acid specific porin, OprD, and the glycine-glutamate specific porin, OpdP, with respect to arginine transport. Thus, members of this family had diverged to take up unique substrates but had also retained some redundancy which may allow them to compensate for one another in the event of a gene loss or mutation. To gain insights regarding the evolution of this family, one homologue, OpdH, was characterized in detail. This porin was specifically induced by citrate, isocitrate and cis-aconitate through the action of the PA0756-757 two-component regulatory system and was involved in the uptake of the latter two compounds. The channel properties of OpdH differed significantly from those of OprD. OpdH demonstrated an average single channel conductance of 0.7 nS in 1 M KCl. The channel was cation selective and did not harbour a tricarboxylate specific binding site. The structural and evolutionary implications of the differences between the two porins are discussed.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Salmonella enterica Outbreaks Linked to the Consumption of Tahini and Tahini-Based Products

Salmonella is a leading cause of bacterial foodborne illness in the world. Although typically associated with foods of animal origin, low-moisture foods, such as tahini, are quickly gaining recognition as an important vehicle of Salmonella exposure. This review offers the Canadian perspective on the issue of Salmonella in tahini and tahini-based products. A summary of several recent food product recalls and foodborne outbreaks related to the presence of Salmonella in tahini and tahini-based products such as halva are presented. The properties of the food vehicles, their production practices, and potential routes of contamination are discussed. Particular focus is placed on the ecology of Salmonella in the tahini production continuum, including its survival characteristics and response to intervention technologies

SarZ Promotes the Expression of Virulence Factors and Represses Biofilm Formation by Modulating SarA and agr in Staphylococcus aureus▿

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

Sérovars de Salmonella enterica associés à des bactériémies au Canada, 2006 à 2019

Contexte : Les membres du genre bactérien Salmonella sont à l’origine de la salmonellose, une maladie dont les manifestations cliniques varient d’une gastro-entérite autolimitée à une bactériémie plus grave, une défaillance organique et une septicémie. Le genre se compose de plus de 2 600 variantes sérologiques (sérovars). On a remarqué des différences importantes dans la pathogenèse des sérovars de Salmonella. Objectif : Le but de cette étude était de déterminer quels sérovars de Salmonella étaient plus susceptibles d’être associés à des bactériémies au Canada. Méthodes : Pour chaque sérovar, on a extrait les informations sur le nombre total d’infections à Salmonella et d’isolats sanguins signalés au Programme national de surveillance des maladies entériques de 2006 à 2019. Le risque (proportion) et la probabilité (cote) de bactériémie ont été calculés pour tous les sérovars. Résultats : Sur les 96 082 cas de salmonellose signalés au Programme national de surveillance des maladies entériques pendant la période d’étude de 14 ans, 4,4 % (IC 95 % : 4,3 %–4,6 %) étaient bactériémiques. Vingt sérovars de Salmonella non typhique ont été associés à des taux plus faibles de bactériémie par rapport à tous les sérovars de Salmonella non typhique, et 19 sérovars de Salmonella non typhique ont été identifiés comme ayant des taux plus élevés, Heidelberg, Oranienburg, Schwarzengrund, Virchow, Panama et Poona comptant parmi les 25 sérovars les plus fréquemment signalés au Canada pendant la période d’étude. Conclusion : L’identification des sérovars associés aux bactériémies à Salmonella au Canada est un premier pas vers la compréhension des différences dans la pathogenèse et la présentation de la maladie

Salmonella enterica serovars associated with bacteremia in Canada, 2006–2019

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

Role of the Novel OprD Family of Porins in Nutrient Uptake in Pseudomonas aeruginosa

To circumvent the permeability barrier of its outer membrane, Pseudomonas aeruginosa has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of Pseudomonas putida. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an opdK mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with opdK. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH, cis-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms

Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates

The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis-aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions

MgrA Represses Biofilm Formation in Staphylococcus aureus▿

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

Overexpression of MazFSa in Staphylococcus aureus Induces Bacteriostasis by Selectively Targeting mRNAs for Cleavage▿

The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazFSa, a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazFSa induction. This finding suggested that the toxin MazFSa induced cell stasis rather than cell death. We also showed that MazFSa selectively cleaves cellular mRNAs in vivo, avoiding “important” transcripts such as recA, gyrB, and sarA mRNAs in MazFSa-induced cells, while these three mRNAs can be cleaved in vitro. The results of Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein. These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon the expression of MazFSa in vivo