Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples.

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a method implementing TruSeq high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant gene targets representing a full-spectrum of antibiotic resistance classes common to environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within complex environmental samples originated from manure, soil, and livestock feces, in addition to a mock-community reference to assess sensitivity and specificity. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate some of the obstacles that many researchers in this area currently face.

Genomic Changes within a Subset of IncI2 Plasmids Associated with Dissemination of mcr-1 Genes and Other Important Antimicrobial Resistance Determinants.

IncI2 plasmids appear to have only recently become associated with resistance genes; however, their tendency to carry resistance to the antibiotics of last resort and their widespread distribution increase their relative importance. In this study, we describe lineages within this plasmid family that have an increased likelihood of acquisition of antimicrobial resistance genes. Globally distributed mcr-1-carrying IncI2 plasmids were found to cluster with other IncI2 plasmids carrying extended-spectrum beta-lactamase genes, and separately from the non-resistant IncI2 plasmids. In addition, insertion sequence (IS) elements with no direct association with the acquired resistance genes also clustered with the resistance plasmids in the phylogenetic tree. In recognition of the biased sequencing of resistant plasmids globally, the analysis was also performed on resistant and non-resistant IncI2 plasmids sequenced in the USA through government surveillance efforts that do not rely on antibiotic selection. This analysis confirmed a distinct clustering associated with both resistance and mobile elements and identified possible genomic changes in core genes that correlate with increased acquisition of foreign DNA. This work highlights a potential genetic mechanism for increased uptake of foreign DNA within this prevalent family of plasmids.

Weaning Age and Its Effect on the Development of the Swine Gut Microbiome and Resistome.

Piglets are often weaned between 19 and 22 days of age in North America, although in some swine operations this may occur at 14 days or less. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. The effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Here, pigs were weaned at either 14, 21, or 28 days of age, and fecal samples were collected 20 times from day 4 (neonatal) through marketing at day 140. The fecal microbiome was characterized using 16S rRNA gene and shotgun metagenomic sequencing. The fecal microbiome of all piglets shifted significantly 3 to 7 days postweaning, with an increase in microbial diversity. Several Prevotella spp. increased in relative abundance immediately after weaning, as did butyrate-producing species such as Butyricicoccus porcorum, Faecalibacterium prausnitzii, and Megasphaera elsdenii. Within 7 days of weaning, the gut microbiome of pigs weaned at 21 and 28 days of age resembled that of pigs weaned at 14 days. Resistance genes to most antimicrobial classes decreased in relative abundance postweaning, with the exception of those conferring resistance to tetracyclines and macrolides-lincosamides-streptogramin B. The relative abundance of microbial carbohydrate-active enzymes (CAZymes) changed significantly in the postweaning period, with an enrichment of CAZymes involved in degradation of plant-derived polysaccharides. These results demonstrate that the pig gut microbiome tends change in a predictable manner postweaning and that weaning age has only a temporary effect on this microbiome. IMPORTANCE Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sow's milk to a plant-based diet. This is the most important period in commercial swine production, yet the effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Metagenomic sequencing allows for a higher-resolution assessment of the pig gut microbiome and enables characterization of the resistome. Here, we used metagenomic sequencing to identify bacterial species that were enriched postweaning and therefore may provide targets for future manipulation studies. In addition, functional profiling of the microbiome indicated that many carbohydrate and metabolic enzymes decrease in relative abundance after weaning. This study also highlights the challenges faced in reducing antimicrobial resistance in pigs, as genes conferring tetracycline and macrolide resistance remained relatively stable from 7 days of age through to market weight at 140 days despite no exposure to antimicrobials.

Antimicrobial resistance in commensal Escherichia coli and Enterococcus spp. isolated from pigs subjected to different antimicrobial administration protocols.

The antimicrobial resistance (AMR) in human and animal pathogens is a global concern, and antimicrobial use (AMU) is considered the most important driver for its increase. The aim of this study was to assess AMR in Escherichia coli and Enterococcus spp. in faecal samples of pigs subjected to four different AMU protocols from birth to finishing: G1, no in-feed antimicrobials; G2: a total average dose 6018 mg antimicrobials/pig; G3: a total average dose 8127 mg antimicrobials/pig; and G4: a total average dose 15,678 mg antimicrobials/pig. Faecal samples were collected at six time points and AMR was assessed in both bacteria. The microbiota composition was assessed by 16S rRNA sequencing. Minor differences on the microbiota profile was observed among groups, but a lower Firmicutes:Bacteroidetes ratio was noted in G4. Escherichia coli and Enterococcus spp. strains isolated from all groups showed a high level of multi-drug resistance (MDR). The amount of antimicrobials used was significantly positively associated with the probability of MDR in both bacteria. Approximately 43% of the variation in MIC90 for colistin could be explained by AMU, and a one-day increase in administration of colistin increased MIC90 by 0.05 µg mL-1. In conclusion, the results suggest that the higher the use of antimicrobials in farms, the higher the MDR frequency and resistance to the highest priority critically important antimicrobials for humans in commensal gut bacteria of pigs.

Toward Antibiotic Stewardship: Route of Antibiotic Administration Impacts the Microbiota and Resistance Gene Diversity in Swine Feces.

Oral antibiotics are a critical tool for fighting bacterial infections, yet their use can have negative consequences, such as the disturbance of healthy gut bacterial communities and the dissemination of antibiotic residues in feces. Altering antibiotic administration route may limit negative impacts on intestinal microbiota and reduce selective pressure for antimicrobial resistance genes (ARG) persistence and mobility. Thus, a study was performed in pigs to evaluate route of therapeutic oxytetracycline (oxytet) administration, an antibiotic commonly used in the U.S. swine industry, on intestinal microbial diversity and ARG abundance. Given that oral antibiotics would be in direct contact with intestinal bacteria, we hypothesized that oral administration would cause a major shift in intestinal bacterial community structure when compared to injected antibiotic. We further postulated that the impact would extend to the diversity and abundance of ARG in swine feces. At approximately 3 weeks-of-age, piglets were separated into three groups (n = 21-22 per group) with two groups receiving oxytet (one via injection and the second via feed) and a third non-medicated group. Oxytet levels in the plasma indicated injected antibiotic resulted in a spike 1 day after administration, which decreased over time, though oxytet was still detected in plasma 14 days after injection. Conversely, in-feed oxytet delivery resulted in lower but less variable oxytet levels in circulation and high concentrations in feces. Similar trends were observed in microbial community changes regardless of route of oxytet administration; however, the impact on the microbial community was more pronounced at all time points and in all samples with in-feed administration. Fecal ARG abundance was increased with in-feed administration over injected, with genes for tetracycline and aminoglycoside resistance enriched specifically in the feces of the in-feed group. Sequencing of plasmid-enriched samples revealed multiple genetic contexts for the resistance genes detected and highlighted the potential role of small plasmids in the movement of antibiotic resistance genes. The findings are informative for disease management in food animals, but also manure management and antibiotic therapy in human medicine for improved antibiotic stewardship.

Recto-Anal Junction (RAJ) and Fecal Microbiomes of Cattle Experimentally Challenged With Escherichia coli O157:H7.

Cattle are the asymptomatic reservoirs of Escherichia coli O157:H7 (O157) that preferentially colonizes the bovine recto-anal junction (RAJ). Understanding the influence of O157 on the diversity of the RAJ microbiota could give insights into its persistence at the RAJ in cattle. Hence, we compared changes in bovine RAJ and fecal microbiota following O157 challenge under experimental conditions. Cattle were either orally challenged (n = 4) with1010 CFU of a streptomycin-resistant O157 strain 86-24, or mock-challenged (n = 4) with phosphate buffered saline. Rectoanal mucosal swab (RAMS) and fecal samples were collected at different time points for analysis. Alpha diversity measures (Chao1 species richness and Shannon diversity index) were found to be significantly different between RAMS and fecal samples but not influenced by O157 challenge. The Firmicutes to Bacteroidetes (F: B) ratio was higher in RAMS samples from O157 colonized animals and this may have influenced the consistent yet decreased O157 colonization at the RAJ. Specific bacterial genera that were present in relative low abundance in fecal and RAMS microbiota did not affect overall microbial diversity but were associated with O157 colonization. Differential abundance analysis (DAA) of genera in samples from O157 shedding cattle indicated significantly higher relative abundance of Paenibacillus and Fusobacterium in RAMS, and Tyzzerella in fecal samples. Mock-challenged cattle showed higher relative abundance of Intestinimonas and Citrobacter in RAMS samples, and Succinivibrio, and Prevotella 1 in fecal samples. These results suggest that O157 challenge exerts transient influence on the intestinal microbial community which in turn might promote O157 colonization in a site-specific manner.

Outer membrane protein A (OmpA) of extraintestinal pathogenic Escherichia coli.

OBJECTIVE: Extraintestinal Pathogenic E. coli (ExPEC), are responsible for host diseases such as Neonatal Meningitis Escherichia coli (NMEC), the second-leading cause of neonatal bacterial meningitis, Avian Pathogenic E. coli (APEC), a cause of extraintestinal disease in poultry, and Uropathogenic E. coli (UPEC), the most common cause of urinary tract infections. Virulence factors associated with NMEC include outer membrane protein A (OmpA) and type I fimbriae (FimH), which also occur in APEC and UPEC. OmpA contributes to NMEC's ability to cross the blood-brain barrier, persist in the bloodstream and has been identified as a potential vaccine target for ExPEC, however the protein has amino acid variants, which may influence virulence of strains or alter vaccine efficacy. Although OmpA is present in virtually all E. coli, differences in its amino acid residues have yet to be surveyed in ExPEC. RESULTS: Here the ompA gene (n = 399) from ExPEC collections were sequenced and translated in silico. Twenty-five different OmpA polymorphism patterns were identified. Seven polymorphism patterns were significantly associated with an ExPEC subpathotype, but chromosomal history most likely accounts for most differences found. The differences in OmpA protein sequences suggest that OmpA may influence variation in virulence and host specificity within ExPEC subpathotypes.

Cattle intestinal microbiota shifts following Escherichia coli O157:H7 vaccination and colonization

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p <0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7.

Investigating the dispersal of antibiotic resistance associated genes from manure application to soil and drainage waters in simulated agricultural farmland systems.

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment.

Shifts in the nasal microbiota of swine in response to different dosing regimens of oxytetracycline administration.

The impacts of antibiotic treatment and dosing regimen of an antibiotic on the swine respiratory microbiota are poorly defined. To begin to address this, this study characterized the impact of oxytetracycline administration, given either parenterally or in feed, on the diversity of the nasal and tonsil microbiotas of post-weaned pigs over a two-week period. One group received a single intramuscular injection (IM) of oxytetracycline, the second was treated with oxytetracycline mixed in feed (IF), and the control group received non-medicated (NON) feed. Nasal samples were collected on days 0 (before start of treatment), 4, 7, 11, and 14. Tonsil tissue samples were collected from a subset of pigs selected for necropsy on days 4, 7, and 14. The results showed that the tonsil microbiota was stable regardless of antibiotic treatment. In contrast, the nasal bacterial diversity decreased for both oxytetracycline-treated groups compared to NON. The IF group also exhibited decreased diversity on more days than the IM group. The nasal bacterial community structures of the antibiotic treatment groups were significantly different from the NON group that persisted from day 4 until day 7 for the IM group, and up until day 11 for the IF group. This included relative increased abundances of Actinobacillus and Streptococcus, and relative decreased abundances of multiple commensal genera. The microbiota of the IF group was also more disturbed than the microbiota of the IM group, relative to NON. This study revealed that short-term exposure to broad-spectrum antibiotics like oxytetracycline can disturb the upper respiratory microbiota, and the dosing regimen has differential effects on the microbiota.

Dietary Resistant Potato Starch Alters Intestinal Microbial Communities and Their Metabolites, and Markers of Immune Regulation and Barrier Function in Swine.

Interactions between diet, the microbiota, and the host set the ecological conditions in the gut and have broad implications for health. Prebiotics are dietary compounds that may shift conditions toward health by promoting the growth of beneficial microbes that produce metabolites capable of modulating host cells. This study's objective was to assess how a dietary prebiotic could impact host tissues via modulation of the intestinal microbiota. Pigs fed a diet amended with 5% resistant potato starch (RPS) exhibited alterations associated with gut health relative to swine fed an unamended control diet (CON). RPS intake increased abundances of anaerobic Clostridia in feces and several tissues, as well as intestinal concentrations of butyrate. Functional gene amplicons suggested bacteria similar to Anaerostipes hadrus were stimulated by RPS intake. The CON treatment exhibited increased abundances of several genera of Proteobacteria (which utilize respiratory metabolisms) in several intestinal locations. RPS intake increased the abundance of regulatory T cells in the cecum, but not periphery, and cecal immune status alterations were indicative of enhanced mucosal defenses. A network analysis of host and microbial changes in the cecum revealed that regulatory T cells positively correlated with butyrate concentration, luminal IgA concentration, expression of IL-6 and DEF1B, and several mucosa-associated bacterial taxa. Thus, the administration of RPS modulated the microbiota and host immune status, altering markers of cecal barrier function and immunological tolerance, and suggesting a reduced niche for bacterial respiration.

Paenibacillus 79R4, a potential rumen probiotic to enhance nitrite detoxification and methane mitigation in nitrate-treated ruminants.

The effects of supplemental nitrate administered alone or with a denitrifying ruminal bacterium, designated Paenibacillus 79R4 (79R4) intentionally selected for enhanced nitrate- and nitrite-metabolizing ability, on select rumen fermentation characteristics was examined in vivo. Rumen and blood samples were collected from cannulated Holstein steers one day prior to and one day after initiation of treatments applied as three consecutive intra-ruminal administrations of nitrate, to achieve the equivalent of 83 mg sodium nitrate/kg body weight day, given alone or with the nitrite-selected 79R4 (provided to achieve 106 cells/mL rumen fluid). Results revealed a day effect on methane-producing activity, with rates of methane production by ruminal microbes being more rapid when collected one day before than one day after initiation of treatments. Nitrate-metabolizing activity of the rumen microbes was unaffected by day, treatment or their interaction. A day by treatment interaction was observed on nitrite-metabolizing activity, with rates of nitrite metabolism by rumen microbes being most rapid in populations collected one day after initiation of treatment from steers treated with nitrate plus 79R4. A day by treatment interaction was also observed on plasma methemoglobin concentrations, with concentrations being lower from steers one day after initiation of treatments than from collected one day prior to treatment initiation and concentrations being lowest in steers treated with nitrate plus 79R4. A major effect of treatment was observed on accumulations of most prominent and branched chain volatile fatty acids produced and amounts of hexose fermented in the rumen of animals administered nitrate, with concentrations being decreased in steers administered nitrate alone when compared to steers treated with nitrate plus the 79R4. These results demonstrate that the nitrite-selected Paenibacillus 79R4 may help prevent nitrite toxicity in nitrate-treated ruminants while maintaining benefits of reduced methane emissions and preventing inhibition of fermentation efficiency by the microbial ecosystem.

Fecal microbiota changes associated with dehorning and castration stress primarily affects light-weight dairy calves.

Gastrointestinal tract (GIT) microbiota and stress can impact animal health. Studies have shown that perturbations in the GIT microbiota can influence host health and productivity by affecting physiological homeostasis, metabolism, hematopoiesis and inflammation. The present study aimed to evaluate possible effects of dehorning and castration stress on the GIT microbiota of dairy calves. Dehorning and castration are routinely performed on over 90% of dairy farms, and analgesics like flunixin meglumine (FLU) are given at the time of these procedures to reduce pain. We analyzed fecal microbiota of 24 weaned male dairy calves at two different stages in their life (at 10 weeks for dehorning and 36 weeks age for castration) to determine any GIT microbiota changes due to these stressful procedures and the FLU treatment. Dehorning was performed using an electrocautery dehorner applied to the horn for 10 seconds, and surgical castration was used as the castration method. Our analysis showed that the Shannon diversity index was significantly higher in animals that were not dehorned compared to dehorned animals. Castration stress also resulted in a significant decrease in Shannon diversity index, which was more pronounced in lower weight calves. Body weight and stress had significant effects on the taxonomic profiles of the GIT microbiota. There was a significant difference in the GIT bacterial community structure between heavy- and light-weight calves at Day 3 after castration but not at Day 0 (prior to castration). Our results indicate that dehorning and castration stress reduced microbial diversity of the GIT microbiota, but only in light-weight calves. This work is important for elucidating biological effects of stress on dairy calves and identifying potential modulation points in the microbiota of these food-producing animals to improve animal health and production.

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.

Complete Genome Sequence of the Multidrug-Resistant Neonatal Meningitis Escherichia coli Serotype O75:H5:K1 Strain mcjchv-1 (NMEC-O75).

Neonatal meningitis Escherichia coli (NMEC) is the second leading cause of neonatal bacterial meningitis worldwide. We report the genome sequence of the multidrug-resistant NMEC serotype O75:H5:K1 strain mcjchv-1, which resulted in an infant's death. The O75 serogroup is rare among NMEC isolates; therefore, this strain is considered an emergent pathogen.

Complete Genome Sequence of Avian Pathogenic Escherichia coli Strain APEC O2-211.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, a disease that affects poultry production worldwide and leads to multimillion-dollar losses annually. Here, we report the genome sequence of APEC O2-211, a sequence type 117 (ST117) strain isolated from a diseased chicken.

Isolation, characterization and strain selection of a Paenibacillus species for use as a probiotic to aid in ruminal methane mitigation, nitrate/nitrite detoxification and food safety.

The effects of dietary nitrate and Paenibacillus 79R4 (79R4), a denitrifying bacterium, when co-administered as a probiotic, on methane emissions, nitrate and nitrite-metabolizing capacity and fermentation characteristics were studied in vitro. Mixed populations of rumen microbes inoculated with 79R4 metabolized all levels of nitrite studied after 24 h in vitro incubation. Results from in vitro simulations resulted in up to 2 log10 colony forming unit reductions in E. coli O157:H7 and Campylobacter jejuni when these were co-cultured with 79R4. Nitrogen gas was the predominant final product of nitrite reduction by 79R4. When tested with nitrate-treated incubations of rumen microbes, 79R4 inoculation (provided to achieve 106 cells/mL rumen fluid volume) complemented the ruminal methane-decreasing potential of nitrate (P < 0.05) while concurrently increasing fermentation efficiency and enhancing ruminal nitrate and nitrite-metabolizing activity (P < 0.05) compared to untreated and nitrate only-treated incubations.

Butyricicoccus porcorum sp. nov., a butyrate-producing bacterium from swine intestinal tract.

A Gram-stain-positive, non-motile, butyrate-producing coccus was cultured from the distal ileum of swine. This organism was isolated on rumen-fluid medium, consumes acetate, and produces butyrate as its major end product when grown on mono- and di-saccharides. A phylogenetic analysis based on near full-length 16S rRNA gene sequences as well as whole-genome phylogenies suggests that this isolate is most closely related to species in the genus Butyricicoccus, with Butyricicoccus pullicaecorum being the closest named relative (93.5 % 16S similarity). The G+C content of this isolate is 54 mol%, and the major cellular fatty acids are C18 : 0 DMA, C14 : 0, C18 : 1ω9c and C16 : 0. These data indicate that this isolate represents a novel species within the genus Butyricicoccus, for which the name Butyricicoccus porcorum sp. nov. is proposed. The type strain of Butyricicoccus porcorum is BB10T (ATCC TSD-102T, DSM 104997T).

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.

Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance.

Use of antibiotics in human and animal medicine has applied selective pressure for the global dissemination of antibiotic-resistant bacteria. Therefore, it is of interest to develop strategies to mitigate the continued amplification and transmission of resistance genes in environmental reservoirs such as farms, hospitals and watersheds. However, the efficacy of mitigation strategies is difficult to evaluate because it is unclear which resistance genes are important to monitor, and which primers to use to detect those genes. Here, we evaluated the diversity of one type of macrolide antibiotic resistance gene (erm) in one type of environment (manure) to determine which primers would be most informative to use in a mitigation study of that environment. We analyzed all known erm genes and assessed the ability of previously published erm primers to detect the diversity. The results showed that all known erm resistance genes group into 66 clusters, and 25 of these clusters (40%) can be targeted with primers found in the literature. These primers can target 74%-85% of the erm gene diversity in the manures analyzed.