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In an attempt to isolate new probiotic bacteria, two Gram-variable, spore-forming, rod-shaped aerobic bacteria designated as strain A4 and A15 were isolated from the feces of Canada geese ( Branta canadensis ). Strain A4 was able to grow in high salt levels and exhibited lipase activity, while A15 did not propagate under these conditions. Both were positive for starch hydrolysis, and they inhibited the growth of Staphylococcus aureus . The strains of the 16S rRNA sequence shared only 94% similarity to previously identified Sporosarcina spp. The ANI (78.08%) and AAI (82.35%) between the two strains were less than the species threshold. Searches for the most similar genomes using the Mash/Minhash algorithm showed the nearest genome to strain A4 and A15 as Sporosarcina sp. P13 (distance of 21%) and S. newyorkensis (distance of 17%), respectively. Sporosarcina spp. strains A4 and A15 contain urease genes, and a fibronectin-binding protein gene indicates that these bacteria may bind to eukaryotic cells in host gastrointestinal tracts. Phenotypic and phylogenetic data, along with low dDDH, ANI, and AAI values for strains A4 and A15, indicate these bacteria are two novel isolates of the Sporosarcina genus: Sporosarcina sp. A4 sp. nov., type strain as Sporosarcina cascadiensis and Sporosarcina sp. A15 sp. nov., type strain Sporosarcina obsidiansis .

Chronic enteropathies pose an important difficulty in the captive management of cheetahs ( Acinonyx jubatus ) because of suspected multifactorial pathogenesis and the complex nature of enteric microbiota dynamics. Enterobacteriaceae, Campylobacter spp., Clostridium perfringens , Helicobacter spp., and Salmonella spp. are enteropathogens of interest because of their zoonotic potential and suspected contribution to enteropathies. This study aimed to determine the presence of these enteropathogens of interest in fecal samples from cheetahs ( N = 48) fed different diets from three different institutions and to investigate the associations between diet, fecal score, and specific enteropathogen presence. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes were used to visualize and quantify putative enteropathogens in each sample concurrent with selective culturing for Salmonella and Clostridium perfringens . From FISH counts, carcass-fed animals had greater numbers of Enterobacteriaceae compared with animals fed low-fat dog food, although this trend was not statistically significant ( P = 0.088). Furthermore, no significant associations were found between fecal score and bacterial load. Abundance of Campylobacter spp., Clostridium perfringens , or Helicobacter spp. as measured by FISH were not correlated with diet or fecal score. On the basis of these data, in agreement with published literature, it is concluded that these microbes may be commensals in the cheetah gastrointestinal tract and do not appear to be a primary cause of abnormal fecal scores.

In chickens, early life exposure to environmental microbes has long-lasting impacts on gastrointestinal (GI) microbiome development and host health and growth, via mechanisms that remain uncharacterized. In this study, we demonstrated that administrating a fecal microbiome transplant (FMT) from adults to day-of-hatch chicks results in significantly higher body mass of birds and decreased residual feed intake (RFI), implying enhanced feed efficiency, at 6 weeks of age. To assess the potential mechanisms through which FMT affects adult bird phenotype, we combined 16 S rRNA gene amplification, metagenomic, and comparative genomic approaches to survey the composition and predicted activities of the resident microbiome of various GI tract segments. Early life FMT exposure had a long-lasting significant effect on the microbial community composition and function of the ceca but not on other GI segments. Within the ceca of 6-week-old FMT birds, hydrogenotrophic microbial lineages and genes were most differentially enriched. The results suggest that thermodynamic regulation in the cecum, in this case via hydrogenotrophic methanogenic and sulfur-cycling lineages, potentially serving as hydrogen sinks, may enhance fermentative efficiency and dietary energy harvest capacity. Our study provides a specific mechanism of action through which early-life microbiome transplants modulate market-relevant phenotypes in poultry and, thereby, may represent a significant advance toward microbiome-focused sustainable agriculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ramírez, Keshri, Vahrson, Garber, Berrang, Cox, González-Cerón, Aggrey and Oakley.)

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics-in addition to the traditional considerations when conducting an animal science study-makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

New World vultures, such as turkey vultures ( Cathartes aura ), are obligate scavengers with large geographic ranges. In a preliminary characterization of the turkey vulture (TV) gastrointestinal microbiome in Southern California, we identified 2 recently described emerging bacterial pathogens not previously known to be associated with this avian species. High-throughput sequencing of broad-range 16S rRNA gene amplicons revealed sequences from TV cloacal swabs that were related closest to Wohlfahrtiimonas chitiniclastica and Ignatzschineria species, both Gammaproteobacteria considered by the United States Centers for Disease Control and Prevention as emerging zoonotic pathogens. None of these bacterial sequence types have been previously identified from samples obtained from the turkey vulture gastrointestinal microbiome. With the use of bioinformatics workflows previously established by our research group, we designed specific and sensitive polymerase chain reaction primer sets that represent novel diagnostic assays for the genera Wohlfahrtiimonas and Ignatzschineria . These primer sets were validated by Sanger sequence confirmation from complex TV samples. Because the genera Wohlfahrtiimonas and Ignatzschineria are both known to have dipteran hosts, the molecular diagnostic tools we present here should be useful for better understanding the role of flies, vultures, and other scavengers in the ecology and epidemiology of the genera Wohlfahrtiimonas and Ignatzschineria from a One Health perspective.

Background: Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP ., Results: We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample., Conclusions: Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine., (© 2021. The Author(s).)

Background: Urine from clinically healthy dogs is not sterile. Characterizing microbial diversity and abundance within this population of dogs is important to define normal reference ranges for healthy urine., Objectives: To establish composition and relative representation of bacterial and fungal microbiomes in urine of clinically healthy dogs., Animals: Fifty clinically healthy dogs., Methods: Analytic study. Urine sampling via cystocentesis. Comprehensive evaluation of urine including standard urinalysis, culture and sensitivity, next-generation sequencing (NGS), and bioinformatics to define bacterial and fungal microbiome., Results: Culture did not yield positive results in any samples. Next-generation sequencing of urine established low presence of bacteria, fungi, or both in all samples. Diversity and abundance of bacterial and fungal communities varied between urine samples from different dogs. Struvite crystals were associated with bacterial community structure (P = .07) and there was a positive correlation between struvite crystals and pH., Conclusions and Clinical Importance: The microbiome in urine of clinically healthy dogs has diverse bacterial and fungal species These findings highlight limitations of conventional culture testing and the need for culture-independent molecular diagnostics to detect microorganisms in urine., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first determine if serial transfers exploiting the ceca in vivo, rather than the external environment or artificial incubations, can produce a stable microbial community. Subsequently, we compared microbiome development between chicks receiving this passaged, i.e. host-selected, cecal material orally, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa identified across treatment types may inform future studies aimed at identifying strains associated with beneficial phenotypes., Competing Interests: The authors have declared that no competing interests exist.

Here, we announce the draft genome sequences of two Clostridium strains, C8-1-8 and C2-6-12, isolated from the cecal contents of commercial broiler chickens (in Athens, GA). These strains may represent potentially novel species within the genus Clostridium , and these draft genomes allow further investigation into potential probiotics for poultry., (Copyright © 2020 Keshri et al.)

Here, we present the draft genome sequences of two Bacillus strains, HF117_J1_D and USDA818B3_A, isolated in Pomona, California, from the gastrointestinal (GI) tract of backyard and commercial broiler chickens, respectively. The draft genomes of both strains appear to represent novel species., (Copyright © 2020 Keshri et al.)

Here, we present the draft genome sequences of two Paenibacillus strains, An7 and USDA918EY, isolated from goose feces (Bend, OR, USA) and chicken ceca (Pomona, CA, USA), respectively. These data may assist with analyses of microorganisms associated with free-ranging and commercial avian species., (Copyright © 2020 Keshri et al.)

Bacillus cereus , a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis , a deadly pathogen for humans, and Bacillus thuringenesis , an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli . Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus , B. anthracis , and other Bacillus species.

Next-generation DNA sequencing is rapidly becoming a powerful tool for food animal management. One valuable use of this technology is to re-examine long-standing observations of performance differences associated with animal husbandry practices to better understand how these differences may be modulated by the gastrointestinal (GI) microbiome. The influences of environmental parameters such as air temperature and relative humidity on broiler chicken performance have commonly been observed, but how the GI microbiome may respond to seasonal environmental changes remains largely unknown. The purposes of this study were therefore to: (1) characterize the cecal microflora of commercial broilers (N = 87) collected at harvest across all 4 seasons, and (2) identify any significant changes of the GI microbiome and specific taxa according to season and Campylobacter status. Finding taxa with significant positive or negative correlations with Campylobacter could be useful by identifying indicator or antagonistic taxa and could also inform inferences regarding the ecological niche of Campylobacter. Whole GI tracts were removed from commercial broilers representing 87 independent flocks between April 2013 and May 2014 in the U.S. state of Georgia. Intact ceca were separated, cultured for Campylobacter and cecal contents were frozen. The cecal microbiome was characterized using barcoded sequencing of 16S rRNA genes on the Illumina MiSeq platform. The composition of the microbiome measured at processing was generally not affected by Campylobacter status but was most significantly affected by season of grow-out. Significantly fewer bacterial genera were found in winter than spring or summer. Bacterial genera with prior evidence for both positive or negative influences on gut health outcomes were significantly less abundant in the fall. Identifying specific members of the GI microbiota that vary according to season may help develop novel interventions to improve husbandry practices and growth performance.

Flea-borne diseases (FBDs) impact both human and animal health worldwide. Because adult fleas are obligately hematophagous and can harbor potential pathogens, fleas act as ectoparasites of vertebrates, as well as zoonotic disease vectors. Cat fleas (Ctenocephalides felis) are important vectors of two zoonotic bacterial genera listed as priority pathogens by the National Institute of Allergy and Infectious Diseases (NIAID-USA): Bartonella spp. and Rickettsia spp., causative agents of bartonelloses and rickettsioses, respectively. In this study, we introduce the first microbiome analysis of C. felis samples from California, determining the presence and abundance of relevant pathogenic genera by characterizing the cat flea microbiome through 16S rRNA next-generation sequencing (16S-NGS). Samples from both northern (NoCal) and southern (SoCal) California were assessed to expand current knowledge regarding FBDs in the state. We identified Rickettsia and Bartonella, as well as the endosymbiont Wolbachia, as the most abundant genera, followed by less abundant taxa. In comparison to our previous study screening Californian cat fleas for rickettsiae using PCR/digestion/sequencing of the ompB gene, the 16S-NGS approach applied herein showed a 95% level of agreement in detecting Rickettsia spp. There was no overall difference in microbiome diversity between NoCal and SoCal samples. Bacterial taxa identified by 16S-NGS in this study may help to improve epidemiological investigations, pathogen surveillance efforts, and clinical diagnostics of FBDs in California and elsewhere.

Clostridium perfringens, a spore-forming anaerobic bacterium, causes food poisoning and gas gangrene in humans and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from C. perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50 and 100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 50°C for 30 min and still retained >60% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential., (Published by Oxford University Press on behalf of FEMS 2018.)

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2 nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

The Mycobacterium genus comprises over one-hundred-and-fifty recognised species, the majority of which reside in the environment and many of which can be pathogenic to mammals. Some species of environmental mycobacteria may interfere with BCG vaccination efficacy and in tuberculin test interpretation. Examining biogeographic trends in the distribution of members of the mycobacteria across a number of physicochemical and spatial gradients in soil and water environments across Ethiopia using oligotyping identified differential distributions of pathogenic and significant species. The tuberculosis complex was identified in more than 90% of water samples and taxonomic groups implicated in lower BCG vaccine efficiency were core in both soil and water Mycobacterium communities. A reservoir of Mycobacterium bovis was identified in water, with up to 7.3×102 genome equivalents per ml. Elevation, temperature, habitat and vegetation type were important predictors of both soil and water Mycobacterium communities. These results represent the first step in understanding the potential risk of exposure to environmental mycobacteria that may undermine efforts to reduce disease incidence.

The objective of this study was to compare subtypes of Campylobacter jejuni and Campylobacter coli detected on three selective Campylobacter plating media to determine whether each medium selected for different subtypes. Fifty ceca and 50 carcasses (representing 50 flocks) were collected from the evisceration line in a commercial broiler processing plant. Campylobacter was cultured and isolated from cecal contents and carcass rinses on Campy-Cefex, Campy Line, and RF Campylobacter jejuni/coli agars. When a positive result was obtained with all three media, one colony of the most prevalent morphology on each medium was selected for further analysis by full genome sequencing and multilocus sequence typing. Sequence types were assigned according to PubMLST. A total of 49 samples were positive for Campylobacter on all three media. Forty samples contained only C. jejuni , three had only C. coli , and both species were detected in six samples. From 71% of samples, Campylobacter isolates of the same sequence type were recovered on all three media. From 81.6% of samples, isolates were all from the same clonal complex. From significantly fewer samples (26%, P < 0.01), one medium recovered an isolate with a sequence type different from the type recovered on the other two media. When multiple sequence types were detected, six times the medium with the odd sequence type was Campy-Cefex, four times it was Campy-Line, and six times it was RF Campylobacter jejuni/coli . From one sample, three sequence types were detected. In most cases, all three plating media allowed detection of the same type of Campylobacter from complex naturally contaminated chicken samples.

To better understand the ecology of the poultry gastrointestinal (GI) microbiome and its interactions with the host, we compared GI bacterial communities by sample type (fecal or cecal), time (1, 3, and 6 weeks posthatch), and experimental pen (1, 2, 3, or 4), and measured cecal mRNA transcription of the cytokines IL18, IL1β, and IL6, IL10, and TGF-β4. The microbiome was characterized by sequencing of 16S rRNA gene amplicons, and cytokine gene expression was measured by a panel of quantitative-PCR assays targeting mRNAs. Significant differences were observed in the microbiome by GI location (fecal versus cecal) and bird age as determined by permutational MANOVA and UniFrac phylogenetic hypothesis tests. At 1-week posthatch, bacterial genera significantly over-represented in fecal versus cecal samples included Gallibacterium and Lactobacillus, while the genus Bacteroides was significantly more abundant in the cecum. By 6-week posthatch, Clostridium and Caloramator (also a Clostridiales) sequence types had increased significantly in the cecum and Lactobacillus remained over-represented in fecal samples. In the ceca, the relative abundance of sequences classified as Clostridium increased by ca. 10-fold each sampling period from 0.1% at 1 week to 1% at 3 week and 18% at 6 week. Increasing community complexity through time were observed in increased taxonomic richness and diversity. IL18 and IL1β significantly (p < 0.05, pairwise t-tests) increased to maximum mean expression levels 1.5 fold greater at week 3 than 1, while IL6 significantly decreased to 0.8- and 0.5-fold expression at 3- and 6-week posthatch, respectively relative to week 1. Transcription of pro-inflammatory cytokines was generally negatively correlated with the relative abundance of various members of the phylum Firmicutes and positively correlated with Proteobacteria. Correlations of the microbiome with specific cytokine mRNA transcription highlight the importance of the GI microbiome for bird health and productivity and may be a successful high-throughput strategy to identify bacterial taxa with specific immune-modulatory properties.

Campylobacter lari is frequently isolated from shore birds and can cause illness in humans. Here, we report the draft whole-genome sequence of a urease-positive strain of C. lari that was isolated in estuarial water on the coast of Delaware, USA., (Copyright © 2015 Meinersmann et al.)

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds ("sentinels") placed on farms revealed colonization by members of the Picornaviridae, Picobirnaviridae, Reoviridae, and Astroviridae that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus families and genera. Members of the avian enteric Reoviridae and Astroviridae have been well-characterized and have historically been implicated in poultry enteric disease; members of the Picobirnaviridae and Picornaviridae have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.

Background: Poultry remains a major source of foodborne bacterial infections. A variety of additives with presumed anti-microbial and/or growth-promoting effects are commonly added to poultry feed during commercial grow-out, yet the effects of these additives on the gastrointestinal microbial community (the GI microbiome) as the bird matures remain largely unknown. Here we compared temporal changes in the cecal microbiome to the effects of formic acid, propionic acid, and medium-chain fatty acids (MCFA) added to feed and/or drinking water., Results: Cecal bacterial communities at day of hatch (n = 5 birds), 7d (n = 32), 21d (n = 27), and 42d (n = 36) post-hatch were surveyed using direct 454 sequencing of 16S rRNA gene amplicons from each bird in combination with cultivation-based recovery of a Salmonella Typhimurium marker strain and quantitative-PCR targeting Clostridium perfringens. Treatment effects on specific pathogens were generally non-significant. S. Typhimurium introduced by oral gavage at day of hatch was recovered by cultivation from nearly all birds sampled across treatments at 7d and 21d, but by 42d, S. Typhimurium was only recovered from ca. 25% of birds, regardless of treatment. Sequencing data also revealed non-significant treatment effects on genera containing known pathogens and on the cecal microbiome as a whole. In contrast, temporal changes in the cecal microbiome were dramatic, highly significant, and consistent across treatments. At 7d, the cecal community was dominated by three genera (Flavonifractor, Pseudoflavonifractor, and a Lachnospiracea sequence type) that accounted for more than half of sequences. By 21d post-hatch, a single genus (Faecalibacterium) accounted for 23-55% of sequences, and the number of Clostridium 16S rRNA gene copies detected by quantitative-PCR reached a maximum., Conclusions: Over the 42 d experiment, the cecal bacterial community changed significantly as measured by a variety of ecological metrics and increases in the complexity of co-occurrence networks. Management of poultry to improve animal health, nutrition, or food safety may need to consider the interactive effects of any treatments with the dramatic temporal shifts in the taxonomic composition of the cecal microbiome as described here.

The domestic chicken is a common model organism for human biological research and of course also forms the basis of a global protein industry. Recent methodological advances have spurred the recognition of microbiomes as complex communities with important influences on the health and disease status of the host. In this minireview, we provide an overview of the current state of knowledge of the chicken gastrointestinal microbiome focusing on spatial and temporal variability, the presence and importance of human pathogens, the influence of the microbiota on the immune system, and the importance of the microbiome for poultry nutrition. Review and meta-analysis of public data showed cecal communities dominated by Firmicutes and Bacteroides at the phylum level, while at finer levels of taxonomic resolution, a phylogenetically diverse assemblage of microorganisms appears to have similar metabolic functions that provide important benefits to the host as inferred from metagenomic data. This observation of functional redundancy may have important implications for management of the microbiome. We foresee advances in strategies to improve gut health in commercial operations through management of the intestinal microbiota as an alternative to in-feed subtherapeutic antibiotics, improvements in pre- and probiotics, improved management of polymicrobial poultry diseases, and better control of human pathogens via colonization reduction or competitive exclusion strategies., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Broilers may carry Salmonella and Campylobacter on inner and outer surfaces upon arrival at the slaughter plant, and carcasses can be further contaminated during commercial processing. A sensitive, nondestructive, repeatable sampling method would be useful to test carcasses for levels of bacteria before and after specific processing steps to measure either contamination or efficacy of intervention techniques. Blending of excised skin is accepted as an effective sampling method but requires damage to the carcass; this makes repeated measurements on the same carcass difficult. Herein we compare sponge sampling to skin excision to recover inoculated Salmonella and Campylobacter from broiler carcasses. In each of three replications, broiler carcass breast skin was inoculated with approximately 6.0 log antimicrobial-resistant Salmonella and Campylobacter, allowed to dry for 60 s, and sampled by either sponge, skin excision, or sponge followed by skin excision. Antimicrobial-resistant Salmonella and Campylobacter were enumerated from all samples. Skin excision allowed recovery of 0.1 to 0.2 log more inoculated bacteria than did sponge sampling. When excision was used on the same skin previously sampled by sponging, the combination of both methods did not significantly improve recovery compared with sponging alone. Skin excision is slightly more sensitive than sponge sampling; however, for repeated nondestructive sampling of broiler carcasses during processing, sponge sampling may be preferable to recover Salmonella and Campylobacter within 60 s of a contamination event.

A better understand the ecology of microbes and their role in the global ecosystem could be achieved if traditional ecological theories can be applied to microbes. In ecology organisms are defined as specialists or generalists according to the breadth of their niche. Spatial distribution is often used as a proxy measure of niche breadth; generalists have broad niches and a wide spatial distribution and specialists a narrow niche and spatial distribution. Previous studies suggest that microbial distribution patterns are contrary to this idea; a microbial generalist genus (Desulfobulbus) has a limited spatial distribution while a specialist genus (Methanosaeta) has a cosmopolitan distribution. Therefore, we hypothesise that this counter-intuitive distribution within generalist and specialist microbial genera is a common microbial characteristic. Using molecular fingerprinting the distribution of four microbial genera, two generalists, Desulfobulbus and the methanogenic archaea Methanosarcina, and two specialists, Methanosaeta and the sulfate-reducing bacteria Desulfobacter were analysed in sediment samples from along a UK estuary. Detected genotypes of both generalist genera showed a distinct spatial distribution, significantly correlated with geographic distance between sites. Genotypes of both specialist genera showed no significant differential spatial distribution. These data support the hypothesis that the spatial distribution of specialist and generalist microbes does not match that seen with specialist and generalist large organisms. It may be that generalist microbes, while having a wider potential niche, are constrained, possibly by intrageneric competition, to exploit only a small part of that potential niche while specialists, with far fewer constraints to their niche, are more capable of filling their potential niche more effectively, perhaps by avoiding intrageneric competition. We suggest that these counter-intuitive distribution patterns may be a common feature of microbes in general and represent a distinct microbial principle in ecology, which is a real challenge if we are to develop a truly inclusive ecology.

Food animal production systems have become more consolidated and integrated, producing large, concentrated animal populations and significant amounts of fecal waste. Increasing use of manure and litter as a more "natural" and affordable source of fertilizer may be contributing to contamination of fruits and vegetables with foodborne pathogens. In addition, human and animal manure have been identified as a significant source of antibiotic resistance genes thereby serving as a disseminator of resistance to soil and waterways. Therefore, identifying methods to remediate human and animal waste is critical in developing strategies to improve food safety and minimize the dissemination of antibiotic resistant bacteria. In this study, we sought to determine whether withdrawing antibiotic growth promoters or using alternatives to antibiotics would reduce the abundance of antibiotic resistance genes or prevalence of pathogens in poultry litter. Terminal restriction fragment length polymorphism (T-RFLP) paired with high throughput sequencing was used to evaluate the bacterial community composition of litter from broiler chickens that were treated with streptogramin growth-promoting antibiotics, probiotics, or prebiotics. The prevalence of resistance genes and pathogens was determined from sequencing results or PCR screens of litter community DNA. Streptogramin antibiotic usage did not elicit statistically significant differences in Shannon diversity indices or correlation coefficients among the flocks. However, T-RFLP revealed that there were inter-farm differences in the litter composition that was independent of antibiotic usage. The litter from all farms, regardless of antibiotic usage, contained streptogramin resistance genes (vatA, vatB, and vatE), macrolide-lincosamide-streptogramin B resistance genes (ermA and ermB), the tetracycline resistance gene tetM and class 1 integrons. There was inter-farm variability in the distribution of vatA and vatE with no statistically significant differences with regards to usage. Bacterial diversity was higher in litter when probiotics or prebiotics were administered to flocks but as the litter aged, diversity decreased. No statistically significant differences were detected in the abundance of class 1 integrons where 3%-5% of the community was estimated to harbor a copy. Abundance of pathogenic Clostridium species increased in aging litter despite the treatment while the abundance of tetracycline-resistant coliforms was unaffected by treatment. However some treatments decreased the prevalence of Salmonella. These findings suggest that withdrawing antibiotics or administering alternatives to antibiotics can change the litter bacterial community and reduce the prevalence of some pathogenic bacteria, but may not immediately impact the prevalence of antibiotic resistance.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

Background: Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine., Methods: We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR., Results: PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×10(7) to 2.7×10(8) gene targets g(-1); slow growers prevalence from 2.9×10(5) to 1.2×10(7) cells g(-1)., Conclusions: This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.

Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni., (Published by Elsevier GmbH.)

The whole carcass rinse (WCR) procedure is routinely used as a sampling method for determining the presence and number of quality-indicator organisms or pathogens associated with broiler chicken carcasses in processing facilities. Collection of a cumulative drip sample by placing collection vessels under the processing line could potentially capture a more representative sample of bacterial populations associated with an entire flock with less labor than individual bird rinses. The purpose of this study was to evaluate a cumulative drip sampling method for recovery of Campylobacter spp. and 3 types of quality indicator organisms from broiler carcasses. Cumulative drip and WCR samples were collected on 14 d from a commercial broiler processing facility over a 3-mo period. No statistically significant difference was demonstrated between the WCR and cumulative drip sampling methods in recovery of Campylobacter spp., total aerobes, Enterobacteriaceae, or Escherichia coli associated with the postevisceration samples (P > 0.01). Analysis of the pyrosequencing census data demonstrated high interbird variability and indicates cumulative sampling may be required to obtain fully representative sampling of a flock. For most bacterial taxa, the relative abundance in individual WCR was correlated with cumulative drip samples, but some taxa were undercounted or missed entirely by individual WCR. Consequently, individual carcass rinses may not be representative of the flock microbial community. The cumulative drip sampling technique may save labor and provide a more representative summary of process control in poultry processing facilities.

Microbial communities associated with agricultural animals are important for animal health, food safety, and public health. Here we combine high-throughput sequencing (HTS), quantitative-PCR assays, and network analysis to profile the poultry-associated microbiome and important pathogens at various stages of commercial poultry production from the farm to the consumer. Analysis of longitudinal data following two flocks from the farm through processing showed a core microbiome containing multiple sequence types most closely related to genera known to be pathogenic for animals and/or humans, including Campylobacter, Clostridium, and Shigella. After the final stage of commercial poultry processing, taxonomic richness was ca. 2-4 times lower than the richness of fecal samples from the same flocks and Campylobacter abundance was significantly reduced. Interestingly, however, carcasses sampled at 48 hr after processing harboured the greatest proportion of unique taxa (those not encountered in other samples), significantly more than expected by chance. Among these were anaerobes such as Prevotella, Veillonella, Leptrotrichia, and multiple Campylobacter sequence types. Retail products were dominated by Pseudomonas, but also contained 27 other genera, most of which were potentially metabolically active and encountered in on-farm samples. Network analysis was focused on the foodborne pathogen Campylobacter and revealed a majority of sequence types with no significant interactions with other taxa, perhaps explaining the limited efficacy of previous attempts at competitive exclusion of Campylobacter. These data represent the first use of HTS to characterize the poultry microbiome across a series of farm-to-fork samples and demonstrate the utility of HTS in monitoring the food supply chain and identifying sources of potential zoonoses and interactions among taxa in complex communities.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

We examined the distribution of uncultured Planctomycetes phylotypes along depth profiles spanning the redox gradient of the Black Sea suboxic zone to gain insight into their respective ecological niches. Planctomycetes phylogeny correlated with depth and chemical profiles, implying similar metabolisms within phylogenetic groups. A suboxic zone sample was split into > 30 and < 30 μm fractions to examine putative aggregate-attached and free-living Planctomycetes. All identified Planctomycetes were present in the > 30 μm fraction except for members of the Scalindua genus, which were apparently free-living. Sequences from Candidatus Scalindua, known to carry out the anammox process, formed two distinct clusters with nonoverlapping depth ranges. One cluster, only 97.1% similar to the named species, was present at high nitrite/nitrate and low ammonium concentrations in the upper suboxic zone. We propose this sequence type be named 'Candidatus Scalindua richardsii'. A second cluster, containing sequences more similar to 'Candidatus Scalindua sorokinii', was present at high ammonium and low nitrite conditions in the lower suboxic zone. Sequences obtained from the sulfidic zone (1000 m depth) yielded Planctomycetes from two uncharacterized Planctomycetacia clusters and three potentially new genera as well as sequences from the uncultured OP3 phylum., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A reductionist ecological approach of using a model genus was adopted in order to understand how microbial community structure is driven by metabolic properties. The distribution along an estuarine gradient of the highly specialised genus Methanosaeta was investigated and compared to the previously determined distribution of the more metabolically flexible Desulfobulbus. Methanosaeta genotypic distribution along the Colne estuary (Essex, UK) was determined by DNA- and RNA-based denaturing gradient gel electrophoresis and 16S rRNA gene sequence analyses. Methanosaeta distribution was monotonic, with a consistently diverse community and no apparent niche partitioning either in DNA or RNA analyses. This distribution pattern contrasts markedly with the previously described niche partitioning and sympatric differentiation of the model generalist, Desulfobulbus. To explain this difference, it is hypothesised that Methanosaeta's strict metabolic needs limit its adaptation potential, thus populations do not partition into spatially distinct groups and so do not appear to be constrained by gross environmental factors such as salinity. Thus, at least for these two model genera, it appears that metabolic flexibility may be an important factor in spatial distribution and this may be applicable to other microbes.

Understanding the ecological principles underlying the structure and function of microbial communities remains an important goal for microbial ecology. We examined two biogeochemically important taxa, the sulfate-reducing bacterial genus, Desulfobulbus, and the methanogenic archaeal genus, Methanosaeta, to compare and contrast niche partitioning by these two taxa that are ecologically linked as anaerobic terminal oxidizers of organic material. An observational approach utilizing functional gene pyrosequencing was combined with a community-based reciprocal incubation experiment and characterization of a novel Desulfobulbus isolate. To analyze the pyrosequencing data, we constructed a data analysis pipeline, which we validated with several control data sets. For both taxa, particular genotypes were clearly associated with certain portions of an estuarine gradient, consistent with habitat or niche partitioning. Methanosaeta genotypes were generally divided between those found almost exclusively in the marine habitat (∼30% of operational taxonomic units (OTUs)), and those which were ubiquitously distributed across all or most of the estuary (∼70% of OTUs). In contrast to this relatively monotonic distribution, for Desulfobulbus, there were many more genotypes, and their distributions represented a wide range of inferred niche widths from specialist genotypes found only at a single site, to ubiquitous or generalist genotypes found in all 10 sites examined along the full estuarine gradient. Incubation experiments clearly showed that, for both taxa, communities from opposite ends of the estuary did not come to resemble one another, regardless of the chemical environment. Growth of a Desulfobulbus isolated into pure culture indicated that the potential niche of this organism is significantly larger than the realized niche. We concluded that niche partitioning can be an important force structuring microbial populations, with biotic and abiotic components having very different effects depending on the physiology and ecology of each taxon.

Bacteriophage ΦCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The ΦCP24R genome described herein is only the second Clostridium perfringens podovirus genome reported to date.

Listeria monocytogenes is a Gram-positive, non-spore forming, catalase-positive rod that is a major bacterial food-borne disease agent associated with uncooked meats, including poultry, uncooked vegetables, soft cheeses, and unpasteurized milk. The bacterium may be carried by animals without signs of disease, can replicate at refrigeration temperatures, and is frequently associated with biofilms. There is a need to discover innovative pathogen intervention technologies for this bacterium. Consequently, bioinformatic analyses were used to identify genes encoding lytic protein sequences in the genomes of L. monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of a putative N-acetylmuramoyl-L-alanine amidase gene from L. monocytogenes strain 4b. The resultant amplification product was cloned into an expression vector, propagated in Escherichia coli Rosetta strains, and the recombinant protein was purified to homogeneity. Gene and protein sequencing confirmed that the predicted and chemically determined amino acid sequence of the recombinant protein designated PlyLM was a putative N-acetylmuramoyl-L-alanine amidase. The recombinant lytic protein was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot and MIC/MIB assays, but was not active against other bacteria beyond the genus. A microtiter plate assay was utilized to assay for the ability of the recombinant lysin protein to potentially aid with digestion of a L. monocytogenes biofilm. Protease or lysozyme digestion alone did not significantly reduce the L. monocytogenes biofilm. Although the recombinant protein alone reduced the biofilm by only 20%, complete digestion of the bacterial monolayer was accomplished in conjunction with a protease.

Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (< 0.04%) of sequences present in the feces, but 88-97% of sequences from each media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes., (Published 2011. This article is a U.S. Government work and is in the public domain in the USA.)

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications., (Published by Elsevier Inc.)

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

An instrument (TEMPO) has been developed to automate the most-probable-number (MPN) technique and reduce the effort required to estimate some bacterial populations. We compared the automated MPN technique with traditional microbiological plating methods and Petrifilm methods for estimating the total viable count of aerobic microorganisms (TVC), total coliforms (CC), and Escherichia coli populations (EC) on freshly processed broiler chicken carcasses (postchill whole carcass rinse [WCR] samples) and cumulative drip-line samples from a commercial broiler processing facility. Overall, 120 broiler carcasses, 36 prechill drip-line samples, and 40 postchill drip-line samples were collected over 5 days (representing five individual flocks) and analyzed by the automated MPN and direct agar plating and Petrifilm methods. The TVC correlation coefficient between the automated MPN and traditional methods was very high (0.972) for the prechill drip samples, which had mean log-transformed values of 3.09 and 3.02, respectively. The TVC correlation coefficient was lower (0.710) for the postchill WCR samples, which had lower mean log values of 1.53 and 1.31, respectively. Correlations between the methods for the prechill CC and EC samples were 0.812 and 0.880, respectively. The estimated number of total aerobes was generally greater than the total number of coliforms or E. coli recovered for all sample types (P < 2e⁻¹⁶). Significantly more bacteria were recovered from the prechill samples than from the postchill WCR or cumulative drip samples (P < 9.5e⁻¹² and P < 2e⁻¹⁶, respectively). When samples below the limit of detection were excluded, 92.1% of the total responses were within a single log difference between the traditional plating or Petrifilm methods and the automated MPN method.

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates. Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units, a richness that precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem, we have developed and validated a free software tool that automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and nontarget sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimized candidate assays derived from primer design software packages. ThermoPhyl derives its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan(®) qPCR) application and potentially for oligonucleotide probes. Here, we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.

Background: Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen., Results: Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical., Conclusions: Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents and ΦCPV1, a virulent bacteriophage, was classified in the family Podoviridae. The purified virus had an icosahedral head and collar of approximately 42nm and 23nm in diameter, respectively, with a structurally complex tail of 37nm lengthwise and a basal plate of 30nm. The ΦCPV1 double-stranded DNA genome was 16,747 base pairs with a GC composition of 30.5%. Twenty-two open reading frames (ORFs) coding for putative peptides containing 30 or more amino acid residues were identified and analyzed in the genome. Amino acid sequences of the predicted proteins from the ΦCPV1 genome ORFs were compared with those from the NCBI database and potential functions of 12 proteins were predicted by sequence homology. Three putative proteins were similar to hypothetical proteins with unknown functions, whereas seven proteins did not have similarity with any known bacteriophage or bacterial proteins. Identified ORFs formed at least four genomic clusters that accounted for predicted proteins involved with replication of the viral DNA, its folding, production of structural components and lytic properties. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl-l-alanine amidases and a second structural lysin was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens., (Published by Elsevier B.V.)

Using multiple lines of evidence from denaturing gradient gel electrophoresis, environmental sequences and TaqMan quantitative PCR assays targeting a functional gene for sulfate respiration (dsr) affiliated with the geochemically important genus Desulfobulbus, we revealed strongly restricted distributions of specific genotypes and populations correlated with sampling position along an estuarine gradient free of dispersal barriers. Evidence of evolutionary divergence of populations was provided by three complementary analyses. First, analysis of molecular variance rejected the null hypothesis that genetic diversity within each sampling site was not significantly different than that of all sites pooled together (P<0.0001). Second, UniFrac and Parsimony tests showed phylogenetic clustering of sampling sites was highly significant (P<0.001). Third, pairwise F(ST) statistics showed significant evolutionary divergence of populations based on the location in the estuary. To test the hypothesis that environmental niche-driven evolutionary divergence can create and maintain microbial biogeography, we used both statistical inference and an experimental manipulation to assess the independent effects of environment and geography. Significant effects of each on genotype distributions and population divergence supported the hypothesis. Our data are consistent with both sympatric and parapatric models of speciation, and suggest niche partitioning can contribute to evolutionary divergence and observable biogeographic patterns in microbial communities even among closely related taxa at limited spatial scales without significant barriers to dispersal.

Methanosaeta spp. are globally important biogenic methane producers with only three strains described due to isolation difficulties. Here, clonal axenic isolates from estuarine sediments were obtained using alternative gelling and reducing agents. It is suggested that more systematic approaches with various combinations of media components will help to cultivate difficult-to-isolate anaerobes., (Copyright 2009 Elsevier B.V. All rights reserved.)