Location: Food Safety and Enteric Pathogens Research
2017 Annual Report
Objectives
Our objectives target three factors that influence Salmonella colonization, pathogenesis, and persistence. These factors include virulence mechanisms of Salmonella, the tactical response from the host, and interactions with the microbiota residing within the host. Our systematic approach integrates these research areas into three complementary objectives:
Objective 1: Investigate the impact of antibiotic usage on influencing Salmonella virulence mechanisms and enhancing antibiotic resistance.
Objective 2: Develop novel non-antibiotic intervention strategies such as beneficial microbes and vaccines to limit Salmonella colonization, persistence and shedding.
Objective 3: Evaluate immune networks and identify porcine genes for their relationship with the host microbiota to reduce Salmonella colonization, persistence, and shedding.
Approach
The common goal of each research objective is to identify targets for the development of novel antibiotic alternatives to reduce both Salmonella transmission through the food chain and antibiotic usage on the farm. To accomplish these objectives, experiments are planned to examine molecular mechanisms in Salmonella that are influenced by antimicrobial resistance and host colonization, elucidate porcine genetic pathways associated with decreased Salmonella colonization, and investigate interactions between Salmonella and host microbiota that affect Salmonella colonization and persistence. We plan to: 1) identify antibiotics that enhance virulence properties in multidrug-resistant (MDR) Salmonella, as well as those antibiotics that have no effect on virulence; this useful information will aide producers and veterinarians when determining antibiotic therapy for the treatment of infectious diseases; 2) genome sequence and transcriptionally analyze MDR Salmonella isolates that phenotypically respond to antibiotic exposure; 3) measure the effect chlortetracycline treatment has on limiting or exacerbating Salmonella shedding and altering the microbiota in swine; 4) evaluate a cross-protective Salmonella vaccine in turkeys for reduction of Salmonella colonization and transmission; 5) systematically characterize changes in the porcine immune response and gastrointestinal microbiota during Salmonella colonization; 6) assess biotherapeutic treatments as alternatives to antibiotics for treatment of swine colonized with Salmonella.
Progress Report
Various antibiotics are routinely administered to swine for treatment of disease, which may have unintended collateral effects on intestinal bacteria, particularly the foodborne pathogen Salmonella. DT104 and DT193 are classifications for two groups of important multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolates that have been associated with foodborne outbreaks. While Salmonella Typhimurium DT104 and DT193 have very similar antibiotic resistance profiles, the genes that encode these resistance factors differ between the two groups, and such genetic differences may lead to unique responses after exposure to antibiotics. To support Objective 1 to investigate the impact of antibiotic usage on influencing Salmonella virulence mechanisms and enhancing antibiotic resistance, isolates of DT104 and DT193 were exposed to sub-lethal concentrations of chlortetracycline and florfenicol, which are two antibiotics used in livestock medicine. Following exposure to chlortetracycline and florfenicol, the full cellular invasive phenotype was induced in a subset of MDR DT193 isolates during early-log growth phase, which is a phase that Salmonella normally has little-to-no invasiveness. In addition, florfenicol decreased invasion in several isolates. Identifying the differences in response to the two antibiotics within and between isolates will be important to better discern mechanisms employed by Salmonella to enhance disease; future work will examine the transcriptional changes of these isolates and how they correlate with the various observed invasive phenotypes. In addition, we have sequenced and annotated a genome of a DT193 isolate as none previously existed, and we will examine the genetic differences compared to published genomes to identify possible virulence-related factors. Fully characterizing the potential collateral effects of antibiotics on Salmonella will provide information to help limit negative consequences of antibiotic therapy by allowing producers and veterinarians to make informed decisions when determining antibiotic therapy for the treatment of other bacterial infections.
The response of the pig’s immune system and changes in the intestinal microbial community (microbiota), which is the site of Salmonella invasion, can directly influence Salmonella colonization and invasion. In support of Objective 2 to develop novel non-antibiotic intervention strategies to limit Salmonella colonization, persistence and shedding, a study was conducted in which pigs were challenged with MDR Salmonella enterica serovar I 4,[5],12:i:- and ileal intestinal samples were collected during the acute stage of infection (0, 1, 2, 3 days post-challenge). This study was conducted to simultaneously evaluate the porcine gene expression and alterations of the intestinal microbiota at the site of Salmonella invasion. Work is ongoing to isolate nucleic acids for sequencing analyses from the collected samples, which will be examined for associations between alterations in the intestinal microbiota and changes in porcine gene expression in response to Salmonella challenge. These data will clarify the role of the intestinal microbiota in regulating and reprogramming the gene expression in the porcine gastrointestinal tract in response to Salmonella invasion. Novel porcine-microbiota-Salmonella interactions will be exploited as potential targets to inhibit Salmonella colonization in the porcine intestinal tract.
Accomplishments
1. Vaccination with live-attenuated vaccine significantly reduces Salmonella levels in turkeys. ARS scientists in Ames, Iowa created a Salmonella vaccine with genetic mutations in the bacterial genome to limit Salmonella serotype-specific immunity (greater than 2,500 Salmonella serotypes exist) and instead, induce an immune response that would be cross-protective against diverse Salmonella serotypes. In addition to designing a vaccine that would protect against multiple Salmonella serotypes, the intent was also for application in multiple food-producing animal species. The researchers have previously shown the effective reduction of Salmonella disease, colonization and fecal shedding in vaccinated swine. To highlight utility of the vaccine, researchers also tested the vaccine in turkeys and showed a reduction in systemic and intestinal colonization of vaccinated turkeys following challenge with multi-drug resistant Salmonella Heidelberg. Pre-harvest control of Salmonella in food-producing animals can protect animal health, limit antibiotic usage, decrease environmental contamination, reduce Salmonella carriage into the human food chain, and diminish the cost of meat product recalls to producers. The results of this study highlight the utility of the ARS designed vaccine for enhancing pre-harvest control of Salmonella.
Review Publications
Lippolis, J.D., Brunelle, B.W., Reinhardt, T.A., Sacco, R.E., Thacker, T.C., Looft, T.P., Casey, T. 2016. Differential gene expression of three mastitis-causing Escherichia coli strains grown under planktonic, swimming, and swarming culture conditions. mSystems. 1(4):e00064-16.
Register, K.B., Nicholson, T.L., Brunelle, B.W. 2016. Comparison of ribotyping and sequence-based typing for discriminating among isolates of Bordetella bronchiseptica. Journal of Microbiological Methods. 129:117-126. doi: 10.1016/j.mimet.2016.08.008.
Sharma, V.K., Bayles, D.O., Alt, D.P., Looft, T.P., Brunelle, B.W., Stasko, J.A. 2017. Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses, and metabolism. BMC Microbiology. 17(1):56. doi: 10.1186/s12866-017-0966-x.
Holman, D.B., Brunelle, B.W., Trachsel, J., Allen, H.K. 2017. Meta-analysis to define a core microbiota in the swine gut. mSystems. 2(3):e00004-17. doi: 10.1128/mSystems.00004-17.
Bearson, B.L., Bearson, S.M., Brunelle, B.W., Bayles, D.O., Lee, I., Kich, J.D. 2017. Salmonella DIVA vaccine reduces disease, colonization and shedding due to virulent S. Typhimurium infection in swine. Journal of Medical Microbiology. 66:651-661. doi: 10.1099/jmm.0.000482.
