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1 - 8 of 8 for "Jay C. D. Hinton"
Microbe Profile: Salmonella Typhimurium: the master of the art of adaptation
Salmonella Typhimurium is a major Salmonella serovar that is found globally. It is responsible for outbreaks of self-limiting gastroenteritis that are broadly linked to the industrialization of food production. S. Typhimurium is a pathogen with a broad host range and remarkable metabolic versatility. The ∼5 Mb genome includes the pSLT virulence plasmid and has a characteristic prophage repertoire. The major virulence determinants are encoded by a variety of pathogenicity islands. Emerging multidrug-resistant lineages of epidemics of S. Typhimurium are currently causing bloodstream infections in sub-Saharan Africa. The versatility and adaptability of S. Typhimurium pose an important public health challenge.
Class 1 integrons in clinical and swine industry isolates of Salmonella Typhimurium from Colombia, dating 1997 to 2017
Background. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) has been linked to outbreaks of foodborne gastroenteritis disease and the emergence of antimicrobial-resistant clones. In Colombia laboratory surveillance of Salmonella spp. between 1997–2018 revealed that S. Typhimurium was the most ubiquitous serovar (27.6 % of all Salmonella isolates) with increasing levels of resistance to several families of antibiotics.
Hypothesis. Resistant isolates of S. Typhimurium recovered from human clinical food and swine samples carry class 1 integrons that are linked to antimicrobial resistance genes.
Aim. Identify class 1 integrons and investigate their association with other mobile genetic elements and their relationship to the antimicrobial resistance of Colombian S. Typhimurium isolates.
Methods. In this study 442 isolates of S. Typhimurium were analysed of which 237 were obtained from blood culture 151 from other clinical sources 4 from non-clinical sources and 50 from swine samples. Class 1 integrons and plasmid incompatibility groups were analysed by PCR and whole-genome sequencing (WGS) and regions flanking integrons were identified by WGS. The phylogenetic relationship was established by multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) distances for 30 clinical isolates.
Results . Overall 39 % (153/392) of the human clinical isolates and 22 % (11/50) of the swine S. Typhimurium isolates carried complete class 1 integrons. Twelve types of gene cassette arrays were identified including dfr7-aac-bla OXA-2 (Int1-Col1) which was the most common one in human clinical isolates (75.2 % 115/153). Human clinical and swine isolates that carried class 1 integrons were resistant to up to five and up to three antimicrobial families respectively. The Int1-Col1 integron was most prevalent in stool isolates and was associated with Tn21. The most common plasmid incompatibility group was IncA/C.
Conclusions. The widespread presence of the IntI1-Col1 integron in Colombia since 1997 was striking. A possible relationship between integrons source and mobile elements that favour the spread of antimicrobial resistance determinants in Colombian S. Typhimurium was identified.
Genome-wide fitness analysis identifies genes required for in vitro growth and macrophage infection by African and global epidemic pathovariants of Salmonella enterica Enteritidis
Salmonella enterica Enteritidis is the second most common serovar associated with invasive non-typhoidal Salmonella (iNTS) disease in sub-Saharan Africa. Previously genomic and phylogenetic characterization of S . enterica Enteritidis isolates from the human bloodstream led to the discovery of the Central/Eastern African clade (CEAC) and West African clade which were distinct from the gastroenteritis-associated global epidemic clade (GEC). The African S . enterica Enteritidis clades have unique genetic signatures that include genomic degradation novel prophage repertoires and multi-drug resistance but the molecular basis for the enhanced propensity of African S . enterica Enteritidis to cause bloodstream infection is poorly understood. We used transposon insertion sequencing (TIS) to identify the genetic determinants of the GEC representative strain P125109 and the CEAC representative strain D7795 for growth in three in vitro conditions (LB or minimal NonSPI2 and InSPI2 growth media) and for survival and replication in RAW 264.7 murine macrophages. We identified 207 in vitro-required genes that were common to both S . enterica Enteritidis strains and also required by S . enterica Typhimurium S . enterica Typhi and Escherichia coli and 63 genes that were only required by individual S . enterica Enteritidis strains. Similar types of genes were required by both P125109 and D7795 for optimal growth in particular media. Screening the transposon libraries during macrophage infection identified 177 P125109 and 201 D7795 genes that contribute to bacterial survival and replication in mammalian cells. The majority of these genes have proven roles in Salmonella virulence. Our analysis uncovered candidate strain-specific macrophage fitness genes that could encode novel Salmonella virulence factors.
A window into lysogeny: revealing temperate phage biology with transcriptomics
Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence stress tolerance or phage resistance and these functions are encoded by ‘accessory’ or ‘moron’ loci. Whilst the majority of phage-encoded genes are repressed during lysogeny accessory loci are often highly expressed. However it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach we identify a novel antisense RNA species in prophage BTP1 STnc6030 which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.
Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium
The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.
Multiple redundant stress resistance mechanisms are induced in Salmonella enterica serovar Typhimurium in response to alteration of the intracellular environment via TLR4 signalling
Toll-like receptor 4 (TLR4) senses bacterial LPS and is required for the control of systemic Salmonella enterica serovar Typhimurium infection in mice. The mechanisms of TLR4 activation and its downstream signalling cascades are well described yet the direct effects on the pathogen of signalling via this receptor remain unknown. To investigate this we used microarray-based transcriptome profiling of intracellular S. Typhimurium during infection of primary bone marrow-derived macrophages from wild-type and TLR4-deficient mice. We identified 17 S. Typhimurium genes that were upregulated in the presence of functional TLR4. Nine of these genes have putative functions in oxidative stress resistance. We therefore examined S. Typhimurium gene expression during infection of NADPH oxidase-deficient macrophages which lack normal oxidative killing mechanisms. We identified significant overlap between the ‘TLR4-responsive’ and ‘NADPH oxidase-responsive’ genes. This is new evidence for a link between TLR4 signalling and NADPH oxidase activity. Interestingly with the exception of a dps mutant S. Typhimurium strains lacking individual TLR4- and/or oxidative stress-responsive genes were not attenuated during intravenous murine infections. Our study shows that TLR4 activity either directly or indirectly induces the expression of multiple stress resistance genes during the intracellular life of S. Typhimurium.
A combination of cytochrome c nitrite reductase (NrfA) and flavorubredoxin (NorV) protects Salmonella enterica serovar Typhimurium against killing by NO in anoxic environments
The enteric bacterium Salmonella enterica serovar Typhimurium is a pathogen that is highly adapted for both intracellular and extracellular survival in a range of oxic and anoxic environments. The cytotoxic radical nitric oxide (NO) is encountered in many of these environments. Protection against NO may involve reductive detoxification in low-oxygen environments and three enzymes flavorubredoxin (NorV) flavohaemoglobin (HmpA) and cytochrome c nitrite reductase (NrfA) have been shown to reduce NO in vitro. In this work we determined the role of these three enzymes in NO detoxification by Salmonella by assessing the effects of all eight possible combinations of norV hmpA and nrfA single double and triple mutations. The mutant strains were cultured and exposed to NO following either glucose fermentation (when nitrite reductase activity is low) or anaerobic respiration (when nitrite reductase activity is high). Wild-type cultures were more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions. Analysis of the mutant strains suggested an important additive role for both NorV and NrfA in both environments since the norV nrfA mutant could not grow after NO addition. The results also suggested a minor role for HmpA in anaerobic detoxification of NO under the two growth conditions and a larger role for HmpA in aerobic NO detoxification was confirmed. Activity assays and measurements of NO consumption showed that increased nitrite reductase activity correlates with an elevated capacity for NO reduction by intact cells. Taken together the results reveal a combined role for NorV and NrfA in NO detoxification under anaerobic conditions and highlight the influence that growth conditions have on the sensitivity to NO of this pathogenic bacterium.
A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium
Fis is a key DNA-binding protein involved in nucleoid organization and modulation of many DNA transactions including transcription in enteric bacteria. The regulon of genes whose expression is influenced by Fis in Salmonella enterica serovar Typhimurium (S. typhimurium) has been defined by DNA microarray analysis. These data suggest that Fis plays a central role in coordinating the expression of both metabolic and type III secretion factors. The genes that were most strongly up-regulated by Fis were those involved in virulence and located in the pathogenicity islands SPI-1 SPI-2 SPI-3 and SPI-5. Similarly motility and flagellar genes required Fis for full expression. This was shown to be a direct effect as purified Fis protein bound to the promoter regions of representative flagella and SPI-2 genes. Genes contributing to aspects of metabolism known to assist the bacterium during survival in the mammalian gut were also Fis-regulated usually negatively. This category included components of metabolic pathways for propanediol utilization biotin synthesis vitamin B12 transport fatty acids and acetate metabolism as well as genes for the glyoxylate bypass of the tricarboxylic acid cycle. Genes found to be positively regulated by Fis included those for ethanolamine utilization. The data reported reveal the central role played by Fis in coordinating the expression of both housekeeping and virulence factors required by S. typhimurium during life in the gut lumen or during systemic infection of host cells.