#16-113

Progress

Date Full Report Received

10/09/2018

Date Abstract Report Received

10/09/2018

Investigation

Institution:
Primary Investigator:
In 2015, a multistate outbreak of Salmonella serovar I 4,[5],12:i:- was associated with pork products from Washington state and resulted in 188 illnesses including 30 individuals being hospitalized. The Salmonella isolates associated with the outbreak were determined to be multidrug-resistant (resistant to ≥3 antimicrobials) and ~523,380 pounds of pork products were recalled. Salmonella serovar I 4,[5],12:i:- has increased globally over the last 10-15 years and as of 2015 serovar I 4,[5],12:i:- was the 4th most common Salmonella serovar associated with human illness in the U.S. Furthermore, ~67% of serovar I 4,[5],12:i:- isolates are considered multidrug-resistant.

The Objectives were 1) Identification of unique characteristics of multidrug-resistant Salmonella serovar I 4,[5],12:i:-, and 2) Determine whether vaccination of swine with an attenuated Salmonella Typhimurium vaccine can provide protection against multidrug-resistant Salmonella serovar I 4,[5],12:i:- transmission.

In 2015 and 2016, the USDA, Food Safety and Inspection Service (FSIS) collected 33 isolates of Salmonella serovar I 4,[5],12:i:- from pork products in 15 states (CA, 2; IL, 1; IN, 2; KY, 2; MD, 1; MI, 1; NC, 2; NE, 1; NY, 3; OK, 2; PA, 1; SC, 2; SD, 1; TX, 1; and WA, 11). Thirty of the serovar I 4,[5],12:i:- isolates were multidrug-resistant including all 11 isolates from WA that were associated with the 2015 Salmonella outbreak. FSIS determined bacterial strain relatedness for the 33 serovar I 4,[5],12:i:- isolates using pulsed-field gel electrophoresis (PFGE) and identified 13 primary PFGE patterns. Of the 33 serovar I 4,[5],12:i:- isolates, 19 isolates share the same primary PFGE pattern JPXX01.1314, 3 isolates share the primary PFGE pattern of JPXX01.2583, and 11 isolates have unique primary PFGE patterns. The 19 serovar I 4,[5],12:i:- isolates with the primary PFGE pattern JPXX01.1314 included all of the outbreak-associated strains from WA state but also isolates from CA, IL, KY, MI, NY, SD, and TX. This data indicates that serovar I 4,[5],12:i:- isolates that are closely related to the 2015 outbreak-associated strains are present in numerous states across the U.S. We obtained the 33 isolates of Salmonella serovar I 4,[5],12:i:- from FSIS for our investigation. FSIS initially performed whole genome sequencing on all of the serovar I 4,[5],12:i:- isolates using the Illumina MiSeq platform. Additionally, we obtained the whole genome sequence for strain FSIS1503788 associated with the WA state outbreak using the PacBio platform to create a serovar I 4,[5],12:i:- genome scaffold and facilitate assembly of the sequencing reads previously generated by FSIS. The genome of serovar I 4,[5],12:i:- strain FSIS1503788 is 5,029,387 bp, a genome size that is similar to other sequenced Salmonella serovars.

Salmonella serovar designations are identified by a combination of lipopolysaccharide (LPS) and flagellar antigens. The LPS antigens for serovar Typhimurium are 4,[5],12 and the flagellar antigens are i:1,2. Salmonella serovars alternate expression of two flagellar antigens [fliC (i) and fljB (1,2)] resulting in a biphasic phenotype. The absence of one of the two flagellar antigens results in a monophasic phenotype. Salmonella serovar I 4,[5],12:i:- is a monophasic variant of serovar Typhimurium that is missing fljB. The fljB genomic region of Salmonella serovar I 4,[5],12:i:- isolate FSIS1503788 is missing an ~15.7 kb portion of DNA that is present in the closely related serovar Typhimurium. However, inserted into the fljB genomic region of serovar I 4,[5],12:i:- isolate FSIS1503788 is an ~28 kb module encoding resistance to the metal mercury and the antimicrobials ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT). Therefore, the 28 kb insertion in the fljB region of isolate FSIS1503788 confers the multidrug-resistance phenotype. The composition of this multidrug-resistance module is quite similar to an insertion in the fljB genomic region of multidrug-resistant serovar I 4,[5],12:i:- strain 07-2006 isolated from a lymph node of a pig in Germany.

Nucleotide sequence analysis also identified the presence of Salmonella Genomic Island 4 (SGI-4) in serovar I 4,[5],12:i:- isolate FSIS1503788. SGI-4 is an ~80 kb genomic island containing multiple genetic operons encoding potential resistance to copper, arsenic, and zinc. An Australian serovar I 4,[5],12:i:- strain TW-Stm6 isolated from swine feces also contains SGI-4 indicating global distribution of Salmonella strains related to isolates from the 2015 pork outbreak. We constructed a derivative of FSIS1503788 with a deletion of the entire 80 kb SGI-4 genomic island and therefore no longer contains the genes for metal resistance to copper, arsenic, and zinc. We performed phenotypic analysis on FSIS1503788 and its SGI-4 derivative to determine microbial sensitivity to copper, arsenic, and zinc. The SGI-4 mutant of serovar I 4,[5],12:i:- had increased sensitivity to arsenic and copper compounds compared to the FSIS1503788 parental wildtype. However, neither wildtype serovar I 4,[5],12:i:- isolate FSIS1503788 nor the SGI-4 mutant derivative had a sensitivity to the various concentrations of zinc present in our phenotypic assay. This indicates that higher concentrations of zinc will need to be assessed to determine the level(s) for growth inhibition for these strains. The phenotypic analysis indicates that the metal resistance genes present in the SGI-4 genetic island confer increased survival for wildtype serovar I 4,[5],12:i:- isolate FSIS1503788 in the presence of copper and arsenic compared to a strain lacking these metal resistance genes. The use of copper, arsenic, and potentially zinc antimicrobials in animal and poultry production may have selected for the ~80 kb SGI-4 genetic island in Salmonella serovar I 4,[5],12:i:- and the presence of SGI-4 in the genome may provide a selective advantage for colonization of swine or survival in the environment when metal containing antimicrobials are used as alternatives to antibiotics during animal production.

A pathogenesis trial using Salmonella serovar I 4,[5],12:i:- isolate FSIS1503788 associated with the 2015 pork outbreak was performed in swine and has recently been published (Foodborne Pathog. Dis. 2018. 15(5):253-261; doi: 10.1089/fpd.2017.2378). Challenge of swine with serovar I 4,[5],12:i:- isolate FSIS1503788 significantly increased the swine body temperature, fecal moisture content, and IFN-γ levels in pigs at certain time points. Furthermore, colonization of swine with serovar I 4,[5],12:i:- resulted in the disruption of the gastrointestinal microbiota. The increases in swine body temperature, fecal moisture content, and IFN-γ levels, and the level of fecal shedding and tissue colonization for serovar I 4,[5],12:i:- following swine challenge is consistent with previous challenge experiments that we have conducted in pigs using Salmonella Typhimurium. Therefore, Salmonella serovar I 4,[5],12:i:- is pathogenic to pigs but its virulence does not appear to be greater than Salmonella Typhimurium strains that we have used in other swine pathogenesis trials.

A swine trial was performed to determine whether vaccination with a live, attenuated Salmonella Typhimurium vaccine could reduce colonization by Salmonella serovar I 4,[5],12:i:- isolate FSIS1503788 associated with the 2015 pork outbreak following transmission from inoculated penmates. Over the 14 day trial, fecal shedding between vaccinated (n=10) and mock-vaccinated (n=10) groups was not significantly different but serovar I 4,[5],12:i:- was significantly reduced in the cecal contents of vaccinated compared to mock-vaccinated pigs following transmission. In combination with other swine management tools, vaccination against Salmonella may assist in reducing Salmonella serovar I 4,[5],12:i:- carriage in pigs.

Characterization of Salmonella serovar I 4,[5],12:i:- associated with a 2015 pork outbreak indicates that these strains are globally distributed and the presence of multiple antimicrobial and metal resistance genes may provide a unique and selective advantage for colonization of swine when either the antimicrobials or metals are used during animal production.

Contact information: brad.bearson@ars.usda.gov