Litter Commensal Bacteria Can Limit the Horizontal Gene Transfer of Antimicrobial Resistance to Salmonella in Chickens.

Fostering a "balanced" gut microbiome through the administration of beneficial microbes that can competitively exclude pathogens has gained a lot of attention and use in human and animal medicine. However, little is known about how microbes affect the horizontal gene transfer of antimicrobial resistance (AMR). To shed more light on this question, we challenged neonatal broiler chicks raised on reused broiler chicken litter-a complex environment made up of decomposing pine shavings, feces, uric acid, feathers, and feed-with Salmonella enterica serovar Heidelberg (S. Heidelberg), a model pathogen. Neonatal chicks challenged with S. Heidelberg and raised on reused litter were more resistant to S. Heidelberg cecal colonization than chicks grown on fresh litter. Furthermore, chicks grown on reused litter were at a lower risk of colonization with S. Heidelberg strains that encoded AMR on IncI1 plasmids. We used 16S rRNA gene sequencing and shotgun metagenomics to show that the major difference between chicks grown on fresh litter and those grown on reused litter was the microbiome harbored in the litter and ceca. The microbiome of reused litter samples was more uniform and enriched in functional pathways related to the biosynthesis of organic and antimicrobial molecules than that in fresh litter samples. We found that Escherichia coli was the main reservoir of plasmids encoding AMR and that the IncI1 plasmid was maintained at a significantly lower copy per cell in reused litter compared to fresh litter. These findings support the notion that commensal bacteria play an integral role in the horizontal transfer of plasmids encoding AMR to pathogens like Salmonella. IMPORTANCE Antimicrobial resistance spread is a worldwide health challenge, stemming in large part from the ability of microorganisms to share their genetic material through horizontal gene transfer. To address this issue, many countries and international organizations have adopted a One Health approach to curtail the proliferation of antimicrobial-resistant bacteria. This includes the removal and reduction of antibiotics used in food animal production and the development of alternatives to antibiotics. However, there is still a significant knowledge gap in our understanding of how resistance spreads in the absence of antibiotic selection and the role commensal bacteria play in reducing antibiotic resistance transfer. In this study, we show that commensal bacteria play a key role in reducing the horizontal gene transfer of antibiotic resistance to Salmonella, provide the identity of the bacterial species that potentially perform this function in broiler chickens, and also postulate the mechanism involved.

Turicibacter bilis sp. nov., a novel bacterium isolated from the chicken eggshell and swine ileum.

Three novel, anaerobic, Gram-positive bacteria were isolated from the eggshell of two separate white leghorn chicken flocks and the ileum of a healthy pig, and designated MMM721T, ISU324 and PIG517 respectively. Cells were pleomorphic and capable of forming long chains of rods or coccoid clusters. Phylogenetic analysis of the 16S rRNA gene sequences identified these strains to be within the genus Turicibacter, of which only one species, Turicibacter sanguinis, has been formally described. However, whole genome sequencing of novel isolates returned a digital DNA-DNA hybridization value of 22.5 % and average nucleotide identity (ANI) values of 76.4 % (ANIb) and 86.0 % (ANIm), indicating divergence between the type strain MMM721T and T. sanguinis, suggesting the strains represented a novel species. The major fatty acid methyl esters of strain MMM721T were C16 : 0, C18 : 1 ω7c and C18 : 0. The strains mainly produced the volatile fatty acid lactate, along with smaller amounts of acetate and butyrate. Together, these data indicate that MMM721T, along with ISU324 and PIG517, represent a novel species within the genus Turicibacter. We propose the name Turicibacter bilis sp. nov. for the species. The type strain is MMM721T (=ATCC TSD-238T=CCUG 74757T).

Horizontal Gene Transfer and Acquired Antibiotic Resistance in Salmonella enterica Serovar Heidelberg following In Vitro Incubation in Broiler Ceca.

The chicken gastrointestinal tract harbors microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta, and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from cecal flora to Salmonella enterica serovar Heidelberg. We used whole-genome sequencing and resistome enrichment to decipher the interactions between S Heidelberg, the gut microbiome, and acquired antibiotic resistance. After 48 h of incubation of ceca under microaerophilic conditions, we recovered one S Heidelberg isolate with an acquired IncK2 plasmid (88 kb) carrying an extended-spectrum-ß-lactamase gene (blaCMY-2). In vitro, this plasmid was transferable between Escherichia coli and S Heidelberg strains but transfer was unsuccessful between S Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of horizontal gene transfer between an important foodborne pathogen and the chicken gut microbiome.IMPORTANCES. Heidelberg is a clinically important serovar, linked to foodborne illness and among the top 5 serovars isolated from poultry in the United States and Canada. Acquisition of new genetic material from the microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding of the interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this report, we show that the native flora in broiler ceca were capable of transferring mobile genetic elements carrying the AmpC ß-lactamase (blaCMY-2) gene to an important foodborne pathogen, S Heidelberg. The potential role for bacteriophage transduction is also discussed.

Nanoparticle microarray for high-throughput microbiome metabolomics using matrix-assisted laser desorption ionization mass spectrometry.

A high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI)-MS-based metabolomics platform was developed using a pre-fabricated microarray of nanoparticles and organic matrices. Selected organic matrices, inorganic nanoparticle (NP) suspensions, and sputter coated metal NPs, as well as various additives, were tested for metabolomics analysis of the turkey gut microbiome. Four NPs and one organic matrix were selected as the optimal matrix set: α-cyano-4-hydroycinnamic acid, Fe3O4 and Au NPs in positive ion mode with 10 mM sodium acetate, and Cu and Ag NPs in negative ion mode with no additive. Using this set of five matrices, over two thousand unique metabolite features were reproducibly detected across intestinal samples from turkeys fed a diet amended with therapeutic or sub-therapeutic antibiotics (200 g/ton or 50 g/ton bacitracin methylene disalicylate (BMD), respectively), or non-amended feed. Among the thousands of unique features, 56 of them were chemically identified using MALDI-MS/MS, with the help of in-parallel liquid chromatography (LC)-MS/MS analysis. Lastly, as a proof of concept application, this protocol was applied to 52 turkey cecal samples at three different time points from the antibiotic feed trial. Statistical analysis indicated variations in the metabolome of turkeys with different ages or treatments. Graphical abstract ᅟ.

Megasphaera stantonii sp. nov., a butyrate-producing bacterium isolated from the cecum of a healthy chicken.

A novel mesophilic, anaerobic, Gram-stain-negative bacterium was isolated from the cecum of a healthy white leghorn chicken, and designated AJH120T. Cells were coccoid or diplococcoid with an average size of 0.8-1.8 µm and were non-motile with no evidence of spores. Phylogenetic analysis of 16S rRNA gene sequences revealed this organism to be a member of the genus Megasphaera, with the closest relatives being Megasphaera elsdenii (95 % sequence identity) and Megasphaera cerevisiae (95 % sequence identity). Growth was observed between 30 and 50 °C and between pH 5.0 and 9.0. AJH120T utilized a variety of carbon sources, including succinate, gluconate, fructose, ribose and pyruvate, as well as many individual amino acids. The DNA G+C content for the genome sequence of AJH120T was 52.1 mol%. Digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) and average amino acid identity (AAI) between AJH120T and close taxonomic relatives, indicated divergence consistent with the strain representing a novel species. The major fatty acid methyl esters of the organism were C12 : 0, C14 : 0 3-OH, C18 : 1ω9c, C16 : 0 and C16 : 1ω9c. AJH120T was able to produce several short chain fatty acids, including butyrate, acetate, propionate and isovalerate. Together, these data indicate that AJH120T represents a novel species within the genus Megasphaera. We propose the name Megasphaerastantonii sp. nov. for the species. The type strain of this species is AJH120T (=DSM 106750T=CCUG 71842T).

Bulk soil bacterial community structure and function respond to long-term organic and conventional agricultural management.

Understanding how soil microbiomes respond to management is essential to maximizing soil health. We contrasted microbiomes in bulk soil under long-term organic and conventional management in a grain production setting. Management category significantly impacted the relative abundances of 17% of the most abundant taxa. Both conventional and organic management favored particular taxa, but these effects were not reflected in summary richness and diversity indices. Management systems also lead to differences in soil edaphic properties, including pH and nutrient status; this may have been the mechanism by which change in the prokaryote community was enacted. Community change between years of sampling was less pronounced, with only 6 taxa differentially abundant among years. Management category also impacted the abundance of functional genes related to the production and consumption of greenhouse gases. Particulate methane monooxygenase genes were more frequent in soil under organic management, while soluble methane monooxygenase genes were more frequent in soil under conventional management in 1 of 2 years. Nitrous oxide reductase genes were significantly less abundant in soils under second-year alfalfa than in soils under corn. This work highlights the ability of agricultural management to enact broad rearrangements to the structure of bulk soil bacterial communities.

Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine.

Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family (but) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCE: Butyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease.

In-feed antibiotic effects on the swine intestinal microbiome.

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in Proteobacteria (1-11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in Escherichia coli populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.

Salmonella Biomapping of a Commercial Broiler Hatchery.

Poultry-associated salmonellosis results in significant costs to poultry producers and consumers. Given the vertically integrated nature of the United States poultry industry, a better understanding of Salmonella ecology throughout all levels of poultry production is essential. One nexus point is the hatchery, where eggs from multiple broiler breeder farms are incubated and hatched, with the chicks being sent to numerous farms; therefore, the hatchery represents an ideal area to understand preharvest Salmonella ecology and flow. To achieve this, a commercial broiler hatchery was biomapped, focusing on Salmonella prevalence and serotype diversity among four major sample type categories (Air, Egg, Water, Facility) across five different places in the prehatch, hatch, and posthatch areas. Following two sets of eggs from broiler breeder farms over two production days, the overall Salmonella prevalence was 26% (48/184). Of the positive samples, the highest prevalence was observed in swabs taken from the floor drains in the facility and transport truck (56%), as well as in the hatch and posthatch hatchery areas (50%). Kentucky (n = 17), Gaminara (n = 12), and Alachua (n = 11) were the dominant Salmonella serotypes, with serotypes of greatest outbreak concern from chickens (Enteritidis) representing only 6.25% (3/48) of all recovered Salmonella isolates. The posthatch transport area, including the underfloor reservoirs of the transport trucks, not only harbored Enteritidis but also the enrichment broths from these Salmonella-positive samples also possessed sequences matching the commercial live-attenuated vaccine Typhimurium strain according to CRISPR SeroSeq analyses. These findings highlight the complex diversity of commercial hatchery Salmonella populations, including identifying facility floor drains and transport trucks as potentially important critical control points for hatchery managers to focus their Salmonella mitigation efforts to reduce loads and serotypes entering live production farms.

Butyrate-producing bacteria, including mucin degraders, from the swine intestinal tract.

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.

Deciphering the Association between Campylobacter Colonization and Microbiota Composition in the Intestine of Commercial Broilers.

Campylobacter is a major food safety concern and is transmitted mainly via poultry meat. We previously found that some commercial broiler farms consistently produced Campylobacter-negative flocks while others were consistently Campylobacter-positive for consecutive production cycles although the farms operated under similar management practices. We hypothesized that this difference in Campylobacter colonization might be associated with the gut microbiota composition. To address this, six commercial broiler farms were selected based on their Campylobacter status (three negative and three positive) to evaluate the microbiota differences between each farm category. For each farm on each production cycle (2-3 cycles), 40 ceca collected from five-week-old broilers were processed for microbiota analysis via 16S rRNA gene sequencing. Cecal microbiota species richness, phylogenetic diversity, community structure, and composition of Campylobacter-positive farms were noticeably different from those of Campylobacter-negative farms. Rikenella, Methanocorpusculum, Barnesiella, Parasutterella, and Helicobacter were significantly more abundant among Campylobacter-positive farms. In contrast, Ruminococcaceae, Streptococcus, Escherichia, Eggerthellaceae, Lactobacillus, Monoglobus, and Blausia were more abundant in Campylobacter-negative farms. Eggerthellaceae, Clostridia, Lachnospiraceae, Lactobacillus, Monoglobus, and Parabacteroides were significantly negatively correlated with Campylobacter abundance. These findings suggest that specific members of cecal microbiota may influence Campylobacter colonization in commercial broilers and may be further explored to control Campylobacter in poultry.

Fecal Microbiota Transplantation Reduces Campylobacter jejuni Colonization in Young Broiler Chickens Challenged by Oral Gavage but Not by Seeder Birds.

Campylobacter spp., particularly C. jejuni and C. coli, are major food safety concerns, transmitted to humans mainly via contaminated poultry meat. In a previous study, we found that some commercial broiler farms consistently produced Campylobacter-free flocks while others consistently reared Campylobacter-colonized flocks, and significant differences in the gut microbiota compositions between the two types of farm categories were revealed. Therefore, we hypothesized that gut microbiota influences Campylobacter colonization in poultry and that the microbiota from Campylobacter-free flocks may confer colonization resistance to Campylobacter in the chicken intestine. In this study, two fecal microbiota transplantation (FMT) trials were performed to test the hypothesis. Newly hatched chicks were given FMT via oral gavage of the cecal content of Campylobacter-free adult chickens (treatment groups) or PBS (control groups) before the feed consumption. Approximately two weeks after the FMT, the birds were challenged with C. jejuni either by oral gavage (trial 1) or by co-mingling with Campylobacter-colonized seeder birds (trial 2) to evaluate the potential protective effect of the FMT. Cecal contents were collected (3 times, 5 days apart) to determine the Campylobacter colonization levels via culture and microbiota compositions via 16S rRNA gene sequencing. FMT reduced cecal Campylobacter colonization significantly (log10 1.2-2.54 CFU/g) in trial 1 but not in trial 2, although FMT significantly impacted the diversity and compositions of the gut microbiota in both trials. Several genera, such as Butyricimonas, Parabacteroides, Parasutterella, Bilophila, Fournierella, Phascolarctobacterium, and Helicobacter, had increased abundance in the FMT-treated groups in both trials. Furthermore, Campylobacter abundance was found to be negatively correlated with the Escherichia and Ruminococcus_torques_group genera. These findings indicate that even though FMT with adult cecal microbiota can positively affect the subsequent development of the gut microbiota in young broilers, its inhibitory effect on Campylobacter colonization varies and appears to be influenced by the challenge models.

On-tissue chemical derivatization of volatile metabolites for matrix-assisted laser desorption/ionization mass spectrometry imaging.

Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix-assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. On-tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide to chemically modify short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization.

Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution.

Campylobacter fetus is a pathogen, which is primarily associated with fertility problems in sheep and cattle. In humans, it can cause severe infections that require antimicrobial treatment. However, knowledge on the development of antimicrobial resistance in C. fetus is limited. Moreover, the lack of epidemiological cut-off values (ECOFFs) and clinical breakpoints for C. fetus hinders consistent reporting about wild-type and non-wild-type susceptibility. The aim of this study was to determine the phenotypic susceptibility pattern of C. fetus and to determine the C. fetus resistome [the collection of all antimicrobial resistance genes (ARGs) and their precursors] to describe the genomic basis of antimicrobial resistance in C. fetus isolates over time. Whole-genome sequences of 295 C. fetus isolates, including isolates that were isolated in the period 1939 till the mid 1940s, before the usage of non-synthetic antimicrobials, were analysed for the presence of resistance markers, and phenotypic antimicrobial susceptibility was obtained for a selection of 47 isolates. C. fetus subspecies fetus (Cff) isolates showed multiple phenotypic antimicrobial resistances compared to C. fetus subspecies venerealis (Cfv) isolates that were only intrinsic resistant to nalidixic acid and trimethoprim. Cff isolates showed elevated minimal inhibitory concentrations for cefotaxime and cefquinome that were observed in isolates from 1943 onwards, and Cff isolates contained gyrA substitutions, which conferred resistance to ciprofloxacin. Resistances to aminoglycosides, tetracycline and phenicols were linked to acquired ARGs on mobile genetic elements. A plasmid-derived tet(O) gene in a bovine Cff isolate in 1999 was the first mobile genetic element observed, followed by detection of mobile elements containing tet(O)-aph(3')-III and tet(44)-ant(6)-Ib genes, and a plasmid from a single human isolate in 2003, carrying aph(3')-III-ant(6)-Ib and a chloramphenicol resistance gene (cat). The presence of ARGs in multiple mobile elements distributed among different Cff lineages highlights the risk for spread and further emergence of AMR in C. fetus. Surveillance for these resistances requires the establishment of ECOFFs for C. fetus.

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.

Temporal dynamics of the cecal and litter microbiome of chickens raised in two separate broiler houses.

In this study, we investigated the dynamics of the ceca and litter microbiome of chickens from post-hatch through pre-harvest. To achieve this, six hundred one-day old Cobb 500 broiler chicks were raised on floor pens for 49 days in two separate houses. We performed short-read and full-length sequencing of the bacterial 16S rRNA gene present in the meconium and in cecal and litter samples collected over the duration of the study. In addition, we determined the antimicrobial resistance (AMR) phenotype of Escherichia coli and Enterococcus spp. isolated from the meconium and the ceca of 49-day old chickens. We monitored the relative humidity, temperature, and ammonia in each house daily and the pH and moisture of litter samples weekly. The overall microbial community structure of the ceca and litter consistently changed throughout the course of the grow-out and correlated with some of the environmental parameters measured (p < 0.05). We found that the ceca and litter microbiome were similar in the two houses at the beginning of the experiment, but over time, the microbial community separated and differed between the houses. When we compared the environmental parameters in the two houses, we found no significant differences in the first half of the growth cycle (day 0-21), but morning temperature, morning humidity, and ammonia significantly differed (p < 0.05) between the two houses from day 22-49. Lastly, the prevalence of AMR in cecal E. coli isolates differed from meconium isolates (p < 0.001), while the AMR phenotype of cecal Enterococcus isolates differed between houses (p < 0.05).

Proteomic response of Turicibacter bilis MMM721 to chicken bile and its bile acids.

OBJECTIVE: Bile and its individual components, mainly bile acids, are important for digestion and drive bacterial community dynamics in the upper gastrointestinal tract of chickens. However, specific responses to bile acids have been characterized in only a few commensal bacteria, and it is unclear how other members of the microbiota respond to biliary stress. Here, we used label-free LC-MS/MS to assess the proteomic response of a common inhabitant of the chicken small intestine, Turicibacter bilis MMM721, to 24 h of growth in anaerobic growth media supplemented with 0.1% whole chicken bile, 0.1% taurochenodeoxycholic acid (TCDCA), or 0.1% taurocholic acid (TCA). RESULTS: Seventy, 46, and 10 differentially expressed proteins were identified in Turicibacter bilis MMM721 cultured with supplements of chicken bile, TCDCA, and TCA, respectively, when compared to unsupplemented controls. Many differentially expressed proteins were predicted to be involved in ribosomal processes, post-translational modifications and chaperones, and modifications to the cell surface. Ultimately, the T. bilis MMM721 response to whole bile and bile acids is complex and may relate to adaptations for small intestine colonization, with numerous proteins from a variety of functional categories being impacted.

Succession patterns of the bacterial community in poultry litter after bird removal and sodium bisulfate application.

Sulfate-based acid amendments are used for treating litter between broiler chicken flocks and during grow-out for in-house ammonia abatement. These amendments reduce litter pH and inhibit ammonia volatilization by converting ammonia to nonvolatile ammonium. Research on the effects of acid amendments on litter microbiota is limited and usually done in microcosms, which do not replicate natural environments. In this study, we determined the changes in bacterial populations present in litter during downtime (the period after a flock was removed and before new broiler chicks were placed) and 24 h before and after the application of a sodium bisulfate (NaHSO4 )-based amendment. We used DNA sequencing technologies to characterize the litter microbiota, elucidating microbial shifts in litter samples with respect to downtime, litter depth, and NaHSO4 application. During downtime (∼18 d), the litter microbiota was dominated by Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Sodium bisulfate affected the microbiota in the top layer (3 cm) of reused litter topdressed with fresh pine shavings and resulted in an increase in Escherichia spp. and Faecalibacterium spp. and a decrease in members of the phylum Acidobacteria. Furthermore, culturable Escherichia coli decreased by 1.5 log units during downtime, but an increase was observed for topdressed litter after NaHSO4 was applied. Although the effect of acidifiers on ammonia reduction, bird performance, and litter performance are well documented, their effect on litter bacteria is not well understood. Our results suggest that acidifiers may perturb litter bacteria when topdressed with fresh pine shavings and that further research is required.

The Acute Host-Response of Turkeys Colonized With Campylobacter coli.

Consumption of contaminated poultry products is one of the main sources of human campylobacteriosis, of which Campylobacter jejuni subsp. jejuni (C. jejuni) and C. coli are responsible for ~98% of the cases. In turkeys, the ceca are an important anatomical site where Campylobacter asymptomatically colonizes. We previously demonstrated that commercial turkey poults colonized by C. jejuni showed acute changes in cytokine gene expression profiles, and histological intestinal lesions at 2 days post-inoculation (dpi). Cecal tonsils (CT) are an important part of the gastrointestinal-associated lymphoid tissue that surveil material passing in and out of the ceca, and generate immune responses against intestinal pathogens. The CT immune response toward Campylobacter remains unknown. In this study, we generated a kanamycin-resistant C. coli construct (CcK) to facilitate its enumeration from cecal contents after experimental challenge. In vitro analysis of CcK demonstrated no changes in motility when compared to the parent isolate. Poults were inoculated by oral gavage with CcK (5 × 107 colony forming units) or sterile-media (mock-colonized), and euthanized at 1 and 3 dpi. At both time points, CcK was recovered from cecal contents, but not from the mock-colonized group. As a marker of acute inflammation, serum alpha-1 acid glycoprotein was significantly elevated at 3 dpi in CcK inoculated poults compared to mock-infected samples. Significant histological lesions were detected in cecal and CT tissues of CcK colonized poults at 1 and 3 dpi, respectively. RNAseq analysis identified 250 differentially expressed genes (DEG) in CT from CcK colonized poults at 3 dpi, of which 194 were upregulated and 56 were downregulated. From the DEG, 9 significantly enriched biological pathways were identified, including platelet aggregation, response to oxidative stress and negative regulation of oxidative stress-induced intrinsic apoptotic signaling pathway. These data suggest that C. coli induced an acute inflammatory response in the intestinal tract of poults, and that platelet aggregation and oxidative stress in the CT may affect the turkey's ability to resist Campylobacter colonization. These findings will help to develop and test Campylobacter mitigation strategies to promote food safety in commercial turkeys.

Natural Horizontal Gene Transfer of Antimicrobial Resistance Genes in Campylobacter spp. From Turkeys and Swine.

Antibiotic-resistant Campylobacter constitutes a serious threat to public health. The clonal expansion of resistant strains and/or the horizontal spread of resistance genes to other strains and species can hinder the clinical effectiveness of antibiotics to treat severe campylobacteriosis. Still, gaps exist in our understanding of the risks of acquisition and spread of antibiotic resistance in Campylobacter. While the in vitro transfer of antimicrobial resistance genes between Campylobacter species via natural transformation has been extensively demonstrated, experimental studies have favored the use of naked DNA to obtain transformants. In this study, we used experimental designs closer to real-world conditions to evaluate the possible transfer of antimicrobial resistance genes between Campylobacter strains of the same or different species (Campylobacter coli or Campylobacter jejuni) and originating from different animal hosts (swine or turkeys). This was evaluated in vitro through co-culture experiments and in vivo with dual-strain inoculation of turkeys, followed by whole genome sequencing of parental and newly emerged strains. In vitro, we observed four independent horizontal gene transfer events leading to the acquisition of resistance to beta-lactams (blaOXA), aminoglycosides [aph(2'')-If and rpsL] and tetracycline [tet(O)]. Observed events involved the displacement of resistance-associated genes by a mutated version, or the acquisition of genomic islands harboring a resistance determinant by homologous recombination; we did not detect the transfer of resistance-carrying plasmids even though they were present in some strains. In vivo, we recovered a newly emerged strain with dual-resistance pattern and identified the replacement of an existing non-functional tet(O) by a functional tet(O) in the recipient strain. Whole genome comparisons allowed characterization of the events involved in the horizontal spread of resistance genes between Campylobacter following in vitro co-culture and in vivo dual inoculation. Our study also highlights the potential for antimicrobial resistance transfer across Campylobacter species originating from turkeys and swine, which may have implications for farms hosting both species in close proximity.