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.

Chlortetracycline Enhances Tonsil Colonization and Fecal Shedding of Multidrug-Resistant Salmonella enterica Serovar Typhimurium DT104 without Major Alterations to the Porcine Tonsillar and Intestinal Microbiota.

Salmonella spp. are estimated to cause 1.2 million cases of human foodborne illness each year in the United States, and pigs can often be asymptomatically colonized with Salmonella spp. (>50% of farms). Recent reports state that 18.3% of Salmonella enterica serovar Typhimurium isolates are resistant to ≥3 antimicrobial classes, and multidrug-resistant (MDR) strains are associated with an increased hospitalization rate and other complications. Chlortetracycline is commonly used in swine production to prevent/treat various diseases; therefore, chlortetracycline treatment of pigs unknowingly colonized with MDR Salmonella may have collateral effects on Salmonella spp. (and other gut bacteria). In this study, we determined the effect of in-feed chlortetracycline (400 g/ton) on shedding and colonization of pigs challenged with the MDR S Typhimurium strain DT104 (n = 11/group). We also assessed the impact on the fecal microbiota over the 12-day experimental period and on the ileum, cecum, and tonsil microbiota at 7 days postinoculation (dpi). In MDR S Typhimurium-inoculated pigs, chlortetracycline administration significantly increased fecal shedding at 2 dpi (+1.4 log10 CFU/g; P < 0.001) and enhanced tonsil colonization (+3.1 log10 CFU/g; P < 0.001). There were few major alterations detected in the gut or tonsillar microbiota of pigs treated with MDR S Typhimurium and/or chlortetracycline. The tonsillar transcriptome was largely unaffected despite increased colonization by MDR S Typhimurium following inoculation of the chlortetracycline-treated pigs. These results highlight the idea that chlortetracycline administration can enhance shedding and colonization of MDR S Typhimurium in pigs, which could increase the risk of environmental dissemination of MDR Salmonella strains.IMPORTANCESalmonella spp. are an important cause of foodborne illness in North America, and pork products are associated with sporadic cases and outbreaks of human salmonellosis. Isolates of Salmonella may be resistant to multiple antibiotics, and infections with multidrug-resistant (MDR) Salmonella spp. are more difficult to treat, leading to increased hospitalization rates. Swine operations commonly use antimicrobials, such as chlortetracycline, to prevent/treat infections, which may have collateral effects on pig microbial populations. Recently, we demonstrated that chlortetracycline induces the expression of genes associated with pathogenesis and invasion in MDR Salmonella enterica serovar Typhimurium in vitro In our current study, we show increased tonsillar colonization and fecal shedding of the MDR S Typhimurium strain DT104 from pigs administered chlortetracycline. Therefore, pigs unknowingly colonized with multidrug-resistant Salmonella spp. and receiving chlortetracycline for an unrelated infection may be at a greater risk for disseminating MDR Salmonella spp. to other pigs and to humans through environmental or pork product contamination.

Genomic and Transcriptomic Analysis of Escherichia coli Strains Associated with Persistent and Transient Bovine Mastitis and the Role of Colanic Acid.

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. It is most often transient in nature, causing an infection that lasts 2 to 3 days. However, E. coli has been shown to cause a persistent infection in a minority of cases. Mechanisms that allow for a persistent E. coli infection are not fully understood. The goal of this work was to determine differences between E. coli strains originally isolated from dairy cattle with transient and persistent mastitis. Using RNA sequencing, we show gene expression differences in nearly 200 genes when bacteria from the two clinical phenotypes are compared. We sequenced the genomes of the E. coli strains and report genes unique to the two phenotypes. Differences in the wca operon, which encodes colanic acid, were identified by DNA as well as RNA sequencing and differentiated the two phenotypes. Previous work demonstrated that E. coli strains that cause persistent infections were more motile than those that cause transient infections. Deletion of genes in the wca operon from a persistent-infection strain resulted in a reduction of motility as measured in swimming and swarming assays. Furthermore, colanic acid has been shown to protect bacteria from complement-mediated killing. We show that transient-infection E. coli strains were more sensitive to complement-mediated killing. The deletion of genes from the wca operon caused a persistent-infection E. coli strain to become sensitive to complement-mediated killing. This work identifies important differences between E. coli strains that cause persistent and transient mammary infections in dairy cattle.

Porcine Response to a Multidrug-Resistant Salmonella enterica serovar I 4,[5],12:i:- Outbreak Isolate.

Salmonella enterica serovar I 4,[5],12:i:- has emerged as a common nontyphoidal Salmonella serovar to cause human foodborne illness. An interesting trait of serovar I 4,[5],12:i:- is that it only expresses the fliC gene for bacterial motility (i.e., monophasic), while most Salmonella strains alternately express two flagellin genes (fliC and fljB). The goal of this study was to characterize the porcine response following inoculation with a multidrug-resistant (MDR) serovar I 4,[5],12:i:- isolate associated with a multistate pork outbreak to determine if the increased prevalence of serovar I 4,[5],12:i:- in swine is due to enhanced pathogenicity. Pigs were inoculated and subsequently evaluated for the ability of the isolate to colonize intestinal tissues, cause clinical symptoms, induce an immune response, and alter the fecal microbiota over a 7-day period. Pigs exhibited a significant increase in rectal temperature (fever) (p < 0.01) and fecal moisture content (diarrhea) (p < 0.05) at 2 days postinoculation (d.p.i.) compared with preinoculation (day 0). Serum analyses revealed significantly increased interferon-gamma (IFN-γ) levels at 2 (p ≤ 0.0001) and 3 (p < 0.01) d.p.i. compared with day 0, and antibodies against Salmonella lipopolysaccharide (LPS) were present in all pigs by 7 d.p.i. Serovar I 4,[5],12:i:- colonized porcine intestinal tissues and was shed in the feces throughout the 7-day study. Analysis of the 16S rRNA gene sequences demonstrated that the fecal microbiota was significantly altered following MDR serovar I 4,[5],12:i:- inoculation, with the largest shift observed between 0 and 7 d.p.i. Our data indicate that the pork outbreak-associated MDR serovar I 4,[5],12:i:- isolate induced transient clinical disease in swine and perturbed the gastrointestinal microbial community. The porcine response to MDR serovar I 4,[5],12:i:- is similar to previous studies with virulent biphasic Salmonella enterica serovar Typhimurium, suggesting that the absence of fljB does not substantially alter acute colonization or pathogenesis in pigs.

Molecular Profiling: Catecholamine Modulation of Gene Expression in Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium.

Investigations of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium have demonstrated that these bacterial pathogens can respond to the presence of catecholamines including norepinephrine and/or epinephrine in their environment by modulating gene expression and exhibiting various phenotypes. For example, one of the most intensively investigated phenotypes following exposure of E. coli and S. Typhimurium to norepinephrine is enhanced bacterial growth in a serum-based medium. Host-pathogen investigations have demonstrated that the mammalian host utilizes nutritional immunity to sequester iron and prevent extraintestinal growth by bacterial pathogens. However, Salmonella and certain E. coli strains have a genetic arsenal designed for subversion and subterfuge of the host. Norepinephrine enhances bacterial growth due, in part, to increased iron availability, and transcriptional profiling indicates differential expression of genes encoding iron acquisition and transport proteins. Bacterial motility of E. coli and S. Typhimurium is also enhanced in the presence of catecholamines and increased flagellar gene expression has been described. Furthermore, epinephrine and norepinephrine are chemoattractants for E. coli O157:H7. In S. Typhimurium, norepinephrine enhances horizontal gene transfer and increases expression of genes involved in plasmid transfer. Exposure of E. coli O157:H7 to norepinephrine increases expression of the genes encoding Shiga toxin and operons within the locus of enterocyte effacement (LEE). Alterations in the transcriptional response of enteric bacteria to catecholamine exposure in vivo are predicted to enhance bacterial colonization and pathogen virulence. This chapter will review the current literature on the transcriptional response of E. coli and S. Typhimurium to catecholamines.

Microbial Community and Chemical Characteristics of Swine Manure during Maturation.

Swine diet formulations have the potential to lower animal emissions, including odor and ammonia (NH). The purpose of this study was to determine the impact of manure storage duration on manure chemical and microbial properties in swine feeding trials. Three groups of 12 pigs were fed a standard corn-soybean meal diet over a 13-wk period. Urine and feces were collected at each feeding and transferred to 12 manure storage tanks. Manure chemical characteristics and headspace gas concentrations were monitored for NH, hydrogen sulfide (HS), volatile fatty acids, phenols, and indoles. Microbial analysis of the stored manure included plate counts, community structure (denaturing gradient gel electrophoresis), and metabolic function (Biolog). All odorants in manure and headspace gas concentrations were significantly ( < 0.01) correlated for length of storage using quadratic equations, peaking after Week 5 for all headspace gases and most manure chemical characteristics. Microbial community structure and metabolic utilization patterns showed continued change throughout the 13-wk trial. Denaturing gradient gel electrophoresis species diversity patterns declined significantly ( < 0.01) with time as substrate utilization declined for sugars and certain amino acids, but functionality increased in the utilization of short chain fatty acids as levels of these compounds increased in manure. Studies to assess the effect of swine diet formulations on manure emissions for odor need to be conducted for a minimum of 5 wk. Efforts to determine the impact of diets on greenhouse gas emissions will require longer periods of study (>13 wk).

Commercial vaccine provides cross-protection by reducing colonization of Salmonella enterica serovars Infantis and Hadar in turkeys.

Human foodborne outbreaks with antibiotic-resistant Salmonella enterica associated with contaminated poultry products have recently involved serogroup C serovars Infantis and Hadar. The current study evaluated a commercially available Salmonella vaccine for cross-protection against Infantis and Hadar serovars in turkeys. The live, attenuated S. Typhimurium (serogroup B) vaccine significantly reduced colonization of intestinal tissues (cecum, cecal tonsils, and cloaca) by serovars Infantis (C1) and Hadar (C2) and significantly limited systemic dissemination to the spleen. S. Infantis, but not S. Hadar, disseminated to bone marrow in non-vaccinated turkeys, but vaccination prevented S. Infantis dissemination to the bone marrow. The S. Infantis challenge strain contained the pESI megaplasmid, and virulence mechanism(s) residing on this plasmid may support dissemination and/or colonization of systemic niches such as myeloid tissue. Collectively, the data indicate that vaccinating turkeys with the serogroup B S. Typhimurium vaccine limited intestinal colonization and systemic dissemination by serogroup C serovars Infantis and Hadar.

Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD.

Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.

Tetracycline accelerates the temporally-regulated invasion response in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium.

BACKGROUND: Multidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates. RESULTS: The isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype. CONCLUSIONS: In this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

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.

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:-.

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.

A mutation in the poxA gene of Salmonella enterica serovar Typhimurium alters protein production, elevates susceptibility to environmental challenges, and decreases swine colonization.

Control of foodborne Salmonella within the farm-retail continuum is a complex issue since over 2500 serovars of Salmonella exist, the host range of Salmonella spp. varies greatly, and Salmonella is environmentally ubiquitous. To identify Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) genes important for pathogen survival, our research group previously screened a signature-tagged mutagenesis bank in an ex vivo swine stomach content assay. A mutation in the poxA gene, a member of the gene family encoding class-II aminoacyl-tRNA synthetases, decreased survival of Salmonella Typhimurium in the ex vivo swine stomach content assay. In the current study, complementation with a plasmid-encoded poxA gene restored survival of the poxA mutant to the level of the parental, wild-type strain. In vivo analysis of the poxA mutant in the natural porcine host revealed significantly reduced fecal shedding of Salmonella, decreased colonization of the tonsils, and decreased detection of the mutant strain in the cecal contents of the pigs at 7 days postinoculation (p < 0.05). Body temperature (fever) of the pigs inoculated with wild-type Salmonella Typhimurium was significantly higher than that of pigs inoculated with the poxA mutant (p < 0.05). Two-dimensional gel electrophoresis revealed characteristic differences in the protein profile of the poxA mutant relative to the wild-type strain, indicating that deletion of poxA in Salmonella Typhimurium exerts selective effects on translation and/or posttranslational modifications of mRNA species that are necessary for stress survival and colonization of the natural swine host.

Transcriptional response of blood leukocytes from turkeys challenged with Salmonella enterica serovar Typhimurium UK1.

Non-typhoidal Salmonella is one of the most common causes of bacterial foodborne disease and consumption of contaminated poultry products, including turkey, is one source of exposure. Minimizing Salmonella colonization of commercial turkeys could decrease the incidence of Salmonella-associated human foodborne illness. Understanding host responses to these bacteria is critical in developing strategies to minimize colonization and reduce food safety risk. In this study, we evaluated bacterial load and blood leukocyte transcriptomic responses of 3-week-old turkeys challenged with the Salmonella enterica serovar Typhimurium (S. Typhimurium) UK1 strain. Turkeys (n = 8/dose) were inoculated by oral gavage with 108 or 1010 colony forming units (CFU) of S. Typhimurium UK1, and fecal shedding and tissue colonization were measured across multiple days post-inoculation (dpi). Fecal shedding was 1-2 log10 higher in the 1010 CFU group than the 108 CFU group, but both doses effectively colonized the crop, spleen, ileum, cecum, colon, bursa of Fabricius and cloaca without causing any detectable clinical signs in either group of birds. Blood leukocytes were isolated from a subset of the birds (n = 3-4/dpi) both pre-inoculation (0 dpi) and 2 dpi with 1010 CFU and their transcriptomic responses assayed by RNA-sequencing (RNA-seq). At 2 dpi, 647 genes had significant differential expression (DE), including large increases in expression of immune genes such as CCAH221, IL4I1, LYZ, IL13RA2, IL22RA2, and ACOD1. IL1ß was predicted as a major regulator of DE in the leukocytes, which was predicted to activate cell migration, phagocytosis and proliferation, and to impact the STAT3 and toll-like receptor pathways. These analyses revealed genes and pathways by which turkey blood leukocytes responded to the pathogen and can provide potential targets for developing intervention strategies or diagnostic assays to mitigate S. Typhimurium colonization in turkeys.

Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7.

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

The Salmonella enterica serovar Typhimurium QseB response regulator negatively regulates bacterial motility and swine colonization in the absence of the QseC sensor kinase.

Salmonella enterica serovar Typhimurium (S. Typhimurium) responds to the catecholamine, norepinephrine by increasing bacterial growth and enhancing motility. In this study, iron with or without the siderophore, ferrioxamine E also enhanced bacterial motility. Iron-enhanced motility was growth-rate dependent, while norepinephrine-enhanced motility was growth-rate independent. The outer membrane catecholate receptors, IroN, FepA and CirA (required for norepinephrine-enhanced growth) were not required for norepinephrine-enhanced motility, nor was ExbD of the energy-transducing TonB-ExbB-ExbD ferri-siderophore uptake system. Examination of the QseBC two-component system revealed that qseB and qseBC mutants have motility phenotypes similar to wild-type S. Typhimurium, while motility of the qseC mutant was significantly decreased (P<0.01). Each mutant of the QseBC system, as well as mutants of qseE and pmrA, responded to norepinephrine with increased motility, suggesting that other genes are involved in norepinephrine-enhanced motility of S. Typhimurium. In the swine host, fecal shedding of the qseBC mutant was similar to wild-type S. Typhimurium, whereas fecal shedding of the qseC mutant was significantly decreased (P<0.01). Our data indicate that, in a qseC mutant, the QseB response regulator decreases motility and swine colonization; inactivation of the qseBC operon restores these bacterial phenotypes, classifying QseB as a negative regulator of bacterial motility and swine colonization.

Detection of Campylobacter jejuni liver dissemination in experimentally colonized turkey poults.

Consumption of contaminated poultry products, including chicken livers, is the main source of human campylobacteriosis and approximately 90% of human cases are caused by Campylobacter jejuni subsp. jejuni (C. jejuni). Recent culinary trends that favor undercooked chicken livers may be responsible for outbreaks. Turkey is an emerging human protein source, and poultry livers are commonly prepared in popular cuisine such as pâté. The mechanism of how Campylobacter disseminates to poultry liver tissue is unknown. We have previously demonstrated that certain strains of C. jejuni persistently colonize turkeys with the highest density in the ceca. Whether C. jejuni disseminates to the liver of turkeys following intestinal colonization is unknown. In this study, 45 D of hatch turkey poults were co-housed for 30 D. Five poults were euthanized to screen for Campylobacter colonization, and were free of detectable Campylobacter. The remaining 40 poults were randomly split into 2 rooms, with 20 poults per room. At 35 D of age, poults were inoculated by oral gavage with 1 × 106 cfu of C. jejuni isolate NCTC 11168 or mock-inoculated with sterile medium. Ten poults from each room were euthanized at 7 and 14 D post-inoculation (dpi), and cecal contents and livers were cultured and/or enriched for Campylobacter. Livers were harvested aseptically. The ceca of C. jejuni-inoculated poults were highly colonized at 7 and 14 dpi with approximately 108 cfu/mL of cecal contents. At 7 and 14 dpi, 3 and 5 of 10 liver samples were positive for C. jejuni culture (8.6 × 103 cfu/g of liver ± 4.43 × 103 and 5.10 × 103 cfu/g of liver ± 1.74 × 103), respectively. At 14 dpi, liver samples were cultured by enrichment, and 6 of 10 were positive for Campylobacter. Some liver samples may be below the limit of detection for direct plate culturing. These data determined that turkey liver is a potential reservoir of C. jejuni following intestinal colonization, and identified a potential food safety consideration when turkey liver is prepared for human or pet food consumption.

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.