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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.

Multidrug-resistant (MDR, resistance to &gt, 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.

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.

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.

Supershedding cattle shed Escherichia coli O157:H7 (O157) at ≥ 104 colony-forming units/g feces. We recently demonstrated that a supershed O157 (SS-O157) strain, SS-17, hyperadheres to the rectoanal junction (RAJ) squamous epithelial (RSE) cells which may contribute to SS-O157 persistence at this site in greater numbers, thereby increasing the fecal O157 load characterizing the supershedding phenomenon. In order to verify if this would be the signature adherence profile of any SS-O157, we tested additional SS-O157 isolates (n = 101; each from a different animal) in the RSE cell adherence assay. Similar to SS-17, all 101 SS-O157 exhibited aggregative adherence on RSE cells, with 56% attaching strongly (>10 bacteria/cell; hyperadherent) and 44% attaching moderately (1–10 bacteria/cells). Strain typing using Polymorphic Amplified Typing Sequences (PATS) analysis assigned the 101 SS-O157 into 5 major clades but not to any predominant genotype. Interestingly, 69% of SS-O157 isolates were identical to human O157 outbreak strains based on pulsed field gel electrophoresis profiles (CDC PulseNet Database), grouped into two clades by PATS distinguishing them from remaining SS-O157, and were hyperadherent on RSE cells. A subset of SS-O157 isolates (n = 53) representing different PATS and RSE cell adherence profiles were analyzed for antibiotic resistance (AR). Several SS-O157 (30/53) showed resistance to sulfisoxazole, and one isolate was resistant to both sulfisoxazole and tetracycline. Minimum inhibitory concentration (MIC) tests confirmed some of the resistance observed using the Kirby–Bauer disk diffusion test. Each SS-O157 isolate carried at least 10 genes associated with AR. However, genes directly associated with AR were rarely amplified: aac (3)-IV in 2 isolates, sul2 in 3 isolates, and tetB in one isolate. The integrase gene, int, linked with integron-based AR acquisition/transmission, was identified in 92% of SS-O157 isolates. Our results indicate that SS-O157 isolates could potentially persist longer at the bovine RAJ but exhibit limited resistance towards clinical antibiotics.

Background Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). Methodology/Principal findings L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Conclusions/Significance Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets., Author summary Leptospirosis is a global zoonotic, neglected tropical disease. Interestingly, a high level of species specificity (both bacteria and host) plays a major role in the severity of disease presentation which can vary from asymptomatic to multi-organ failure. Pathogenic Leptospira colonize the kidneys of infected individuals and are shed in urine into the environment where they can survive until they are contracted by another host. This study looks at two strains of L. borgpetersenii, HB203 and JB197 which are genetically very similar, and identical by serotyping as serovar Hardjo, yet HB203 causes a chronic infection in the hamster while JB197 causes organ failure and mortality. To better characterize bacterial factors causing different disease outcomes, we examined the gene expression profile of these strains in the context of temperatures that would reflect natural Leptospira life cycles (environmentally similar 29°C and 37°C which is more indicative of host environment). We found vast differences in gene expression both between the strains and within strains between temperatures. Characterization of the transcriptome of L. borgpetersenii serovar Hardjo strains JB197 and HB203 provides insights into factors that can determine acute versus chronic disease in the hamster model of infection. Additionally, these studies highlight strain to strain variability within the same species, and serovar, at different growth temperatures, which needs to be considered when serovars are selected and propagated for use as bacterin vaccines used to immunize domestic animal species.

PvuII ribotyping and MLST are each highly discriminatory methods for genotyping Bordetella bronchiseptica, but a direct comparison between these approaches has not been undertaken. The goal of this study was to directly compare the discriminatory power of PvuII ribotyping and MLST, using a single set of geographically and genetically diverse strains, and to determine whether subtyping based on repeat region sequences of the pertactin gene (prn) provides additional resolution. One hundred twenty-two isolates were analyzed, representing 11 mammalian or avian hosts, sourced from the United States, Europe, Israel and Australia. Thirty-two ribotype patterns were identified; one isolate could not be typed. In comparison, all isolates were typeable by MLST and a total of 30 sequence types was identified. An analysis based on Simpson's Index of Diversity (SID) revealed that ribotyping and MLST are nearly equally discriminatory, with SIDs of 0.920 for ribotyping and 0.919 for MLST. Nonetheless, for ten ribotypes and eight MLST sequence types, the alternative method discriminates among isolates that otherwise type identically. Pairing prn repeat region typing with ribotyping yielded 54 genotypes and increased the SID to 0.954. Repeat region typing combined with MLST resulted in 47 genotypes and an SID of 0.944. Given the technical and practical advantages of MLST over ribotyping, and the nominal difference in their SIDs, we conclude MLST is the preferred primary typing tool. We recommend the combination of MLST and prn repeat region typing as a high-resolution, objective and standardized approach valuable for investigating the population structure and epidemiology of B. bronchiseptica.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glasser's disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.

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.

Highlights • The KM22 genome sequence is similar to other B. bronchiseptica strains isolated from animals. • bopN, bvgA, fimB, and fimC were the most highly conserved BvgAS-regulated genes present in all strains analyzed. • BvgAS-regulated genes with the highest sequence divergence werefimN, fim2, fhaL, and fhaS. • A total of eight major fimbrial subunit genes were identified in KM22. • qPCR data demonstrated that seven of the eight fimbrial subunit genes are expressed and regulated by BvgAS., The well-characterized Bordetella bronchiseptica strain KM22, originally isolated from a pig with atrophic rhinitis, has been used to develop a reproducible swine respiratory disease model. The goal of this study was to identify genetic features unique to KM22 by comparing the genome sequence of KM22 to the laboratory reference strain RB50. To gain a broader perspective of the genetic relationship of KM22 among other B. bronchiseptica strains, selected genes of KM22 were then compared to five other B. bronchiseptica strains isolated from different hosts. Overall, the KM22 genome sequence is more similar to the genome sequences of the strains isolated from animals than the strains isolated from humans. The majority of virulence gene expression in Bordetella is positively regulated by the two-component sensory transduction system BvgAS. bopN, bvgA, fimB, and fimC were the most highly conserved BvgAS-regulated genes present in all seven strains analyzed. In contrast, the BvgAS-regulated genes present in all seven strains with the highest sequence divergence werefimN, fim2, fhaL, andfhaS. A total of eight major fimbrial subunit genes were identified in KM22. Quantitative real-time PCR data demonstrated that seven of the eight fimbrial subunit genes identified in KM22 are expressed and regulated by BvgAS. The annotation of the KM22 genome sequence, coupled with the comparative genomic analyses reported in this study, can be used to facilitate the development of vaccines with improved efficacy towards B. bronchiseptica in swine to decrease the prevalence and disease burden caused by this pathogen.

Fluoroquinolones are broad-spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity, which can cause DNA damage and result in bacterial cell death. In response to DNA damage, bacteria induce an SOS response to stimulate DNA repair. However, the SOS response may also induce prophage with production of infectious virions. Salmonella strains typically contain multiple prophages, and certain strains including phage types DT120 and DT104 contain prophage that upon induction are capable of generalised transduction. In this study, strains of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium DT120 and DT104 were exposed to fluoroquinolones important for use in human and veterinary disease therapy to determine whether prophage(s) are induced that could facilitate phage-mediated gene transfer. Cultures of MDR S. Typhimurium DT120 and DT104 containing a kanamycin resistance plasmid were lysed after exposure to fluoroquinolones (ciprofloxacin, enrofloxacin and danofloxacin). Bacterial cell lysates were able to transfer the plasmid to a recipient kanamycin-susceptible Salmonella strain by generalised transduction. In addition, exposure of DT120 to ciprofloxacin induced the recA gene of the bacterial SOS response and genes encoded in a P22-like generalised transducing prophage. This research indicates that fluoroquinolone exposure of MDR Salmonella can facilitate horizontal gene transfer, suggesting that fluoroquinolone usage in human and veterinary medicine may have unintended consequences, including the induction of phage-mediated gene transfer from MDR Salmonella. Stimulation of gene transfer following bacterial exposure to fluoroquinolones should be considered an adverse effect, and clinical decisions regarding antibiotic selection for infectious disease therapy should include this potential risk.

We report here the genome sequences of two strains of Escherichia coli (ECA-B and ECC-M) that cause bovine mastitis. These strains are known to be associated with persistent and transient mastitis; strain ECA-B causes a transient infection, and ECC-M leads to a persistent infection.

Purpose. Non-host-adapted Salmonella serovars, including the common human food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), are opportunistic pathogens that can colonize food-producing animals without causing overt disease. Interventions against Salmonella are needed to enhance food safety, protect animal health and allow the differentiation of infected from vaccinated animals (DIVA). Methodology. An attenuated S. Typhimurium DIVA vaccine (BBS 866) was characterized for the protection of pigs following challenge with virulent S. Typhimurium. The porcine transcriptional response to BBS 866 vaccination was evaluated. RNA-Seq analysis was used to compare gene expression between BBS 866 and its parent; phenotypic assays were performed to confirm transcriptional differences observed between the strains. Results. Vaccination significantly reduced fever and interferon-gamma (IFNγ) levels in swine challenged with virulent S. Typhimurium compared to mock-vaccinated pigs. Salmonella faecal shedding and gastrointestinal tissue colonization were significantly lower in vaccinated swine. RNA-Seq analysis comparing BBS 866 to its parental S. Typhimurium strain demonstrated reduced expression of the genes involved in cellular invasion and bacterial motility; decreased invasion of porcine-derived IPEC-J2 cells and swimming motility for the vaccine strain was consistent with the RNA-Seq analysis. Numerous membrane proteins were differentially expressed, which was an anticipated gene expression pattern due to the targeted deletion of several regulatory genes in the vaccine strain. RNA-Seq analysis indicated that genes involved in the porcine immune and inflammatory response were differentially regulated at 2 days post-vaccination compared to pre-vaccination. Conclusion. Evaluation of the S. Typhimurium DIVA vaccine indicates that vaccination will provide both swine health and food safety benefits.

The results of this meta-analysis demonstrate that “study” and GI sample location are the most significant factors in shaping the swine gut microbiota. However, in comparisons of results from different studies, some biological factors may be obscured by technical variation among studies. Nonetheless, there are some bacterial taxa that appear to form a core microbiota within the swine GI tract regardless of country of origin, diet, age, or breed. Thus, these results provide the framework for future studies to manipulate the swine gut microbiota for potential health benefits., The swine gut microbiota encompasses a large and diverse population of bacteria that play a significant role in pig health. As such, a number of recent studies have utilized high-throughput sequencing of the 16S rRNA gene to characterize the composition and structure of the swine gut microbiota, often in response to dietary feed additives. It is important to determine which factors shape the composition of the gut microbiota among multiple studies and if certain bacteria are always present in the gut microbiota of swine, independently of study variables such as country of origin and experimental design. Therefore, we performed a meta-analysis using 20 publically available data sets from high-throughput 16S rRNA gene sequence studies of the swine gut microbiota. Next to the “study” itself, the gastrointestinal (GI) tract section that was sampled had the greatest effect on the composition and structure of the swine gut microbiota (P = 0.0001). Technical variation among studies, particularly the 16S rRNA gene hypervariable region sequenced, also significantly affected the composition of the swine gut microbiota (P = 0.0001). Despite this, numerous commonalities were discovered. Among fecal samples, the genera Prevotella, Clostridium, Alloprevotella, and Ruminococcus and the RC9 gut group were found in 99% of all fecal samples. Additionally, Clostridium, Blautia, Lactobacillus, Prevotella, Ruminococcus, Roseburia, the RC9 gut group, and Subdoligranulum were shared by >90% of all GI samples, suggesting a so-called “core” microbiota for commercial swine worldwide. IMPORTANCE The results of this meta-analysis demonstrate that “study” and GI sample location are the most significant factors in shaping the swine gut microbiota. However, in comparisons of results from different studies, some biological factors may be obscured by technical variation among studies. Nonetheless, there are some bacterial taxa that appear to form a core microbiota within the swine GI tract regardless of country of origin, diet, age, or breed. Thus, these results provide the framework for future studies to manipulate the swine gut microbiota for potential health benefits.

Background Escherichia coli O157:H7 (O157) strain 86–24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR−) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms. Results To identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR− parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR− isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR− parent restored parental phenotypes to the CR+ isolate. Conclusions The results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.

Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature, causing an infection that lasts 2-3days. However, in a minority of cases, E. coli has been shown to cause a persistent infection. The mechanisms that allow for a persistent E. coli infection are not fully understood. The goal of this work was to determine protein expression differences between E. coli strains isolated from dairy cattle with transient and persistent mastitis infections. Three persistent and three transient mastitis-derived strains of E. coli were compared using iTRAQ in a shotgun proteomics experiment. Expression data for 1127 proteins were determined. Of these, 28 proteins were associated with expression changes correlated with a difference in disease phenotype. Of particular interest were proteins that have been shown to be essential for bacterial swimming and swarming. Bacterial swimming and swarming assays showed that the strains from the persistent mastitis cases were significantly better in these motility assays than the strains from the transient cases. This work identifies important protein expression differences between E. coli strains that cause a persistent versus a transient infection as well as demonstrates a corresponding difference in the associated bacterial motility phenotypes.The significance of this study is that proteins associated with bacterial swimming and swarming are more highly expressed in the E. coli strains that cause persistent mastitis infections. These findings point to swimming and swarming as important mechanisms involved in how a pathogen establishes a persistent infection in the mammary gland. The role of swimming and swarming in clinical mastitis clearly requires further experimentation.

Haemophilus parasuis causes Glässer's disease and pneumonia in swine. Serotyping is often used to classify isolates but requires reagents that are costly to produce and not standardized or widely available. Sequence-based methods, such as multilocus sequence typing (MLST), offer many advantages over serotyping. An MLST scheme was previously proposed for H. parasuis but genome sequence data only recently available reveals the primers recommended, based on sequences of related bacteria, are not optimal. Here we report modifications to enhance the original method, including primer redesign to eliminate mismatches with H. parasuis sequences and to avoid regions of high sequence heterogeneity, standardization of primer T(m)s and identification of universal PCR conditions that result in robust and reproducible amplification of all targets. The modified typing method was applied to a collection of 127 isolates from North and South America, Europe and Asia. An alignment of the concatenated sequences obtained from seven target housekeeping genes identified 278 variable nucleotide sites that define 116 unique sequence types. A comparison of the original and modified methods using a subset of 86 isolates indicates little difference in overall locus diversity, discriminatory power or in the clustering of strains within Neighbor-Joining trees. Data from the optimized MLST were used to populate a newly created and publicly available H. parasuis database. An accompanying database designed to capture provenance and epidemiological information for each isolate was also created. The modified MLST scheme is highly discriminatory but more robust, reproducible and user-friendly than the original. The MLST database provides a novel resource for investigation of H. parasuis outbreaks and for tracking strain evolution.

Bacteria can exhibit various types of motility. It is known that different types of motilities can be associated with virulence. In this work, we compare gene expression levels in bacteria that were grown under conditions that promoted three different types of E. coli motility. Better understanding of the mechanisms of how bacteria can cause an infection is an important first step to better diagnostics and therapeutics., Bacterial motility is thought to play an important role in virulence. We have previously shown that proficient bacterial swimming and swarming in vitro is correlated with the persistent intramammary infection phenotype observed in cattle. However, little is known about the gene regulation differences important for different motility phenotypes in Escherichia coli. In this work, three E. coli strains that cause persistent bovine mastitis infections were grown in three media that promote different types of motility (planktonic, swimming, and swarming). Using whole-transcriptome RNA sequencing, we identified a total of 935 genes (~21% of the total genome) that were differentially expressed in comparisons of the various motility-promoting conditions. We found that approximately 7% of the differentially expressed genes were associated with iron regulation. We show that motility assays using iron or iron chelators confirmed the importance of iron regulation to the observed motility phenotypes. Because of the observation that E. coli strains that cause persistent infections are more motile, we contend that better understanding of the genes that are differentially expressed due to the type of motility will yield important information about how bacteria can become established within a host. Elucidating the mechanisms that regulate bacterial motility may provide new approaches in the development of intervention strategies as well as facilitate the discovery of novel diagnostics and therapeutics. IMPORTANCE Bacteria can exhibit various types of motility. It is known that different types of motilities can be associated with virulence. In this work, we compare gene expression levels in bacteria that were grown under conditions that promoted three different types of E. coli motility. Better understanding of the mechanisms of how bacteria can cause an infection is an important first step to better diagnostics and therapeutics.

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.

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 ). 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 ). 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.

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spon- giform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic

Salmonella enterica serovar Typhimurium is one of the most common serovars isolated from humans and livestock, and over 35% of these isolates are resistant to three or more antibiotics. Multidrug-resistant (MDR) Salmonella is a public health concern as it is associated with increased morbidity in patients compared to antibiotic sensitive strains, though it is unknown how the antibiotic resistant isolates lead to a more severe infection. Cellular invasion is temporally regulated in Salmonella and normally occurs during late-log and stationary growth. However, our previous work determined that a 30 minute exposure to a sub-inhibitory concentration of tetracycline can induce the full invasion phenotype during early-log growth in certain MDR S. Typhimurium isolates. The current study examined whether sub-inhibitory concentrations of other antibiotics could also induce the invasiveness in the same set of isolates. Ampicillin and streptomycin had no effect on invasion, but certain concentrations of chloramphenicol were found to induce invasion in a subset of isolates. Two of the isolates induced by chloramphenicol were also inducible by tetracycline. RNA-seq analyses demonstrated that chloramphenicol and tetracycline both down-regulated motility gene expression, while up-regulating genes associated with attachment, invasion, and intracellular survival. Eleven fimbrial operons were up-regulated, which is notable as only three fimbrial operons were thought to be inducible in culture; six of these up-regulated operons have been reported to play a role in Salmonella persistence in mice. Overall, these data show that the normal progression of the genetic pathways that regulate invasion can be expedited to occur within 30 minutes due to antibiotic exposure. This altered invasion process due to antibiotics may play a role in the increased intensity and duration of infection observed in patients with MDR Salmonella.

Strains ofChlamydia trachomatisare classified into serovars based on nucleotide sequence differences inompA, the gene that encodes the major outer membrane protein. Phylogenetic characterization of strains based onompA, however, results in serovar groupings that are inconsistent with the distinguishing features ofC. trachomatispathobiology, e.g., tissue tropisms and disease presentation. We have compared nucleotide sequences at multiple sites distributed around the chlamydial genome from 18 strains representing 16 serovars; sampled regions included genes encoding housekeeping enzymes (totaling 2,073 bp), intergenic noncoding segments (1,612 bp), and a gene encoding a second outer membrane protein (porB; 1,023 bp), with theompAsequence (1,194 bp) used for reference. These comparative analyses revealed substantial variation in nucleotide substitution patterns among the sampled regions, with average pairwise sequence differences ranging from 0.15% for the housekeeping genes to 12.1% forompA. Phylogenetic characterization of the sampled genomic sequences yielded a strongly supported tree that divides the strains into groupings consistent withC. trachomatisbiology and which has a topology quite distinct from theompAtree. This phylogenetic incongruity can be accounted for by recombination of theompAgene between different genomic backgrounds. We found, however, no evidence of recombination within or between any of the sampled regions around theC. trachomatisgenome apart fromompA. Parallel analysis of published sequence data on four members of thepmpgene family are consistent with the phylogenetic analyses reported here.

Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the US during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli (STEC) and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness genes in the environment.

Previous investigations aimed at determining whether the mammalian prion protein actually facilitates tangible molecular aspects of either a discrete or pleiotropic functional niche have been debated, especially given the apparent absence of overt behavioral or physiological phenotypes associated with several mammalian prion gene (PRNP) knockout experiments. Moreover, a previous evaluation of PRNP knockout cattle concluded that they were normal, suggesting that the bovine prion protein is physiologically dispensable. Herein, we examined the frequency and distribution of nucleotide sequence variation within the coding regions of bovine PRNP and the adjacent Doppel (PRND) gene, a proximal paralogue to PRNP on BTA13. Evaluation of PRND variation demonstrated that the gene does not depart from a strictly neutral model of molecular evolution, and would therefore not be expected to influence tests of selection within PRNP. Collectively, our analyses confirm that intense purifying selection is indeed occurring directly on bovine PRNP, which is indicative of a protein with an important role. These results suggest that the lack of observed fitness effects may not manifest in the controlled environmental conditions used to care for and raise PRNP knockout animals.

Background Serovars of the human pathogen Chlamydia trachomatis occupy one of three specific tissue niches. Genomic analyses indicate that the serovars have a phylogeny congruent with their pathobiology and have an average substitution rate of less than one nucleotide per kilobase. In contrast, the gene that determines serovar specificity, ompA, has a phylogenetic association that is not congruent with tissue tropism and has a degree of nucleotide variability much higher than other genomic loci. The ompA gene encodes the major surface-exposed antigenic determinant, and the observed nucleotide diversity at the ompA locus is thought to be due to recombination and host immune selection pressure. The possible contribution of a localized increase in mutation rate, however, has not been investigated. Results Nucleotide diversity and phylogenetic relationships of the five constant and four variable domains of the ompA gene, as well as several loci surrounding ompA, were examined for each serovar. The loci flanking the ompA gene demonstrated that nucleotide diversity increased monotonically as ompA is approached and that their gene trees are not congruent with either ompA or tissue tropism. The variable domains of the ompA gene had a very high level of non-synonymous change, which is expected as these regions encode the surface-exposed epitopes and are under positive selection. However, the synonymous changes are clustered in the variable regions compared to the constant domains; if hitchhiking were to account for the increase in synonymous changes, these substitutions should be more evenly distributed across the gene. Recombination also cannot entirely account for this increase as the phylogenetic relationships of the constant and variable domains are congruent with each other. Conclusions The high number of synonymous substitutions observed within the variable domains of ompA appears to be due to an increased mutation rate within this region of the genome, whereas the increase in nucleotide substitution rate and the lack of phylogenetic congruence in the regions flanking ompA are characteristic motifs of gene conversion. Together, the increased mutation rate in the ompA gene, in conjunction with gene conversion and positive selection, results in a high degree of variability that promotes host immune evasion.

The incidence of multi-drug resistant Salmonella has increased globally over the past several decades and has become a major public health concern. Isolates of Salmonella enterica serovar Typhimurium DT104 are resistant to five or more antibiotics, including florfenicol, and have been associated with enhanced virulence in livestock and humans. Because sub-inhibitory concentrations of some antibiotics have been found to modulate invasion of certain bacteria under specific conditions, the effect of florfenicol on S. Typhimurium DT104 invasion was evaluated. Three clinical bovine isolates were exposed to sub-inhibitory concentrations of florfenicol for 30 minutes to establish the initial response to the antibiotic. HEp-2 cellular invasion assays, as well as expression analyses of invasion-related genes, demonstrated that the invasiveness of the S. Typhimurium DT104 isolates was not enhanced after exposure to sub-inhibitory concentrations of florfenicol. These results suggest that cattle and swine can be treated with florfenicol for respiratory illness without exacerbating Salmonella Typhimurium DT104 virulence in carrier animals.

BACKGROUND: Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle. Classical BSE is associated with ingestion of BSE-contaminated feedstuffs. H- and L-type BSE, collectively known as atypical BSE, differ from classical BSE by displaying a different disease phenotype and they have not been linked to the consumption of contaminated feed. Interestingly, the 2006 US H-type atypical BSE animal had a polymorphism at codon 211 of the bovine prion gene resulting in a glutamic acid to lysine substitution (E211K). This substitution is analogous a human polymorphism associated with the most prevalent form of heritable TSE in humans, and it is considered to have caused BSE in the 2006 US atypical BSE animal. In order to determine if this amino acid change is a heritable trait in cattle, we sequenced the prion alleles of the only known offspring of this animal, a 2-year-old heifer. PRINCIPAL FINDINGS: Sequence analysis revealed that both the 2006 US atypical BSE animal and its 2-year-old heifer were heterozygous at bovine prion gene nucleotides 631 through 633 for GAA (glutamic acid) and AAA (lysine). Both animals carry the E211K polymorphism, indicating that the allele is heritable and may persist within the cattle population. CONCLUSIONS: This is the first evidence that the E211K polymorphism is a germline polymorphism, not a somatic mutation, suggesting BSE may be transmitted genetically in cattle. In the event that E211K proves to result in a genetic form of BSE, this would be the first indication that all 3 etiologic forms of TSEs (spontaneous, hereditary, and infectious) are present in a non-human species. Atypical BSE arising as both genetic and spontaneous disease, in the context of reports that at least some forms of atypical BSE can convert to classical BSE in mice, suggests a cattle origin for classical BSE.

Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease of cattle caused by abnormally folded prion proteins. Two regulatory region polymorphisms in the bovine prion gene are associated with resistance to classical BSE disease: a 23-bp region in the promoter that contains a binding site for the repressor protein RP58, and a 12-bp region in intron 1 that has a binding site for the transcription factor SP1. The presence of these binding sites enhances BSE resistance in cattle, whereas cattle that lack these regions are more susceptible to the disease. The present study examined the allele, genotype, and haplotype frequencies for the 23-bp and 12-bp polymorphisms in Holstein cattle from 9 different US states, and these frequencies were compared with data previously established for Holstein cattle from the United Kingdom, Germany, and Japan. Additionally, the coding region of the prion gene was sequenced from the US samples. Finally, archival samples from US Holstein sires born between 1953 and 1957 were analyzed. We found that the resistant allele and genotype frequencies for the US Holstein cattle were as high, or higher, relative to that observed in other countries. Furthermore, the current US frequencies were comparable to those determined in the archival samples from the 1950s. Based on the frequencies of these regulatory region polymorphisms, the US Holstein population is not at a greater risk for BSE than Holsteins worldwide.

DNA microarrays can be used to detect polymorphic loci in addition to identifying genes or regions that are absent within a genome. A survey such as this offers greater insight into the level of diversification within a species or population, which is useful in organisms that have near-identical genomic content but differ in phenotype. The identification of such variable loci can then lead to the characterization of genes linked to unique biological attributes. Here, we describe a competitive hybridization assay using DNA microarrays as a comparative genomics tool to identify nucleotide polymorphisms among closely related strains of Chlamydia trachomatis.

DNA microarrays can be used to detect polymorphic loci in addition to identifying genes or regions that are absent within a genome. A survey such as this offers greater insight into the level of diversification within a species or population, which is useful in organisms that have near-identical genomic content but differ in phenotype. The identification of such variable loci can then lead to the characterization of genes linked to unique biological attributes. Here, we describe a competitive hybridization assay using DNA microarrays as a comparative genomics tool to identify nucleotide polymorphisms among closely related strains of Chlamydia trachomatis.

Analysis of two genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays revealed a logarithmic correlation between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes., By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis.

Background Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect several mammalian species. At least three factors related to the host prion protein are known to modulate susceptibility or resistance to a TSE: amino acid sequence, atypical number of octapeptide repeats, and expression level. These factors have been extensively studied in breeds of Bos taurus cattle in relation to classical bovine spongiform encephalopathy (BSE). However, little is currently known about these factors in Bos indicus purebred or B. indicus × B. taurus composite cattle. The goal of our study was to establish the frequency of markers associated with enhanced susceptibility or resistance to classical BSE in B. indicus purebred and composite cattle. Results No novel or TSE-associated PRNP-encoded amino acid polymorphisms were observed for B. indicus purebred and composite cattle, and all had the typical number of octapeptide repeats. However, differences were observed in the frequencies of the 23-bp and 12-bp insertion/deletion (indel) polymorphisms associated with two bovine PRNP transcription regulatory sites. Compared to B. taurus, B. indicus purebred and composite cattle had a significantly lower frequency of 23-bp insertion alleles and homozygous genotypes. Conversely, B. indicus purebred cattle had a significantly higher frequency of 12-bp insertion alleles and homozygous genotypes in relation to both B. taurus and composite cattle. The origin of these disparities can be attributed to a significantly different haplotype structure within each species. Conclusion The frequencies of the 23-bp and 12-bp indels were significantly different between B. indicus and B. taurus cattle. No other known or potential risk factors were detected for the B. indicus purebred and composite cattle. To date, no consensus exists regarding which bovine PRNP indel region is more influential with respect to classical BSE. Should one particular indel region and associated genotypes prove more influential with respect to the incidence of classical BSE, differences regarding overall susceptibility and resistance for B. indicus and B. taurus cattle may be elucidated.

By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis.