Craig Altier

Intestinal short‐chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA

Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI‐1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI‐1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl‐phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl‐phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Δ(ackA–pta) mutation alone had no effect on virulence but, in combination with Δ(barA), it increased the oral LD50 24‐fold. Thus, the combined loss of the BarA‐ and acetate‐dependent pathways is required to reduce virulence. Two other short‐chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild‐type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild‐type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella, whereas those of the large intestine inhibit invasion.

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI‐1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI‐1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non‐motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2‐propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co‐ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2‐propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co‐ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Characterization of two novel regulatory genes affecting Salmonella invasion gene expression.

A Salmonella typhimurium chromosomal deletion removing ≈19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype. As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation. We find that the deletion includes two unrelated regulatory genes. One is the Salmonella homologue of Escherichia coli barA (airS ), which encodes a member of the multistep phosphorelay subgroup of two‐component sensor kinases. The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA‐dependent and HilA‐independent components. The other regulatory gene removed by the deletion is the Salmonella homologue of E. coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E. coli. These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.

SdiA, an N-acylhomoserine lactone receptor, becomes active during the transit of Salmonella enterica through the gastrointestinal tract of turtles

Background LuxR-type transcription factors are typically used by bacteria to determine the population density of their own species by detecting N-acylhomoserine lactones (AHLs). However, while Escherichia and Salmonella encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species. Methodology/Principal Findings In this report, we tested the hypothesis that SdiA detects the AHL-production of other bacterial species within the animal host. SdiA did not detect AHLs during the transit of Salmonella through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during the transit of Salmonella through turtles. All turtles examined were colonized by the AHL-producing species Aeromonas hydrophila. Conclusions/Significance We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals.

Molecular epidemiology and diversity of Salmonella serovar Typhimurium in pigs using phenotypic and genotypic approaches.

For epidemiological investigations of the most common and non-host-adapted Salmonella serotypes, such as Typhimurium, highly discriminatory approaches are essential. In the present study, we evaluated three genotyping methods; amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and repetitive palindromic extragenic-PCR (Rep-PCR) using 40 isolates. AFLP showed the highest discriminatory index (0.939), resolution and throughput. To determine clonality of Salmonella Typhimurium isolates and epidemiological relatedness in different commercial pig production units, we employed AFLP in combination with antimicrobial resistance pattern and phage typing. Salmonella serovar Typhimurium isolates (n=196) obtained from a longitudinal study of 18 pig farms over a 3-year period were studied. Using this approach, 16 distinct clonal types were identified. We found two common multidrug- resistant patterns including AmCmStSuTe and AmKmStSuTe. Two commonly multidrug- resistant phage types that are of known public health importance, DT104 and DT193, were also common. AFLP differentiated distinct clones within DT104, a phage type previously reported to be clonal. Fourteen of the clonal types were unique to one of the two production systems, showing diversity between independent commercial pig production systems located in the same geographical area. Clonal types obtained from nursery farms and corresponding finishing units were, however, similar.

Salmonella enterica serovars from pigs on farms and after slaughter and validity of using bacteriologic data to define herd Salmonella status

The primary objective of this study was to evaluate the validity of using data obtained from slaughtered pigs for farm-level epidemiologic studies of Salmonella. The study involved groups of pigs from five farms. Salmonella isolates were obtained from on-farm samples, and a total of 370 on-farm and an additional 486 isolates from samples collected after commercial slaughter were subsequently tested. Preharvest samples included feces of individual animals from defined groups of nursery and finishing pigs on commercial farms and swabs from trucks. Postslaughter samples were cecal contents and mesenteric lymph node samples. The concordance between Salmonella serovars isolated from on-farm samples and those serovars isolated after slaughter varied widely among farms. Results of paired lymph node and cecal cultures were strongly associated (odds ratio, 7.0), but the agreement between on-farm and postslaughter results at the pig level was poor (kappa = 0.34). The results support recent findings that risk of exposure to Salmonella during transport and lairage remains a concern under contemporary industry conditions. The findings further imply that slaughter plant studies based on phenotyping of Salmonella alone (such as serovars) may not reliably indicate the Salmonella status of commercial swine farms.

Characterization of Antimicrobial-Resistant Phenotypes and Genotypes among Salmonella enterica Recovered from Pigs on Farms, from Transport Trucks, and from Pigs after Slaughter

The main objectives of this study were to determine antimicrobial resistance patterns among Salmonella serotypes and to evaluate the role of transport trucks in dissemination of antimicrobial-resistant strains of Salmonella. Salmonella from groups of nursery and finishing pigs on farms, from trucks, and from pigs after slaughter were compared using serotyping, patterns of antimicrobial resistance, and pulsed-field gel electrophoresis patterns. The five farms included in the study yielded 858 isolates representing 27 Salmonella serovars. The most common resistance observed (80% of all isolates) was to tetracycline; resistance to ampicillin (42%), chloramphenicol (31%), amoxicillin/clavulanic acid (30%), and piperacillin (31%) also were common. We found a correlation between serovar and antimicrobial resistance. High correlation was found between Salmonella Typhimurium var. Copenhagen and chloramphenicol resistance (Spearman rank correlation, rho = 0.7). Multidrug resistance was observed primarily in Salmonella Typhimurium var. Copenhagen (94%) and Salmonella Typhimurium (93%) and was much less common in the other common serovars, including Salmonella Derby (7%) and Salmonella Heidelberg (8%). Of the 225 isolates exhibiting the most common pentaresistance pattern in this study, amoxicillin/clavulanic acid-ampicillin-chloramphenicol-piperacillin-tetracycline, 220 (98%) were Salmonella Typhimurium var. Copenhagen, and 86% of the isolates of this serovar had this pattern. Isolates from the trucks were similar, based on pulsed-field gel electrophoresis patterns, to those from the cecum and mesenteric lymph nodes of pigs on two of the farms, suggesting the probable infection of pigs during transport. Class I integrons were also common among various serovars.