Salmonella enterica serovar Typhimurium (STM) is a major cause of gastroenteritis and transmitted by consumption of contaminated food. STM is associated to food originating from animals (pork, ...chicken, eggs) or plants (vegetables, fruits, nuts, and herbs). Infection of warm-blooded mammalian hosts by STM and the underlying complex regulatory network of virulence gene expression depend on various environmental conditions encountered in hosts. However, less is known about the proteome and possible regulatory networks for gene expression of STM outside the preferred host. Nutritional limitations and changes in temperature are the most obvious stresses outside the native host. Thus, we analyzed the proteome profile of STM grown in rich medium (LB medium) or minimal medium (PCN medium) at temperatures ranging from 8 °C to 37 °C. LB medium mimics the nutritional rich environment inside the host, whereas minimal PCN medium represents nutritional limitations outside the host, found during growth of fresh produce (field conditions). Further, the range of temperatures analyzed reflects conditions within natural hosts (37 °C), room temperature (20 °C), during growth under agricultural conditions (16 °C and 12 °C), and during food storage (8 °C). Implications of altered nutrient availability and growth temperature on STM proteomes were analyzed by HPLC/MS-MS and label-free quantification. Our study provides first insights into the complex adaptation of STM to various environmental temperatures, which allows STM not only to infect mammalian hosts but also to enter new infection routes that have been poorly studied so far. With the present dataset, global virulence factors, their impact on infection routes, and potential anti-infective strategies can now be investigated in detail. Especially, we were able to demonstrate functional flagella at 12 °C growth temperature for STM with an altered motility behavior.
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•Systematic proteome analyses of STM grown at 37 °C compared to 20, 16, 12, 8 °C was performed.•Altered gene ontology classes reveal global adaptation of environmental temperatures.•Virulence proteins for interaction with mammalian hosts increased at 37 °C.•STM build functional flagella during growth at 12 °C with altered motility.
We performed systematic proteome analyses of Salmonella enterica serovar Typhimurium (STM) grown at human host body temperature (37 °C) compared to environmental temperatures relevant of agriculture (20, 16, 12, 8 °C). Altered abundance of members of various gene ontology classes reveal global adaptation of environmental temperatures. Virulence proteins for interaction with mammalian hosts were increased at 37 °C, while metabolic proteins were increased at lower temperatures. During growth at 12 °C, STM build functional flagella and showed altered motility.
In Salmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and ...cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology.
Bacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis.
Summary
Spi/Ssa is a unique type three secretion system that functions exclusively when Salmonella enterica is inside eukaryotic cells. Expression of the Spi/Ssa system and its secreted effectors is ...dependent on SsrB/SpiR, a two‐component regulatory system encoded in the SPI‐2 pathogenicity island that also harbours the spi/ssa genes. Here we determine that the PhoP/PhoQ two‐component system controls the intramacrophage expression of spi/ssa genes by regulating the SsrB/SpiR system. We establish that PhoP regulates transcription of the response regulator SsrB and demonstrate binding of the PhoP protein to the ssrB promoter both in vivo using chromatin immunoprecipitation in Salmonella‐infected macrophages, and in vitro using purified PhoP protein. We show that PhoP controls the SpiR sensor post‐transcriptionally and identify a region in the 5′ untranslated region of the spiR message that is required for this effect. The demonstration that the PhoP/PhoQ system is directly involved in the regulation of the SPI‐2 pathogenicity island highlights PhoP/PhoQ's central role in Salmonella virulence. We suggest that different regulatory systems convey distinct signals over time to produce the SsrB/SpiR system, which then modulates expression of the Spi/Ssa apparatus and secreted effectors.
Enterobacteriaceae producing β-lactamases have spread rapidly worldwide and pose a serious threat to human-animal-environment interface. In this study, we present the presence of Salmonella enterica ...(1.3%) and commensal Escherichia coli (96.3%) isolated from 400 environmental fecal dairy cattle samples over 20 farms in Uganda. Among E. coli isolates, 21% were resistant to at least one antimicrobial tested and 7% exhibited multidrug resistance. Four E. coli isolates displayed extended-spectrum beta-lactamase (ESBL)-producing genes, including bla
(n = 2/4), bla
(n = 1/4), bla
(n = 1/4), and bla
(n = 2/4). Whole genome sequencing confirmed the presence of the plasmid-mediated quinolone resistance qnrS1 gene among three ESBL isolates. No statistically significant differences in seasonal prevalence for E. coli and S. enterica among dairy cattle sampling periods were observed. Furthermore, to our knowledge, this is the first report of E. coli carrying bla
, bla
, bla
, or qnrS1 isolated from dairy cattle in Uganda. We conclude that the presence of globally disseminated bla
and bla
warrants further study to prevent further spread. In addition, the presence of fluoroquinolone resistant ESBL-producing E. coli on dairy farms highlights the potential risk among the human-livestock-environment interaction. This study can be used as a baseline for implementation of a more robust national integrated surveillance system throughout Uganda.
Abstract
Background
Salmonella enterica is a leading cause of human gastroenteritis. S. enterica strains that produce ESBLs (ESBL-Salm) remain rare in Europe and North America, but less is known ...about their prevalence among animal-derived foods in countries with weaker food safety practices and unregulated veterinary antibiotic use.
Objectives
To examine the prevalence and characteristics of ESBL-Salm from retail meats in Phnom Penh, Cambodia.
Methods
We tested fish, pork and chicken from two markets for ESBL- and carbapenemase-producing Salmonella from September–December 2016, using cefotaxime- and ertapenem-supplemented media, respectively. ESBL-Salm were sequenced and their genomes characterized. We performed plasmid conjugation experiments to assess the co-transferability of ESBL-encoding genes and MDR phenotypes.
Results
Twenty-six of 150 fish and meat samples (17%) were positive for ESBL-Salm, including 10/60 fish (17%), 15/60 pork (25%) and 1/30 chicken (3%). Carbapenemase-producing Salmonella strains were not detected. Pork-origin ESBL-Salm were primarily serotypes Rissen (10/15) or a monophasic variant of Typhimurium 4,5,12:i:− (3/15), whereas Saintpaul (3/10) and Newport (4/10) were more common among fish. Most ESBL enzymes were encoded by blaCTX-M-55 genes (24/26) harboured on conjugative IncA/C2 (n = 14) or IncHI2 (n = 10) plasmids. Resistance to up to six additional drug classes was co-transferred by each plasmid type. ESBL-Salm were resistant to almost every antibiotic recommended for severe salmonellosis treatment.
Conclusions
CTX-M-55-type S. enterica are highly prevalent among pork and fish from Phnom Penh markets and their spread appears to be mediated by MDR IncA/C2 and IncHI2 plasmids. Food safety must be improved and veterinary antibiotic use should be regulated to protect public health.
The purpose of this study was to determine the occurrence and genotypic relatedness of Salmonella enterica isolates recovered from feed and fecal samples in commercial swine production units. Of 275 ...feed samples, Salmonella was detected in 10 feed samples that originated from 8 of 36 (22.2%) barns, with a prevalence of 3.6% (10/275 samples). In fecal samples, a prevalence of 17.2% was found at the early finishing stage (1,180/6,880 samples), with a significant reduction in prevalence (7.4%) when pigs reached market age (392/5,321 samples). Of the 280 Salmonella isolates systematically selected for further characterization, 50% of the feed isolates and 55.3% of the isolates of fecal origin showed similar phenotypes based on antimicrobial resistance patterns and serogrouping. About 44% of the isolates were multidrug resistant. Pulsed-field gel electrophoresis (PFGE) genotyping grouped the 46 representative isolates into five genotypic clusters, of which four of the clusters consisted of genotypically related isolates recovered from feed and fecal samples. The occurrence of genotypically related and, in some cases, clonal strains, including multidrug-resistant isolates in commercially processed feed and fecal samples, suggests the high significance of commercial feed as a potential vehicle of Salmonella transmission.
Sixteen
Salmonella strains resistant to nalidixic acid isolated from kimbab, the most popular ready-to-eat (RTE) food in Korea, and chicken meat were selected for this study. The resistant strains ...were shown to have high minimal inhibitory concentrations (MICs) against nalidixic acid (512
~
4096
μg/mL). Among them, 4
Salmonella enterica serovar Haardt isolates showed multi-drug resistance (MDR) patterns with reduced susceptibility to fluoroquinolone (0.5
μg/mL of ciprofloxacin MICs). The mechanisms of quinolone resistance in the nalidixic acid resistant strains were characterized by PCR and sequence analysis. The presence of plasmid-mediated quinolone resistance (PMQR) genes and amino acid changes in the quinolone resistance determining region (QRDR) were investigated by PCR-based detection and sequencing, and the efflux pump inhibition test was also done using phe-arg-β-naphthylamide (PAβN). Although PMQR genes were not detected in any of the tested strains, the QRDR mutations were found in this study: single mutation in
gyrA (Asp87Tyr, Asp87Gly, and Asp87Asn), double mutations in
gyrA (Ser83Thr) and
parC (Thr57Ser), and single mutation in
parC (Thr57Ser). MICs of nalidixic acid were reduced by 2- to 32-folds by the efflux pump inhibitor, PAβN. Pulsed-field gel electrophoresis (PFGE) was carried out to confirm the epidemiological relationship between the nalidixic acid resistant strains. The PFGE patterns were classified into 6 groups at cutoff level of 70
~
100% correlation on the dendrogram. Some strains of serotype Haardt and Enteritidis showed several values of genomic identity in accordance with strains, sources, and isolation year. We suggest that point mutation on QRDR and efflux pump systems involved in antimicrobials had independent effects on drug-resistance regardless of bacterial genomic variation.
► The PMQR genes were not detected in any of the tested strains. ► QRDR in
gyrA and
parC had a key role of quinolone resistance in the resistant strains. ► Efflux inhibition of PAßN could not reduce MIC of NA below the breakpoint. ► QRDR and efflux had independent effects on resistance regardless of genomic variation.
Marine-derived fungi constitute an interesting source of bioactive compounds, several of which exhibit antibacterial activity. These acquire special importance, considering that antimicrobial ...resistance is becoming more widespread. The overexpression of efflux pumps, capable of expelling antimicrobials out of bacterial cells, is one of the most worrisome mechanisms. There has been an ongoing effort to find not only new antimicrobials, but also compounds that can block resistance mechanisms which can be used in combination with approved antimicrobial drugs. In this work, a library of nineteen marine natural products, isolated from marine-derived fungi of the genera
and
, was evaluated for their potential as bacterial efflux pump inhibitors as well as the antimicrobial-related mechanisms, such as inhibition of biofilm formation and quorum-sensing. Docking studies were performed to predict their efflux pump action. These compounds were also tested for their cytotoxicity in mouse fibroblast cell line NIH/3T3. The results obtained suggest that the marine-derived fungal metabolites are a promising source of compounds with potential to revert antimicrobial resistance and serve as an inspiration for the synthesis of new antimicrobial drugs.
The reactive enamine 2-aminoacrylate (2AA) is a metabolic stressor capable of damaging cellular components. Members of the broadly conserved Rid (RidA/YER057c/UK114) protein family mitigate 2AA ...stress in vivo by facilitating enamine and/or imine hydrolysis. Previous work showed that 2AA accumulation in ridA strains of Salmonella enterica led to the inactivation of multiple target enzymes, including serine hydroxymethyltransferase (GlyA). However, the specific cause of a ridA strain's inability to grow during periods of 2AA stress had yet to be determined. Work presented here shows that glycine supplementation suppressed all 2AA-dependent ridA strain growth defects described to date. Depending on the metabolic context, glycine appeared to suppress ridA strain growth defects by eliciting a GcvB small RNA-dependent regulatory response or by serving as a precursor to one-carbon units produced by the glycine cleavage complex (GCV). In either case, the data suggest that GlyA is the most physiologically sensitive target of 2AA inactivation in S. enterica. The universally conserved nature of GlyA among free-living organisms highlights the importance of RidA in mitigating 2AA stress.
The RidA stress response prevents 2-aminoacrylate (2AA) damage from occurring in prokaryotes and eukaryotes alike. 2AA inactivation of serine hydroxymethyltransferase (GlyA) from Salmonella enterica restricts glycine and one-carbon production, ultimately reducing fitness of the organism. The cooccurrence of genes encoding 2AA production enzymes and serine hydroxy-methyltransferase (SHMT) in many genomes may in part underlie the evolutionary selection for Rid proteins to maintain appropriate glycine and one-carbon metabolism throughout life.
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