Genomic and phenotypic comparison of two variants of multidrug-resistant Salmonella enterica serovar Heidelberg isolated during the 2015-2017 multi-state outbreak in cattle.

Salmonella enterica subspecies enterica serovar Heidelberg (Salmonella Heidelberg) has caused several multistate foodborne outbreaks in the United States, largely associated with the consumption of poultry. However, a 2015-2017 multidrug-resistant (MDR) Salmonella Heidelberg outbreak was linked to contact with dairy beef calves. Traceback investigations revealed calves infected with outbreak strains of Salmonella Heidelberg exhibited symptoms of disease frequently followed by death from septicemia. To investigate virulence characteristics of Salmonella Heidelberg as a pathogen in bovine, two variants with distinct pulse-field gel electrophoresis (PFGE) patterns that differed in morbidity and mortality during the multistate outbreak were genotypically and phenotypically characterized and compared. Strain SX 245 with PFGE pattern JF6X01.0523 was identified as a dominant and highly pathogenic variant causing high morbidity and mortality in affected calves, whereas strain SX 244 with PFGE pattern JF6X01.0590 was classified as a low pathogenic variant causing less morbidity and mortality. Comparison of whole-genome sequences determined that SX 245 lacked ~200 genes present in SX 244, including genes associated with the IncI1 plasmid and phages; SX 244 lacked eight genes present in SX 245 including a second YdiV Anti-FlhC(2)FlhD(4) factor, a lysin motif domain containing protein, and a pentapeptide repeat protein. RNA-sequencing revealed fimbriae-related, flagella-related, and chemotaxis genes had increased expression in SX 245 compared to SX 244. Furthermore, SX 245 displayed higher invasion of human and bovine epithelial cells than SX 244. These data suggest that the presence and up-regulation of genes involved in type 1 fimbriae production, flagellar regulation and biogenesis, and chemotaxis may play a role in the increased pathogenicity and host range expansion of the Salmonella Heidelberg isolates involved in the bovine-related outbreak.

Effects of ß-glucan on Salmonella enterica serovar Typhimurium swine colonization and microbiota alterations.

BACKGROUND: The 2017 Veterinary Feed Directive eliminated the use of medically important antibiotics for growth promotion of food animals; thus, alternative growth promoters are highly desirable by food animal producers to enhance animal health and reduce pathogen colonization, including the human foodborne pathogen Salmonella. ß(1-3)(1-6)-D-glucan (ß-glucan) is a soluble fiber with prebiotic characteristics; it has been shown to modulate immune and intestinal functions that strengthen swine resistance to health challenges such as bacterial infections when supplemented in the diets of growing pigs. The current study evaluated the effects of a ß-glucan product on gut microbial community structure as well as Salmonella shedding and intestinal colonization. RESULTS: Five-week-old pigs were fed a ß-glucan amended diet at 500 g/ton (n = 13) or a non-amended control diet (n = 14) for three weeks, followed by inoculation of the 27 pigs with 1 × 109 colony forming units of Salmonella enterica serovar Typhimurium strain UK1. While remaining on the respective diets, fecal samples collected at 2, 4, 7, and 16 days post-inoculation (dpi) were similar for Salmonella shedding counts between the two diets. At 16 dpi, Salmonella counts were significantly lower in the cecal contents of the ß-glucan-fed pigs (P = 0.0339) and a trend towards a reduction was observed in the Peyer's patches region of the ileum (P = 0.0790) compared to the control pigs. Pigs fed ß-glucan for three weeks exhibited an increase in members of the Clostridia class in their fecal microbial communities, and after inoculation with Salmonella, several potentially beneficial microorganisms were enriched in the microbiota of ß-glucan-fed pigs (Lactobacillus, Ruminococcaceae, Prevotellaceae, Veillonellaceae, Bifidobacterium and Olsenella). CONCLUSION: Administration of ß-glucan altered the swine gut microbiome and reduced Salmonella colonization in the cecal contents.

Effect of dietary ß-glucan on intestinal microbial diversity and Salmonella vaccine immunogenicity and efficacy in pigs.

Alternatives to antibiotics to improve animal performance, limit the negative impact of infectious disease, and/or reduce colonization with foodborne pathogens is a major focus of animal agricultural research. ß-glucans, a generally-recognized-as-safe (GRAS) product derived from various sources, are used in swine and can serve as both a prebiotic and/or stimulant of the immune system given the expression of ß-glucan receptors on immune cells. When supplied in the diet of nursery pigs, it is unclear how dietary additives, particularly those known to modulate immune status, impact immunogenicity and efficacy of mucosal-delivered vaccines. Salmonellosis is one of the most common bacterial foodborne infections in the United States, and consumption of contaminated pork is a major source of human infection. Reduction of foodborne Salmonella in pigs via vaccination is one strategy to reduce contamination risk and subsequently reduce human disease. We examined the ability of dietary ß-glucan to modulate fecal microbial diversity, and immunogenicity and efficacy of a mucosally-delivered, live-attenuated Salmonella vaccine during the nursery period. While dietaryß-glucan did modulate fecal alpha diversity, it did not alter the induction of peripheral Salmonella-specific IFN-γ secreting Tcells or Salmonella-specific IgA in oral fluids. In addition, vaccination reduced Salmonella enterica serovar Typhimurium fecal shedding and tissue colonization. Overall, addition of ß-glucan to the nursery diet of pigs impacted the microbiota but did not alter mucosal vaccine immunogenicity and efficacy.

Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak.

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella-associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading (S. Reading) linked to 358 human infections in 42 US states and Canada. Since S. Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S. Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S. Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S. Reading human isolates. Of the 41 S. Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S. Reading isolates for colicin and cefiderocol sensitivities (CirA) and ß-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S. Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence.

Evaluation of digestively resistant or soluble fibers, short- and medium-chain fatty acids, trace minerals, and antibiotics in nonchallenged nursery pigs on performance, digestibility, and intestinal integrity.

Three experiments (EXP) were conducted to determine the effect of feed additives on performance, intestinal integrity, gastrointestinal volatile fatty acids (VFA), and energy and nutrient digestion in nonchallenged nursery pigs. In EXP 1, 480 pigs (6.36-kg body weight, BW) were placed into 96 pens with 5 pigs/pen, and allotted to 1 of 10 dietary treatments: 1) negative control containing no feed additive (NC), 2) NC + 44 mg chlortetracycline and 38.5 mg tiamulin/kg diet (CTsb), 3) NC + 5% resistant potato starch (RSpo), 4) NC + 5% soluble corn fiber (SCF), 5) NC + 5% sugar beet pulp (SBP), 6) NC + 0.30% fatty acid mix (FAM), 7) NC + 0.10% phytogenic blend of essential oils and flavoring compounds (PHY), 8) NC + 50 mg Cu and 1,600 mg zinc oxide/kg diet (CuZn), 9) NC + 5% resistant corn starch (RScn), and 10) NC + 0.05% ß-glucan (BG) for 28 d. There was no impact of dietary treatment on BW gain or feed intake (P ≥ 0.22). Pigs fed diets containing SCF, CTsb, and RSpo resulted in microbial community differences compared to pigs fed the NC (P < 0.05). In EXP 2, 48 barrows (12.8 kg BW) were selected at the end of EXP 1 and fed the same dietary treatments they had previously received: 1) NC, 2) NC + 5% RScn, 3) NC + 5% SCF, and 4) NC + FAM for 8 d. There was no effect of feeding diets containing RScn, SCF, or FAM on in vivo intestinal permeability (P ≤ 0.21). Ileal or colon pH, concentrations of VFA did not differ due to dietary treatment (P ≥ 0.36), but pigs fed diets containing FAM resulted in a greater butyric acid concentration in the cecum compared to pigs fed the NC (P ≤ 0.05). In EXP 3, 156 pigs (6.11 kg BW) were placed into 52 pens with 3 pigs/pen and allotted to 1 of 4 dietary treatments arranged in a factorial manner: 1) NC, 2) NC + 5% RSpo, 3) NC + 0.30% FAM, and 4) NC + 5% RSpo + 0.30% FAM for 24 d. Feeding pigs diets containing RSpo did not affect BW gain (P = 0.91) while pigs fed diets containing FAM grew improved BW gain (P = 0.09). Colonic butyric acid concentrations were greater in pigs fed diets containing RSpo (P = 0.03), while pigs fed diets containing FAM exhibited reduced total VFA concentrations (P = 0.11). The results indicate that supplementing diets with digestively resistant but fermentable fibers, short- and medium-chain fatty acids, or antibiotics do not have a consistent effect, positive or negative, on markers of intestinal integrity or barrier function, intestinal VFA patterns, ATTD of energy and nutrients, or on pig performance.

In-feed antimicrobials have been an important technology in swine production for protecting health and supporting growth. However, with legislative restrictions on the use of most antibiotics for growth promotion, research is needed to evaluate in-feed additives in replacing this growth promoting technology. Thus, strategies to enhance energy and nutrient digestibility, intestinal function and integrity, gastrointestinal volatile fatty acid concentrations, and microbial ecology in nursery pigs are desirable targets. The results of the three experiments conducted herein do not indicate that supplementing diets with digestively resistant but fermentable fibers, short-medium-chain fatty acids, or antibiotics have a consistent positive or negative effect on markers of intestinal integrity or barrier function, VFA patterns (ileal, cecal, or colon), ATTD of energy and nutrients, or pig performance.

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells.

Lymphocytes can heavily influence intestinal health, but resolving intestinal lymphocyte function is challenging as the intestine contains a vastly heterogeneous mixture of cells. Pigs are an advantageous biomedical model, but deeper understanding of intestinal lymphocytes is warranted to improve model utility. Twenty-six cell types were identified in the porcine ileum by single-cell RNA sequencing and further compared with cells in human and murine ileum. Though general consensus of cell subsets across species was revealed, some porcine-specific lymphocyte subsets were identified. Differential tissue dissection and in situ analyses conferred spatial context, revealing similar locations of lymphocyte subsets in Peyer's patches and epithelium in pig-to-human comparisons. Like humans, activated and effector lymphocytes were abundant in the ileum but not periphery of pigs, suggesting tissue-specific and/or activation-associated gene expression. Gene signatures for peripheral and ileal innate lymphoid cells newly discovered in pigs were defined and highlighted similarities to human innate lymphoid cells. Overall, we reveal novel lymphocyte subsets in pigs and highlight utility of pigs for intestinal research applications.

Short Chain Fatty Acids and Bacterial Taxa Associated with Reduced Salmonella enterica serovar I 4,[5],12:i:- Shedding in Swine Fed a Diet Supplemented with Resistant Potato Starch.

Salmonella enterica serovar I 4,[5],12:i:- is a foodborne pathogen of concern because many isolates are multidrug-resistant (resistant to ≥3 antimicrobial classes) and metal tolerant. In this study, three in-feed additives were individually tested for their ability to reduce Salmonella I 4,[5],12:i:- shedding in swine: resistant potato starch (RPS), high amylose corn starch, and a fatty acid blend, compared with a standard control diet over 21 days. Only RPS-fed pigs exhibited a reduction in Salmonella fecal shedding, different bacterial community compositions, and different cecal short chain fatty acid (SCFA) profiles relative to control animals. Within the RPS treatment group, pigs shedding the least Salmonella tended to have greater cecal concentrations of butyrate, valerate, caproate, and succinate. Additionally, among RPS-fed pigs, several bacterial taxa (Prevotella_7, Olsenella, and Bifidobacterium, and others) exhibited negative relationships between their abundances of and the amount of Salmonella in the feces of their hosts. Many of these same taxa also had significant positive associations with cecal concentrations of butyrate, valerate, caproate, even though they are not known to produce these SCFAs. Together, these data suggest the RPS-associated reduction in Salmonella shedding may be dependent on the establishment of bacterial cross feeding interactions that result in the production of certain SCFAs. However, directly feeding a fatty acid mix did not replicate the effect. RPS supplementation could be an effective means to reduce multidrug-resistant (MDR) S. enterica serovar I 4,[5],12:i:- in swine, provided appropriate bacterial communities are present in the gut. IMPORTANCE Prebiotics, such as resistant potato starch (RPS), are types of food that help to support beneficial bacteria and their activities in the intestines. Salmonella enterica serovar I 4,[5],12:i:- is a foodborne pathogen that commonly resides in the intestines of pigs without disease, but can make humans sick if unintentionally consumed. Here we show that in Salmonella inoculated pigs, feeding them a diet containing RPS altered the colonization and activity of certain beneficial bacteria in a way that reduced the amount of Salmonella in their feces. Additionally, within those fed RPS, swine with higher abundance of these types of beneficial bacteria had less Salmonella I 4,[5],12:i:- in their feces. This work illustrates likely synergy between the prebiotic RPS and the presence of certain gut microorganisms to reduce the amount of Salmonella in the feces of pigs and therefore reduce the risk that humans will become ill with MDR Salmonella serovar I 4,[5],12:i:-.

Relationship and distribution of Salmonella enterica serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database.

BACKGROUND: Of the > 2600 Salmonella serovars, Salmonella enterica serovar I 4,[5],12:i:- (serovar I 4,[5],12:i:-) has emerged as one of the most common causes of human salmonellosis and the most frequent multidrug-resistant (MDR; resistance to ≥3 antimicrobial classes) nontyphoidal Salmonella serovar in the U.S. Serovar I 4,[5],12:i:- isolates have been described globally with resistance to ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT) and an integrative and conjugative element with multi-metal tolerance named Salmonella Genomic Island 4 (SGI-4). RESULTS: We analyzed 13,612 serovar I 4,[5],12:i:- strain sequences available in the NCBI Pathogen Detection database to determine global distribution, animal sources, presence of SGI-4, occurrence of R-type ASSuT, frequency of antimicrobial resistance (AMR), and potential transmission clusters. Genome sequences for serovar I 4,[5],12:i:- strains represented 30 countries from 5 continents (North America, Europe, Asia, Oceania, and South America), but sequences from the United States (59%) and the United Kingdom (28%) were dominant. The metal tolerance island SGI-4 and the R-type ASSuT were present in 71 and 55% of serovar I 4,[5],12:i:- strain sequences, respectively. Sixty-five percent of strain sequences were MDR which correlates to serovar I 4,[5],12:i:- being the most frequent MDR serovar. The distribution of serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database suggests that swine-associated strain sequences were the most frequent food-animal source and were significantly more likely to contain the metal tolerance island SGI-4 and genes for MDR compared to all other animal-associated isolate sequences. CONCLUSIONS: Our study illustrates how analysis of genomic sequences from the NCBI Pathogen Detection database can be utilized to identify the prevalence of genetic features such as antimicrobial resistance, metal tolerance, and virulence genes that may be responsible for the successful emergence of bacterial foodborne pathogens.

The Role of Salmonella Genomic Island 4 in Metal Tolerance of Salmonella enterica Serovar I 4,[5],12:i:- Pork Outbreak Isolate USDA15WA-1.

Multidrug-resistant (MDR; resistance to >3 antimicrobial classes) Salmonella enterica serovar I 4,[5],12:i:- strains were linked to a 2015 foodborne outbreak from pork. Strain USDA15WA-1, associated with the outbreak, harbors an MDR module and the metal tolerance element Salmonella Genomic Island 4 (SGI-4). Characterization of SGI-4 revealed that conjugational transfer of SGI-4 resulted in the mobile genetic element (MGE) replicating as a plasmid or integrating into the chromosome. Tolerance to copper, arsenic, and antimony compounds was increased in Salmonella strains containing SGI-4 compared to strains lacking the MGE. Following Salmonella exposure to copper, RNA-seq transcriptional analysis demonstrated significant differential expression of diverse genes and pathways, including induction of at least 38 metal tolerance genes (copper, arsenic, silver, and mercury). Evaluation of swine administered elevated concentrations of zinc oxide (2000 mg/kg) and copper sulfate (200 mg/kg) as an antimicrobial feed additive (Zn+Cu) in their diet for four weeks prior to and three weeks post-inoculation with serovar I 4,[5],12:i:- indicated that Salmonella shedding levels declined at a slower rate in pigs receiving in-feed Zn+Cu compared to control pigs (no Zn+Cu). The presence of metal tolerance genes in MDR Salmonella serovar I 4,[5],12:i:- may provide benefits for environmental survival or swine colonization in metal-containing settings.

Intraepithelial T Cells Diverge by Intestinal Location as Pigs Age.

T cells resident within the intestinal epithelium play a central role in barrier integrity and provide a first line of immune defense. Intraepithelial T cells (IETs) are among the earliest immune cells to populate and protect intestinal tissues, thereby giving them an important role in shaping gut health early in life. In pigs, IETs are poorly defined, and their maturation in young pigs has not been well-studied. Given the importance of IETs in contributing to early life and long-term intestinal health through interactions with epithelial cells, the microbiota, and additional environmental factors, a deeper characterization of IETs in pigs is warranted. The objective of this study was to analyze age- and intestinal location-dependent changes in IETs across multiple sites of the small and large intestine in pigs between 4- and 8-weeks of age. IETs increased in abundance over time and belonged to both γδ and αß T cell lineages. Similar compositions of IETs were identified across intestinal sites in 4-week-old pigs, but compositions diverged between intestinal sites as pigs aged. CD2+CD8α+ γδ T cells and CD4-CD8α+ αß T cells comprised >78% of total IETs at all intestinal locations and ages examined. Greater percentages of γδ IETs were present in large intestine compared to small intestine in older pigs. Small intestinal tissues had greater percentages of CD2+CD8α- γδ IETs, while CD2+CD8α+ γδ IET percentages were greater in the large intestine. Percentages of CD4-CD8α+ αß IETs increased over time across all intestinal sites. Moreover, percentages of CD27+ cells decreased in ileum and large intestine over time, indicating increased IET activation as pigs aged. Percentages of CD27+ cells were also higher in small intestine compared to large intestine at later timepoints. Results herein emphasize 4- to 8-weeks of age as a critical window of IET maturation and suggest strong associations between intestinal location and age with IET heterogeneity in pigs.

Complete Genome Sequence of Multidrug-Resistant Salmonella enterica Serovar I 4,[5],12:i:- 2015 U.S. Pork Outbreak Isolate USDA15WA-1.

The genome of a multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar I 4,[5],12:i:- isolate from the 2015 U.S. pork outbreak was sequenced. The complete nucleotide sequence of USDA15WA-1 is 5,031,277 bp, including Salmonella genomic island 4 encoding tolerance to multiple metals and an MDR module inserted in the fljB region.

Evaluating nursery pig responses to in-feed sub-therapeutic antibiotics.

Antibiotics have been used for over 60 years by the swine industry to improve growth performance and feed efficiency. With rising concerns over antimicrobial resistance and government restrictions such as the Veterinary Feed Directive on usage of in-feed antibiotics, alternatives to feeding antibiotic growth promoters (AGPs) to nursery pigs are needed. However, the mechanism of action by which AGPs work is poorly understood. Thus, the objective of this study was to investigate the mechanisms of action by which AGPs increase nursery pig performance. Over two replicates, 24 weaned pigs (6.75 ± 0.75 kg body weight) were randomly allotted to either control (CON, n = 12) or sub-therapeutic antibiotic (sCTC, n = 12) treatments and housed individually. A 2-phase corn-soybean-based nursery diet was fed, with the sCTC diets containing 40 ppm feed-grade chlortetracycline. Individual pig average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were calculated weekly for 5 weeks. Thereafter, all pigs were euthanized and necropsied for tissue collection. The overall performance data indicated that sCTC pigs had increased ADG (0.43 vs. 0.32 kg/d, P = 0.001) and ADFI (0.51 vs. 0.37 kg/d, P = 0.002) compared with CON pigs; however, G:F was not different as a result of dietary treatment (0.85 vs. 0.88, P = 0.617). Intestinal barrier permeability, ileal active nutrient transport, and cecal short chain fatty acid concentrations did not differ (P > 0.10) due to dietary treatment, however changes in several ileum mRNA transcripts suggest that inflammation may be reduced in sCTC pigs. Further, the changes observed in the proteomes of the ileum, colon, skeletal muscle, and liver suggest that the sub-therapeutic mode of action of AGPs may include post-absorptive changes and warrants further investigation.