Shawn M. D. Bearson

The prevalence of Salmonella enterica serovar Enteritidis encoding a GyrA D87Y substitution, associated with decreased susceptibility to ciprofloxacin, has increased since 2018 in the United States. Here, we provide three Salmonella Enteritidis reference genomes belonging to an SNP cluster of interest with ( n = 2) or without (n = 1) the GyrA D87Y substitution.

Background: Salmonella enterica subspecies enterica is an important source of human foodborne illness, frequently via contaminated food animal products. Vaccination is a promisingly effective intervention to lower Salmonella loads in food animals, thus reducing food chain transmission. Currently-available commercial vaccines have limited cross protection across Salmonella serovars (> 2,600), indicating a need for novel vaccines with broad efficacy. In this study, an in silico reverse vaccinology (RV) pipeline comprehensively screened the proteome of Salmonella enterica serovar Typhimurium strain UK-1 for conserved proteins from poultry-associated, human-relevant Salmonella serovars and identified epitopes with predicted broad-spectrum protection for the design of a multiepitope vaccine construct (MEVC). Results: The UK-1 proteome, excluding lipopolysaccharide-, flagellin-, and plasmid-associated proteins, was screened for proteins with pertinent properties including homology to five poultry-associated, human-relevant serovars (Enteritidis, Hadar, Infantis, Kentucky, and Uganda), representing Salmonella serogroups B-E. The resulting 101 proteins were evaluated for cytotoxic and helper T lymphocyte epitopes with strong binding to chicken-like human major histocompatibility complex alleles, high antigenicity, and 100% identity to ≥ 99% of the NCBI proteomes of the selected serovars ( n = 90,800). Of the resulting 93 epitopes, 28 epitopes derived from 24 proteins were incorporated in an MEVC with epitope-type-associated linkers and a Salmonella flagellin adjuvant. In silico tools predicted immunological functions of the MEVC including TLR1/TLR2 binding, induction of classical cellular and humoral immune responses, and sequence homology (i.e. potential cross-protection) to a Salmonella outbreak dataset. Conclusions: A modified RV pipeline using a whole proteome approach targeting human-relevant, poultry-associated serovars identified previously recognized proteins with immunogenic and/or immunoprotective properties (suggestive of in vivo protein expression and immune stimulation), as well as eight novel proteins for vaccine target exploration. Our RV pipeline was further corroborated by 25 of the 28 MEVC epitopes demonstrating 100% sequence identity to > 90% of a PulseNet dataset of 135 outbreak-associated Salmonella serovars from various food sources. Although efficacy validation of the proposed MEVC requires in vivo or in vitro evaluation, the current study illustrates the utility of pairing target organisms with relevant datasets to in silico screen, assemble, and assess a multiepitope vaccine construct designed for predicted broad-spectrum protection.

Salmonella enterica serovar Enteritidis ( S . Enteritidis) is a major cause of foodborne illness linked to poultry across the globe, and its decreased susceptibility to quinolone antibiotics such as ciprofloxacin poses a significant threat to public health. Yet, the ecological and genetic pressures that drive the fitness of this pathogen in poultry environments remain poorly defined. In this study, we combined broiler chicken litter microcosm experiments, whole-genome sequencing, and machine learning to investigate factors that shape the fitness and antimicrobial resistance development of S . Enteritidis. We analyzed more than 10,000 genomes to assess the role bacteriophages, plasmids, and chromosomal mutations play in driving S . Enteritidis fitness. Our findings reveal that an underreported bacteriophage named ESSI-2 and IncX1 plasmids strongly influenced whether strains carried the gyrA mutations associated with decreased ciprofloxacin susceptibility. Strains of S . Enteritidis lacking both ESSI-2 phage and IncX1 plasmid harbored parallel mutations in genes linked to DNA topology, antimicrobial peptide resistance, and virulence. We found that ESSI-2 phage persistence in litter was shaped by distinct ecological community structures rather than random occurrence and that reused litter reduced S . Enteritidis survival compared to fresh pine wood shavings, suggesting that microbiome-based interventions could be leveraged to limit the spread of antibiotic-resistant Enteritidis strains. IMPORTANCE: Foodborne illnesses linked to Salmonella enterica serovar Enteritidis strains with decreased susceptibility to quinolones, including ciprofloxacin, are on the rise in the United States, Canada, and Europe. In this study, we report for the first time that the gyrA mutation responsible for this phenotype is associated with a bacteriophage and a particular plasmid group. We demonstrated that when either of the two mobile genetic elements is carried by a S. Enteritidis strain, the likelihood of having gyrA mutation was negligible. Using broiler chicken litter microcosms, we provided insights into how the microbiome could be harnessed to limit the continued spread of these fitter lineages of S. Enteritidis strains. Our study shows that the survival and evolution of S. Enteritidis in poultry environments are strongly shaped by interactions among bacteriophages, plasmids, and the microbial community.

Background Non-typhoidal Salmonella enterica subspecies enterica (NTS) is an important source of human foodborne illness, frequently via contaminated food animal products. Vaccination is a promisingly effective intervention to lower NTS loads in food animals, thus reducing food chain transmission. Currently available commercial vaccines have limited cross protection across Salmonella serovars (> 2,600), indicating a need for improved vaccine design. Multiepitope vaccines designed using reverse vaccinology tools are created with statistically selected, antigenic epitopes and evaluated in silico . In the current study, a modified reverse vaccinology pipeline was employed to screen for epitopes in the Salmonella enterica serovar Typhimurium strain UK-1 proteome for design of a cross-protective, multiepitope vaccine construct (MEVC) against Salmonella for poultry. Results The UK-1 proteome, excluding immunovariable and immunodominant lipopolysaccharide- and flagellin-associated proteins and plasmid-associated proteins, was screened for proteins with relevant properties including homology to five poultry-associated and human-relevant serovars (Enteritidis, Hadar, Infantis, Kentucky, and Uganda), representing Salmonella serogroups B-E. The resulting 101 proteins were evaluated for cytotoxic and helper lymphocyte epitopes with strong binding to chicken-like human major histocompatibility complex alleles, high antigenicity, and 100% identity to ≥ 99% of the NCBI proteomes (n = 90,800) for each of the selected serovars. Twenty-eight epitopes representing 24 proteins were incorporated in a MEVC with epitope-type-associated linkers and a Salmonella flagellin adjuvant. Immunological predictions for the MEVC included TLR1/TLR2 binding, induction of classical cellular and humoral immune responses, and sequence homology (i.e. potential cross-protection) to a Salmonella outbreak dataset. Conclusions A modified reverse vaccinology pipeline using a whole genome approach targeting clinically-relevant, poultry-associated serovars was validated by not only identifying previously recognized proteins with immunogenic, immunoprotective and/or attenuation properties (n = 16), but also unique proteins (n = 8) for vaccine target exploration. Our reverse vaccinology pipeline was further corroborated by 25/28 MEVC epitopes demonstrating 100% sequence identity to > 90% of a PulseNet dataset of 135 outbreak-associated Salmonella serovars from various food animal sources, thereby illustrating the utility of reverse vaccinology to identify, assemble, assess, and validate predicted efficacy of a vaccine design in silico when target organisms are paired with relevant validation datasets.

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 β-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.

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.

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. β(1-3)(1-6)-d-glucan (β-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 β-glucan product on gut microbial community structure as well as Salmonella shedding and intestinal colonization. Results: Five-week-old pigs were fed a β-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 β-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 β-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 β-glucan-fed pigs (Lactobacillus, Ruminococcaceae, Prevotellaceae, Veillonellaceae, Bifidobacterium and Olsenella). Conclusion: Administration of β-glucan altered the swine gut microbiome and reduced Salmonella colonization in the cecal contents.

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.