Nonredundant Dimethyl Sulfoxide Reductases Influence Salmonella enterica Serotype Typhimurium Anaerobic Growth and Virulence.

Facultative anaerobic enteric pathogens can utilize a diverse array of alternate electron acceptors to support anaerobic metabolism and thrive in the hypoxic conditions within the mammalian gut. Dimethyl sulfoxide (DMSO) is produced by methionine catabolism and can act as an alternate electron acceptor to support anaerobic respiration. The DMSO reductase complex consists of three subunits, DmsA, DmsB, and DmsC, and allows bacteria to grow anaerobically with DMSO as an electron acceptor. The genomes of nontyphoidal Salmonella enterica encode three putative dmsABC operons, but the impact of the apparent genetic redundancy in DMSO reduction on the fitness of nontyphoidal S. enterica during infection remains unknown. We hypothesized that DMSO reduction would be needed for S. enterica serotype Typhimurium to colonize the mammalian gut. We demonstrate that an S. Typhimurium mutant with loss of function in all three putative DMSO reductases (ΔdmsA3) poorly colonizes the mammalian intestine when the microbiota is intact and when inflammation is absent. DMSO reduction enhances anaerobic growth through nonredundant contributions of two of the DMSO reductases. Furthermore, DMSO reduction influences virulence by increasing expression of the type 3 secretion system 2 and reducing expression of the type 3 secretion system 1. Collectively, our data demonstrate that the DMSO reductases of S. Typhimurium are functionally nonredundant and suggest DMSO is a physiologically relevant electron acceptor that supports S. enterica fitness in the gut.

YeiE Regulates Motility and Gut Colonization in Salmonella enterica Serotype Typhimurium.

Regulation of flagellum biosynthesis is a hierarchical process that is tightly controlled to allow for efficient tuning of flagellar expression. Flagellum-mediated motility directs Salmonella enterica serovar Typhimurium toward the epithelial surface to enhance gut colonization, but flagella are potent activators of innate immune signaling, so fine-tuning flagellar expression is necessary for immune avoidance. In this work, we evaluate the role of the LysR transcriptional regulator YeiE in regulating flagellum-mediated motility. We show that yeiE is necessary and sufficient for swimming motility. A ΔyeiE mutant is defective for gut colonization in both the calf ligated ileal loop model and the murine colitis model due to its lack of motility. Expression of flagellar class 2 and 3 but not class 1 genes is reduced in the ΔyeiE mutant. We linked the motility dysregulation of the ΔyeiE mutant to repression of the anti-FlhD4C2 factor STM1697. Together, our results indicate that YeiE promotes virulence by enhancing cell motility, thereby providing a new regulatory control point for flagellar expression in Salmonella Typhimurium. IMPORTANCE The ability to finely tune virulence factor gene expression is required for bacterial pathogens to successfully colonize a host. Flagellum-mediated motility is critical for many gut pathogens to establish productive infections. However, flagella activate the immune system, leading to bacterial clearance; therefore, tight control of flagellar gene expression enhances bacterial fitness in the host. Here, we demonstrate that the transcriptional regulator YeiE acts as a control point for flagellar gene expression and is required for Salmonella Typhimurium to establish a productive infection in mammals. The expression of an inhibitor of flagellar biogenesis is repressed in the absence of yeiE. Our work adds a new layer to the tightly controlled cascade regulating control of flagellar gene expression to facilitate the fitness of an enteric pathogen.

Sulfate Import in Salmonella Typhimurium Impacts Bacterial Aggregation and the Respiratory Burst in Human Neutrophils.

During enteric salmonellosis, neutrophil-generated reactive oxygen species alter the gut microenvironment, favoring survival of Salmonella Typhimurium. While type 3 secretion system 1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone is responsible for stimulation of a maximal respiratory burst. To identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in coculture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single-gene deletion mutants representing particular regions under selection. A subset of single-gene deletion mutants was selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal medium and adhered to surfaces in rich medium, suggesting a role for sulfur homeostasis in the regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

Genetic Determinants of Salmonella Resistance to the Biofilm-Inhibitory Effects of a Synthetic 4-Oxazolidinone Analog.

Biofilms formed by Salmonella enterica are a frequent source of food supply contamination. Since biofilms are inherently resistant to disinfection, new agents capable of preventing biofilm formation are needed. Synthetic analogs of 4-oxazolidinone containing natural products have shown promise as antibiofilm compounds against Gram-positive bacteria. The purpose of our study was 2-fold: to establish the antibiofilm effects and mechanism of action of a synthetic 4-oxazolidinone analog (JJM-ox-3-70) and to establish mechanisms of resistance to this compound in Salmonella enterica serovar Typhimurium (S Typhimurium). JJM-ox-3-70 inhibited biofilm formation but had no effect on cell growth. The antibiofilm effects were linked to disruption of curli fimbriae and flagellar gene expression and alteration in swimming motility, suggesting an effect on multiple cellular processes. Using a 2-step screening approach of defined multigene and single-gene deletion mutant libraries, we identified 3 mutants that produced less biofilm in the presence of JJM-ox-3-70 than the isogenic WT, with phenotypes reversed by complementation in trans Genes responsible for S Typhimurium resistance to the compound included acrB, a component of the major drug efflux pump AcrAB-TolC, and two genes of unknown function (STM0437 and STM1292). The results of this study suggest that JJM-ox-3-70 inhibits biofilm formation by indirect inhibition of extracellular matrix production that may be linked to disruption of flagellar motility. Further work is needed to establish the role of the newly characterized genes as potential mechanisms of biofilm intrinsic antimicrobial resistance.IMPORTANCE Biofilms are resistant to killing by disinfectants and antimicrobials. S. enterica biofilms facilitate long-term host colonization and persistence in food processing environments. Synthetic analogs of 4-oxazolidinone natural products show promise as antibiofilm agents. Here, we show that a synthetic 4-oxazolidinone analog inhibits Salmonella biofilm through effects on both motility and biofilm matrix gene expression. Furthermore, we identify three genes that promote Salmonella resistance to the antibiofilm effects of the compound. This work provides insight into the mechanism of antibiofilm effects of a synthetic 4-oxazolidinone analog in Gram-negative bacteria and demonstrates new mechanisms of intrinsic antimicrobial resistance in Salmonella biofilms.

Linearized Siderophore Products Secreted via MacAB Efflux Pump Protect Salmonella enterica Serovar Typhimurium from Oxidative Stress.

Nontyphoidal salmonellae (NTS) are exposed to reactive oxygen species (ROS) during their residency in the gut. To survive oxidative stress encountered during infection, salmonellae employ several mechanisms. One of these mechanisms involves the multidrug efflux pump MacAB, although the natural substrate of this pump has not been identified. MacAB homologs in pseudomonads secrete products of nonribosomal peptide synthesis (NRPS). In Salmonella enterica serovar Typhimurium, the siderophore enterobactin is produced by NRPS in response to iron starvation and this molecule can be processed into salmochelin and several linear metabolites. We found that Salmonella mutants lacking the key NRPS enzyme EntF are sensitive to peroxide mediated killing and cannot detoxify extracellular H2O2 Moreover, EntF and MacAB function in a common pathway to promote survival of Salmonella during oxidative stress. We further demonstrated that S. Typhimurium secretes siderophores in iron-rich media when peroxide is present and that these MacAB-secreted metabolites participate in protection of bacteria against H2O2 We showed that secretion of anti-H2O2 molecules is independent of the presence of the known siderophore efflux pumps EntS and IroC, well-described efflux systems involved in secretion of enterobactin and salmochelin. Both salmochelin and enterobactin are dispensable for S. Typhimurium protection against ROS; however, linear metabolites of enterobactin produced by esterases IroE and Fes are needed for bacterial survival in peroxide-containing media. We determined that linearized enterobactin trimer protects S. Typhimurium against peroxide-mediated killing in a MacAB-dependent fashion. Thus, we suggest that linearized enterobactin trimer is a natural substrate of MacAB and that its purpose is to detoxify extracellular reactive oxygen species.IMPORTANCE Nontyphoidal Salmonella bacteria induce a classic inflammatory diarrhea by eliciting a large influx of neutrophils, producing a robust oxidative burst. Despite substantial progress understanding the benefits to the host of the inflammatory response to Salmonella, little is known regarding how Salmonella can simultaneously resist the damaging effects of the oxidative burst. The multidrug efflux pump MacAB is important for survival of oxidative stress both in vitro and during infection. We describe a new pathway used by Salmonella Typhimurium to detoxify extracellular reactive oxygen species using a multidrug efflux pump (MacAB) to secrete a linear siderophore, a metabolite of enterobactin. The natural substrates of many multidrug efflux pumps are unknown, and functional roles of the linear metabolites of enterobactin are unknown. We bring two novel discoveries together to highlight an important mechanism used by Salmonella to survive under the oxidative stress conditions that this organism encounters during the classic inflammatory diarrhea that it also induces.

Effect of butorphanol on thermal nociceptive threshold in healthy pony foals.

REASONS FOR PERFORMING STUDY: Pain management is an important component of foal nursing care, and no objective data currently exist regarding the analgesic efficacy of opioids in foals. OBJECTIVES: To evaluate the somatic antinociceptive effects of 2 commonly used doses of intravenous (i.v.) butorphanol in healthy foals. Our hypothesis was that thermal nociceptive threshold would increase following i.v. butorphanol in a dose-dependent manner in both neonatal and older pony foals. METHODS: Seven healthy neonatal pony foals (age 1-2 weeks), and 11 healthy older pony foals (age 4-8 weeks). Five foals were used during both age periods. Treatments, which included saline (0.5 ml), butorphanol (0.05 mg/kg bwt) and butorphanol (0.1 mg/kg bwt), were administered i.v. in a randomised crossover design with at least 2 days between treatments. Response variables included thermal nociceptive threshold, skin temperature and behaviour score. Data within each age period were analysed using a 2-way repeated measures ANOVA, followed by a Holm-Sidak multiple comparison procedure if warranted. RESULTS: There was a significant (P<0.05) increase in thermal threshold, relative to Time 0, following butorphanol (0.1 mg/kg bwt) administration in both age groups. No significant time or treatment effects were apparent for skin temperature. Significant time, but not treatment, effects were evident for behaviour score in both age groups. CONCLUSIONS: Butorphanol (0.1 mg/kg bwt, but not 0.05 mg/kg bwt) significantly increased thermal nociceptive threshold in neonatal and older foals without apparent adverse behavioural effects. POTENTIAL RELEVANCE: Butorphanol shows analgesic potential in foals for management of somatic painful conditions.

Prevalence of gastric and duodenal ulceration in 691 nonsurviving foals (1995-2006).

REASON FOR PERFORMING STUDY: Gastric ulcer disease is reported to be a significant cause of morbidity in foals, but the prevalence of ulcers in this population has not recently been evaluated. OBJECTIVES: To determine the prevalence of gastric and duodenal ulceration in nonsurviving foals, and the association of ulceration with the body system of primary diagnosis. Secondary objectives were to evaluate a potential association between age and ulcer prevalence and to evaluate the use of antacid medication in the neonatal hospital population during the study years. METHODS: Necropsy records were searched for all equine accessions from 1995 to 2006. Foals aged from one day to 6 months were included. Year, age, breed, sex, diagnosis and the presence of glandular, nonglandularand/or duodenal ulceration were recorded. Diagnoses were divided into groups based on the body system of primary diagnosis, with multiple diagnoses possible. A computerised database was searched for antacid treatment of all neonatal admissions. RESULTS: The overall prevalence of ulcers was 22%, with nonglandular ulcers predominating. Ulceration was significantly associated with gastrointestinal disease. There was no significant change in ulcer prevalence over time, although there was a significant decrease in the use of antacid medications in the later study years. Neonatal foals (< 1 month) had a lower ulcer prevalence than olderfoals. CONCLUSIONS: The prevalence of ulcers in foals, although low, has remained stable over time. Gastric or duodenal ulcers are associated with a primary diagnosis of gastrointestinal disease and are less prevalent in neonates. POTENTIAL RELEVANCE: The prevalence of ulcers in nonsurviving foals is low. Foals with gastrointestinal disease are more likely to have ulcers than foals with other primary diagnoses, and older foals are more likely to have ulceration than neonates. The prevalence of ulceration did not appear to be related to hospital-wide antacid medication use in neonates.

Systemic effects of a prolonged continuous infusion of ketamine in healthy horses.

BACKGROUND: Ketamine as continuous rate infusion (CRI) provides analgesia in hospitalized horses. OBJECTIVE: Determine effects of prolonged CRI of ketamine on gastrointestinal transit time, fecal weight, vital parameters, gastrointestinal borborygmi, and behavior scores in healthy adult horses. ANIMALS: Seven adult Thoroughbred or Thoroughbred cross horses, with permanently implanted gastric cannulae. METHODS: Nonblinded trial. Random assignment to 1 of 2 crossover designed treatments. Ketamine (0.55 mg/kg IV over 15 minutes followed by 1.2 mg/kg/h) or lactated Ringer's solution (50 mL IV over 15 minutes followed by 0.15 mL/kg/h) treatments. Two hundred 3 × 5 mm plastic beads administered by nasogastric tube before drug administration. Every 2 hours vital parameters, behavior scores recorded, feces collected and weighed, and beads retrieved. Every 6 hours gastrointestinal borborygmi scores recorded. Study terminated upon retrieval of 180 beads (minimum 34 hours) or maximum 96 hours. Nontransit time data analyzed between hours 0 and 34. RESULTS: No significant (P < .05) differences detected between treatments in vital signs or gastrointestinal borborygmi. Significant (P = .002) increase in behavior score during ketamine infusion (0.381) from hours 24-34 compared with placebo (0). Ketamine caused significant delay in passage of 25, 50, and 75% of beads (ketamine = 30.6 ± 5.3, 41.4 ± 8.4, 65.3 ± 13.5 hours versus placebo = 26.8 ± 7.9, 34.3 ± 11.1, 45.8 ± 19.4 hours), and significant (P < .05) decrease in fecal weight from hours 22 (12.6 ± 3.2 versus 14.5 ± 3.8 kg) through 34 (18.5 ± 3.9 versus 12.8 ± 6.4 kg) of infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Ketamine CRI delayed gastrointestinal transit time in healthy horses without effect on vital parameters.

The effects of deferoxamine mesylate on iron elimination after blood transfusion in neonatal foals.

BACKGROUND: Hepatic failure is one of the more common complications in foals requiring blood transfusion to treat neonatal isoerythrolysis. Iron intoxication is likely the cause of hepatic injury. OBJECTIVES: To determine the effects of deferoxamine on iron elimination in normal foals. ANIMALS: Thirteen neonatal foals. METHODS: Randomized-controlled trial. At 1-3 days of age, foals received either 3 L of washed packed dam's red blood cells (RBC) or 3 L of saline IV once. Foals were treated with deferoxamine (1 g) or saline (5 mL) SC twice daily for 14 days. Foals were randomly assigned to 1 of 3 groups: RBC/deferoxamine (deferoxamine), RBC/saline (placebo), or saline/saline (control). Blood and urine samples and liver biopsy specimens were collected for measurement of hematological, biochemical, and iron metabolism variables. RESULTS: There was a significant (P<.05) increase in hematocrit, RBC count, and hemoglobin in the groups transfused with packed RBC as compared with controls at all times. Biochemical variables and liver biopsy scores were not significantly different between groups at any time. Urine iron concentrations and fractional excretion of iron were significantly higher in deferoxamine treated foals. By 14 days after transfusion, liver iron concentrations in foals treated with deferoxamine (79.9±30.9 ppm) were significantly lower than that of foals receiving placebo (145±53.0 ppm) and similar to that of controls (44.8±4.09 ppm). CONCLUSIONS AND CLINICAL IMPORTANCE: Deferoxamine enhances urinary iron elimination and decreases hepatic iron accumulation after blood transfusion in foals.