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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Food Safety and Enteric Pathogens Research » Research » Research Project #430777

Research Project: Analysis of Virulence and Antibiotic Resistance Mechanisms of Salmonella and Development of Intervention Strategies

Location: Food Safety and Enteric Pathogens Research

2016 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Exposure of bacteria to inhibitory levels of antibiotics can select for bacteria with increased antibiotic resistance. Chlortetracycline is an antibiotic commonly used in swine. To ‘investigate the impact of antibiotic usage on influencing Salmonella virulence mechanisms and enhancing antibiotic resistance’ (Objective 1), we evaluated if inhibitory concentrations select for increased antibiotic resistance in isolates of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium. We found that chlortetracycline exposure can select for increased resistance; this enhanced resistance was achieved by increasing the number of chlortetracycline resistance genes 5-10 fold within the genome. We also found that chlortetracycline exposure can increase resistance to florfenicol, an entirely different antibiotic. Overall, these data indicate that chlortetracycline treatment at therapeutic doses could select for even more antibiotic resistant, and potentially virulent, MDR Salmonella. Many foodborne disease agents are transferred to humans from animals and animal products, and effective food safety control strategies should include methods to reduce foodborne agents in the animal. Salmonella is a bacterium that often resides in the gastrointestinal tract of food animals including pigs, poultry and cattle without causing disease, but can cause significant disease in humans. In support of Objective 2 to ‘develop novel non-antibiotic intervention strategies to limit Salmonella colonization, persistence and shedding’, a Salmonella vaccine originally designed by our research group, and shown to be effective in reducing Salmonella in pigs, was tested in turkeys. Upon exposure to Salmonella enterica serovars Typhimurium or Heidelberg, turkeys previously administered the vaccine had reduced Salmonella in their internal organs (spleen and gastrointestinal tissues) compared to turkeys that were not vaccinated. Work is ongoing to evaluate the immune response to the vaccine. This vaccine will support food safety and public health by reducing the dissemination of human foodborne Salmonella via food animals.

4. Accomplishments
1. Enhancing innate immune defenses reduces Salmonella in swine. Efforts to reduce asymptomatic Salmonella carriage in food animals are needed due to the possibility of farm-to-fork contamination. Development of intervention strategies that are effective against diverse Salmonella serovars are desired to limit cases of foodborne illness. Neutrophils are cells of the immune system that play an important role in combatting bacterial infection, and the immune protein that induces neutrophil production in the animal is called granulocyte-colony stimulating factor (G-CSF). ARS scientists in Ames, Iowa completed a study testing the ability G-CSF to increase pig immune neutrophil defenses and subsequently decrease Salmonella. An engineered vector expressing porcine G-CSF (Ad5-G-CSF) was given to pigs prior to Salmonella exposure. Pigs that received Ad5-G-CSF had significantly reduced Salmonella fecal shedding and tissue colonization, and less Salmonella-induced disturbance of their gastrointestinal bacteria (microbiota) compared to the pigs that were exposed to Salmonella but not Ad5-G-CSF. Collectively, these data suggest that delivery of the targeted immunostimulant G-CSF may be a strategy to reduce Salmonella colonization. These data will benefit producers and pharmaceutical companies working towards development of non-antibiotic methods to decrease shedding of foodborne Salmonella.

Review Publications
Bearson, B.L., Brunelle, B.W. 2015. Fluoroquinolone induction of phage-mediated gene transfer in multidrug-resistant Salmonella. International Journal of Antimicrobial Agents. 46(2):201-204.

Bao, H., Kommadath, A., Liang, G., Sun, X., Arantes, A.S., Tuggle, C.K., Plastow, G.S., Bearson, S.M., Stothard, P., Guan, L. 2015. Genome-wide whole blood microRNAome and transcriptome analyses reveal miRNA-mRNA regulated host response to foodborne pathogen Salmonella infection in swine. Scientific Reports. 5:12620. doi: 10.1038/srep12620.

Bearson, B.L., Bearson, S.M., Kich, J.D. 2016. A DIVA vaccine for cross-protection against Salmonella. Vaccine. 34:1241-1246.

Nicholson, T.L., Shore, S., Register, K.B., Bayles, D.O., Kingsley, R.A., Brunelle, B.W. 2016. Comparative genomic analysis of the swine pathogen Bordetella bronchiseptica strain KM22. Veterinary Microbiology. 182:87-94.

Brunelle, B.W., Hannen, A.M., Nicholson, E.M., Seabury, C.M. 2016. Disparate modes of evolution shaped modern prion (PRNP) and prion-related doppel (PRND) variation in domestic cattle. PLoS One. 11(5):e0155924. doi: 10.1371/journal.pone.0155924.