Evolution of higher torque in Campylobacter-type bacterial flagellar motors.

Understanding the evolution of molecular machines underpins our understanding of the development of life on earth. A well-studied case are bacterial flagellar motors that spin helical propellers for bacterial motility. Diverse motors produce different torques, but how this diversity evolved remains unknown. To gain insights into evolution of the high-torque ε-proteobacterial motor exemplified by the Campylobacter jejuni motor, we inferred ancestral states by combining phylogenetics, electron cryotomography, and motility assays to characterize motors from Wolinella succinogenes, Arcobacter butzleri and Bdellovibrio bacteriovorus. Observation of ~12 stator complexes in many proteobacteria, yet ~17 in ε-proteobacteria suggest a "quantum leap" evolutionary event. Campylobacter-type motors have high stator occupancy in wider rings of additional stator complexes that are scaffolded by large proteinaceous periplasmic rings. We propose a model for motor evolution wherein independent inner- and outer-membrane structures fused to form a scaffold for additional stator complexes. Significantly, inner- and outer-membrane associated structures have evolved independently multiple times, suggesting that evolution of such structures is facile and poised the ε-proteobacteria to fuse them to form the high-torque Campylobacter-type motor.

Distinct Campylobacter fetus lineages adapted as livestock pathogens and human pathobionts in the intestinal microbiota.

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have "jumped" into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.

The vaginal microbiome of pregnant women is less rich and diverse, with lower prevalence of Mollicutes, compared to non-pregnant women.

The vaginal microbiome plays an important role in maternal and neonatal health. Imbalances in this microbiota (dysbiosis) during pregnancy are associated with negative reproductive outcomes, such as pregnancy loss and preterm birth, but the underlying mechanisms remain poorly understood. Consequently a comprehensive understanding of the baseline microbiome in healthy pregnancy is needed. We characterized the vaginal microbiomes of healthy pregnant women at 11-16 weeks of gestational age (n = 182) and compared them to those of non-pregnant women (n = 310). Profiles were created by pyrosequencing of the cpn60 universal target region. Microbiome profiles of pregnant women clustered into six Community State Types: I, II, III, IVC, IVD and V. Overall microbiome profiles could not be distinguished based on pregnancy status. However, the vaginal microbiomes of women with healthy ongoing pregnancies had lower richness and diversity, lower prevalence of Mycoplasma and Ureaplasma and higher bacterial load when compared to non-pregnant women. Lactobacillus abundance was also greater in the microbiomes of pregnant women with Lactobacillus-dominated CSTs in comparison with non-pregnant women. This study provides further information regarding characteristics of the vaginal microbiome of low-risk pregnant women, providing a baseline for forthcoming studies investigating the diagnostic potential of the microbiome for prediction of adverse pregnancy outcomes.

A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.

BACKGROUND: Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary. RESULTS: We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels. CONCLUSION: This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

Effect of sample pooling and transport conditions on the clinical sensitivity of a real-time polymerase chain reaction assay for Campylobacter fetus subsp. venerealis in preputial samples from bulls.

The diagnosis of bovine genital campylobacteriosis (BGC) presents significant challenges, as traditional methods lack sensitivity when prolonged transport of samples is required. Assays of preputial samples by means of real-time polymerase chain reaction (PCR) provide good sensitivity and high throughput capabilities. However, there is limited information on the acceptable duration of transport and temperature during transport of samples. In addition, the use of pooled samples has proven to be a valuable strategy for the diagnosis of other venereal diseases in cattle. The objectives of the present study were to determine the effect of sample pooling and of transport time and temperature on the clinical sensitivity of a real-time quantitative PCR (qPCR) assay for Campylobacter fetus subsp. venerealis in preputial samples from beef bulls. Eight infected bulls and 176 virgin yearling bulls were used as the source of samples. The qPCR sensitivity was comparable for unpooled samples and pools of 5 samples, whereas sensitivity was decreased for pools of 10 samples. Sensitivity for the various pool sizes improved with repeated sampling. For shorter-term transport (2 and 48 h), sensitivity was greatest when the samples were stored at 4°C and 30°C, whereas for longer-term transport (96 h) sensitivity was greatest when the samples were stored at -20°C. The creation of pools of 5 samples is therefore a good option to decrease costs when screening bulls for BGC with the qPCR assay of direct preputial samples. Ideally the samples should be stored at 4°C and arrive at the laboratory within 48 h of collection, but when that is not possible freezing at -20°C could minimize the loss of sensitivity.

Le diagnostic de la campylobactériose génitale bovine (CGB) présente des défis significatifs, étant donné que les méthodes traditionnelles manquent de sensibilité lorsqu'un transport prolongé des échantillons est requis. Les épreuves utilisant des échantillons prépuciaux dans des épreuves de réaction d'amplification en chaine par la polymérase en temps réel (PCR) ont une bonne sensibilité et une capacité de rendement élevée. Toutefois, il y a peu d'information sur la durée acceptable du transport et de la température durant le transport des échantillons. De plus, l'utilisation d'échantillons regroupés s'est avéré être une stratégie valable pour le diagnostic d'autres maladies vénériennes chez les bovins. Les objectifs de la présente étude étaient de déterminer l'effet du regroupement d'échantillons et du temps de transport et de la température sur la sensibilité clinique d'une épreuve PCR quantitative en temps réel (qPCR) pour Campylobacter fetus ssp. venerealis dans des échantillons prépuciaux provenant de taureaux. Huit taureaux infectés et 176 bouvillons vierges ont été utilisés comme source des échantillons. La sensibilité du qPCR était comparable pour des échantillons non-regroupés et des regroupements de 5 échantillons, mais diminuée pour des regroupements de 10 échantillons. La sensibilité pour les différentes tailles de regroupement s'améliorait suite à des échantillonnages répétés. Pour des transport de courte durée (2 et 48 h), la sensibilité était plus élevée lorsque les échantillons étaient entreposés à 4 °C et 30 °C, alors que pour le transport de longue durée (96 h) la sensibilité était plus élevée lorsque les échantillons étaient entreposés à −20 °C. La création de regroupement de 5 échantillons est une bonne option pour diminuer les coûts lors du tamisage de taureaux pour CGB avec le qPCR effectué directement sur des échantillons prépuciaux. Idéalement, les échantillons devraient être entreposés à 4 °C et arriver au laboratoire au plus tard 48 h après le prélèvement, si ce n'est pas possible, la congélation à −20 °C pourrait minimiser la perte de sensibilité.(Traduit par Docteur Serge Messier).

Comparative Genomics of cpn60-Defined Enterococcus hirae Ecotypes and Relationship of Gene Content Differences to Competitive Fitness.

Natural microbial communities undergo selection-driven succession with changes in environmental conditions and available nutrients. In a previous study of the pig faecal Enterococcus community, we demonstrated that cpn60 universal target (UT) sequences could resolve phenotypically and genotypically distinct ecotypes of Enterococcus spp. that emerged over time in the faecal microbiome of growing pigs. In this study, we characterized genomic diversity in the identified Enterococcus hirae ecotypes in order to define further the nature and degree of genome content differences between taxa resolved by cpn60 UT sequences. Genome sequences for six representative isolates (two from each of three ecotypes) were compared. Differences in phosphotransferase systems and amino acid metabolism pathways for glutamine, proline and selenocysteine were observed. Differences in the lac family phosphotransferase system corresponded to lactose utilization phenotypes of the isolates. Competitive fitness of the E. hirae ecotypes was evaluated by in vitro growth competition assays in pig faecal extract medium. Isolates from E. hirae-1 and E. hirae-2 ecotypes were able to out-compete isolates from the E. hirae-3 ecotype, consistent with the relatively low abundance of E. hirae-3 relative to E. hirae-1 and E. hirae-2 previously observed in the pig faecal microbiome, and with observed differences between the ecotypes in gene content related to biosynthetic capacity. Results of this study provide a genomic basis for the definition of ecotypes within E. hirae and confirm the utility of the cpn60 UT sequence for high-resolution profiling of complex microbial communities.

The flagellum in bacterial pathogens: For motility and a whole lot more.

The bacterial flagellum is an amazingly complex molecular machine with a diversity of roles in pathogenesis including reaching the optimal host site, colonization or invasion, maintenance at the infection site, and post-infection dispersal. Multi-megadalton flagellar motors self-assemble across the cell wall to form a reversible rotary motor that spins a helical propeller - the flagellum itself - to drive the motility of diverse bacterial pathogens. The flagellar motor responds to the chemoreceptor system to redirect swimming toward beneficial environments, thus enabling flagellated pathogens to seek out their site of infection. At their target site, additional roles of surface swimming and mechanosensing are mediated by flagella to trigger pathogenesis. Yet while these motility-related functions have long been recognized as virulence factors in bacteria, many bacteria have capitalized upon flagellar structure and function by adapting it to roles in other stages of the infection process. Once at their target site, the flagellum can assist adherence to surfaces, differentiation into biofilms, secretion of effector molecules, further penetration through tissue structures, or in activating phagocytosis to gain entry into eukaryotic cells. Next, upon onset of infection, flagellar expression must be adapted to deal with the host's immune system defenses, either by reduced or altered expression or by flagellar structural modification. Finally, after a successful growth phase on or inside a host, dispersal to new infection sites is often flagellar motility-mediated. Examining examples of all these processes from different bacterial pathogens, it quickly becomes clear that the flagellum is involved in bacterial pathogenesis for motility and a whole lot more.

A Study of the Vaginal Microbiome in Healthy Canadian Women Utilizing cpn60-Based Molecular Profiling Reveals Distinct Gardnerella Subgroup Community State Types.

The vaginal microbiota is important in women's reproductive and overall health. However, the relationships between the structure, function and dynamics of this complex microbial community and health outcomes remain elusive. The objective of this study was to determine the phylogenetic range and abundance of prokaryotes in the vaginal microbiota of healthy, non-pregnant, ethnically diverse, reproductive-aged Canadian women. Socio-demographic, behavioural and clinical data were collected and vaginal swabs were analyzed from 310 women. Detailed profiles of their vaginal microbiomes were generated by pyrosequencing of the chaperonin-60 universal target. Six community state types (CST) were delineated by hierarchical clustering, including three Lactobacillus-dominated CST (L. crispatus, L. iners, L. jensenii), two Gardnerella-dominated (subgroups A and C) and an "intermediate" CST which included a small number of women with microbiomes dominated by seven other species or with no dominant species but minority populations of Streptococcus, Staphylococcus, Peptoniphilus, E. coli and various Proteobacteria in co-dominant communities. The striking correspondence between Nugent score and deep sequencing CST continues to reinforce the basic premise provided by the simpler Gram stain method, while additional analyses reveal detailed cpn60-based phylogeny and estimated abundance in microbial communities from vaginal samples. Ethnicity was the only demographic or clinical characteristic predicting CST, with differences in Asian and White women (p = 0.05). In conclusion, this study confirms previous work describing four cpn60-based subgroups of Gardnerella, revealing previously undescribed CST. The data describe the range of bacterial communities seen in Canadian women presenting with no specific vaginal health concerns, and provides an important baseline for future investigations of clinically important cohorts.

Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

BACKGROUND: The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment. FINDINGS: Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome. CONCLUSIONS: These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies.

Prevalence and diversity of Campylobacter species in Saskatchewan retail ground beef.

The primary objective of this study was to investigate the prevalence of Campylobacter spp. DNA by PCR in retail ground beef sold in Saskatchewan, Canada, and to identify the presence of individual Campylobacter species (C. coli, C. curvus, C. fetus, C. hyointestinalis, C. jejuni, C. rectus, and C. upsaliensis) using real-time quantitative PCR (qPCR). Secondary objectives were to assess potential differences in the prevalence of Campylobacter between ground beef offered for sale during cold and warm seasons as well as that offered for sale fresh and frozen, to investigate any association between the presence of Campylobacter spp. DNA and E. coli and/or aerobic bacterial counts, and finally to compare the prevalence of Campylobacter spp. DNA in ground beef originating from different production and retail environments. Out of the 309 total samples included in the study, 50 (16.2%) samples tested positive for Campylobacter spp. DNA, while 49 (15.9%) samples were determined positive for up to five individual species. Collectively, these assays determined that 14 (4.5%) samples were positive for C. coli, 11 (3.6%) for C. curvus, 6 (1.9%) for C. fetus, 24 (7.8%) for C. hyointestinalis, 12 (3.9%) for C. jejuni, 6 (1.9%) for C. rectus, and 9 (2.9%) for C. upsaliensis. There were 27 (8.7%) samples that were positive at the genus level that did not test positive for any of the seven Campylobacter species investigated (suggesting an alternate Campylobacter species). Also, 26 (8.4%) samples generated positive results by one of the species-specific qPCR assays, but returned no product in the conventional genus-level assay (suggesting a higher sensitivity for the species-specific qPCR assays). There was no significant association between the presence of Campylobacter spp. in Saskatchewan retail ground beef and any of the investigated risk factors.

Characterization of the fecal microbiota of pigs before and after inoculation with "Brachyspira hampsonii".

"Brachyspira hampsonii" causes disease indistinguishable from swine dysentery, and the structure of the intestinal microbiome likely plays a role in determining susceptibility of individual pigs to infection and development of clinical disease. The objectives of the current study were to determine if the pre-inoculation fecal microbiota differed between inoculated pigs that did (INOC MH) or did not (INOC non-MH) develop mucohaemorrhagic diarrhea following challenge with "B. hampsonii", and to quantify changes in the structure of the microbiome following development of clinical disease. Fecal microbiota profiles were generated based on amplification and sequencing of the cpn60 universal target sequence from 89 samples from 18 pigs collected at -8, -5, -3 and 0 days post-inoculation, and at termination. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of INOC MH and INOC non-MH pigs. However, the development of bloody diarrhea in inoculated pigs was associated with perturbation of the microbiota relative to INOC non-MH or sham-inoculated control pigs. Specifically, the fecal microbiota of INOC MH pigs was less dense (fewer total 16S rRNA copies per gram of feces), and had a lower Bacteroidetes:Firmicutes ratio. Further investigation of the potential long-term effects of Brachyspira disease on intestinal health and performance is warranted.

Clinical sensitivity and specificity of a real-time PCR assay for Campylobacter fetus subsp venerealis in preputial samples from bulls.

OBJECTIVE: To determine clinical sensitivity and specificity of a quantitative real-time PCR (qRT-PCR) assay for Campylobacter fetus subsp venerealis (Cfv) in preputial samples of bulls. ANIMALS: 313 beef bulls. PROCEDURES: Preputial samples were collected from 300 virgin bulls and 13 Cfv-infected bulls. Specificity of the qRT-PCR assay, determined on the basis of results for samples collected from virgin bulls, was compared with specificity of bacteriologic culture performed with transport enrichment medium (TEM). Sensitivity of the qRT-PCR assay, determined on the basis of results for multiple samples collected at weekly intervals from infected bulls, was compared with sensitivity of the direct fluorescent antibody test (DFAT), bacteriologic culture, and bacteriologic culture with TEM. RESULTS: Specificity was 85% for the qRT-PCR assay and 100% for bacteriologic culture; results were significantly different. Mean sensitivity was 85.4% for the qRT-PCR assay, 82.3% for direct culture in blood agar, 72.1% for the DFAT, 32.7% for direct culture in Skirrow agar, 30% for bacteriologic culture with TEM and blood agar, and 38.1% for bacteriologic culture with TEM and Skirrow agar. Differences in sensitivity among tests varied with ambient outdoor temperature. Repeated sampling significantly increased sensitivity of the qRT-PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the qRT-PCR assay as a screening test on direct preputial samples had comparable sensitivity to bacteriologic culture, and repeated sampling improved sensitivity. Although improved performance of the qRT-PCR assay, compared with direct bacteriologic culture, was dependent on temperature, transport times that allow direct culture are unlikely under field conditions. The qRT-PCR assay would provide a fast and sensitive screening method for Cfv in bulls.

Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle.

BACKGROUND: The vaginal microbial community plays a vital role in maintaining women's health. Understanding the precise bacterial composition is challenging because of the diverse and difficult-to-culture nature of many bacterial constituents, necessitating culture-independent methodology. During a natural menstrual cycle, physiological changes could have an impact on bacterial growth, colonization, and community structure. The objective of this study was to assess the stability of the vaginal microbiome of healthy Canadian women throughout a menstrual cycle by using cpn60-based microbiota analysis. Vaginal swabs from 27 naturally cycling reproductive-age women were collected weekly through a single menstrual cycle. Polymerase chain reaction (PCR) was performed to amplify the universal target region of the cpn60 gene and generate amplicons representative of the microbial community. Amplicons were pyrosequenced, assembled into operational taxonomic units, and analyzed. Samples were also assayed for total 16S rRNA gene content and Gardnerella vaginalis by quantitative PCR and screened for the presence of Mollicutes by using family and genus-specific PCR. RESULTS: Overall, the vaginal microbiome of most women remained relatively stable throughout the menstrual cycle, with little variation in diversity and only modest fluctuations in species richness. Microbiomes between women were more different than were those collected consecutively from individual women. Clustering of microbial profiles revealed the expected groupings dominated by Lactobacillus crispatus, Lactobacillus iners, and Lactobacillus jensenii. Interestingly, two additional clusters were dominated by either Bifidobacterium breve or a heterogeneous mixture of nonlactobacilli. Direct G. vaginalis quantification correlated strongly with its pyrosequencing-read abundance, and Mollicutes, including Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum, were detected in most samples. CONCLUSIONS: Our cpn60-based investigation of the vaginal microbiome demonstrated that in healthy women most vaginal microbiomes remained stable through their menstrual cycle. Of interest in these findings was the presence of Bifidobacteriales beyond just Gardnerella species. Bifidobacteriales are frequently underrepresented in 16S rRNA gene-based studies, and their detection by cpn60-based investigation suggests that their significance in the vaginal community may be underappreciated.

Comparison of baseline bacterial levels in retail ground beef originating from different regulatory, processing, and packaging environments.

The objectives of this study were to collect baseline measures of bacteria present in retail ground beef offered for sale in Saskatchewan and to assess differences associated with the licensing or regulatory environment of the packaging and processing facilities as indicated by package labeling. Packages of ground beef (n = 309) were purchased from May 2011 to May 2012. Retail samples were categorized as originating from facilities regulated by the federal government or licensed by the provincial government (n = 126), originating from facilities licensed by local health regions (n = 80), or having no inspection or source information on the package label (n = 103). Total aerobic plate counts and total Escherichia coli plate counts were determined using 3M Petrifilm methods. Total bacterial load was estimated using real-time quantitative PCR. The data were analyzed on a log scale using multivariable linear regression, accounting for season and whether the samples were fresh or frozen at purchase. Total aerobic plate counts and Escherichia coli plate counts were lower in samples from federally regulated or provincially licensed facilities than in samples from locally licensed facilities (P < 0.001 and P = 0.002, respectively) or in samples with no inspection information on the label (P < 0.001 and P = 0.011, respectively). Frozen ground beef from federally regulated or provincially licensed facilities had the lowest total bacterial load. Samples clearly labeled as packaged at federally regulated or provincially licensed facilities consistently had the lowest estimated bacterial levels.

Application of a new diagnostic approach to a bovine genital campylobacteriosis outbreak in a Saskatchewan beef herd.

A new real-time quantitative polymerase chain reaction (qPCR) test was used to diagnose Campylobacter fetus subsp. venerealis infection associated with dramatic reproductive losses in a commercial cow-calf herd. The results were verified with repeated culture, phenotypic characterization of the organism and DNA sequencing. This case demonstrates the need for a practical field test for C. fetus subsp. venerealis and the importance of considering this organism as a potential cause of pregnancy failure in beef herds.

Application d'une nouvelle approche diagnostique lors d'une éclosion de campylobactériose génitale bovine dans un troupeau bovin de la Saskatchewan. Un nouveau test quantitatif d'amplification en chaîne par la polymérase en temps réel (qACP) a été utilisé pour diagnostiquer une infection par Campylobacter fetus sous-espèce venerealis associée à une baisse spectaculaire de la reproduction dans un troupeau commercial de vaches-veaux. Les résultats ont été vérifiés à l'aide de cultures répétées, d'une caractérisation phénotypique de l'organisme et du séquençage de l'ADN. Ce cas démontre le besoin d'un test sur le terrain pratique pour C. fetus sous-espèce venerealis et l'importance de considérer cet organisme comme une cause potentielle d'échec de la gestation dans les troupeaux bovins.(Traduit par Isabelle Vallières).

Characterization of the upper respiratory tract microbiomes of patients with pandemic H1N1 influenza.

The upper respiratory tract microbiome has an important role in respiratory health. Influenza A is a common viral infection that challenges that health, and a well-recognized sequela is bacterial pneumonia. Given this connection, we sought to characterize the upper respiratory tract microbiota of individuals suffering from the pandemic H1N1 influenza A outbreak of 2009 and determine if microbiome profiles could be correlated with patient characteristics. We determined the microbial profiles of 65 samples from H1N1 patients by cpn60 universal target amplification and sequencing. Profiles were examined at the phylum and nearest neighbor "species" levels using the characteristics of patient gender, age, originating health authority, sample type and designation (STAT/non-STAT). At the phylum level, Actinobacteria-, Firmicutes- and Proteobacteria-dominated microbiomes were observed, with none of the patient characteristics showing significant profile composition differences. At the nearest neighbor "species" level, the upper respiratory tract microbiomes were composed of 13-20 "species" and showed a trend towards increasing diversity with patient age. Interestingly, at an individual level, most patients had one to three organisms dominant in their microbiota. A limited number of discrete microbiome profiles were observed, shared among influenza patients regardless of patient status variables. To assess the validity of analyses derived from sequence read abundance, several bacterial species were quantified by quantitative PCR and compared to the abundance of cpn60 sequence read counts obtained in the study. A strong positive correlation between read abundance and absolute bacterial quantification was observed. This study represents the first examination of the upper respiratory tract microbiome using a target other than the 16S rRNA gene and to our knowledge, the first thorough examination of this microbiome during a viral infection.

Isolation rates of Campylobacter fetus subsp venerealis from bovine preputial samples via passive filtration on nonselective medium versus selective medium, with and without transport medium.

OBJECTIVE: To compare the recovery rates of Campylobacter fetus subsp venerealis (Cfv) from preputial scrapings of infected bulls with passive filtration on selective medium versus nonselective medium, with and without transport medium. SAMPLES: 217 preputial scrapings from 12 bulls (4 naturally and 8 artificially infected with Cfv). PROCEDURES: Preputial scrapings were collected in 2 mL of PBS solution and bacteriologically cultured directly on Skirrow medium or passively filtered through 0.65-µm filters onto blood agar, with or without 24 hour preincubation in modified Weybridge transport enrichment medium (TEM). After 72 hours, plates were examined for Cfv and bacterial and fungal contamination or overgrowth. RESULTS: Passive filtration of fresh preputial scrapings onto blood agar yielded significantly higher recovery rates of Cfv (86%) than direct plating on Skirrow medium (32%), whereas recovery from TEM was poor for both media (35% and 40%, respectively). Skirrow cultures without TEM were significantly more likely to have fungal contamination than were cultures performed with any other technique, and fungal contamination was virtually eliminated by passive filtration onto blood agar. Bacterial contamination by Pseudomonas spp was significantly more common with Skirrow medium versus passive filtration on blood agar, regardless of TEM use. CONCLUSIONS AND CLINICAL RELEVANCE: The use of transport medium and the choice of culture medium had significant effects on Cfv recovery and culture contamination rates from clinical samples. Both factors should be considered when animals are tested for this pathogen.

CRISPRs of Enterococcus faecalis and E. hirae isolates from pig feces have species-specific repeats but share some common spacer sequences.

Clustered regularly interspaced short palindromic repeats (CRISPR) are currently a topic of interest in microbiology due to their role as a prokaryotic immune system. Investigations of CRISPR distribution and characterization to date have focused on pathogenic bacteria, while less is known about CRISPR in commensal bacteria, where they may have a significant role in the ecology of the microbiota of humans and other animals, and act as a recorder of interactions between bacteria and viruses. A combination of PCR and sequencing was used to determine prevalence and distribution of CRISPR arrays in Enterococcus faecalis and Enterococcus hirae isolates from the feces of healthy pigs. Both type II CRISPR-Cas and Orphan CRISPR (without Cas genes) were detected in the 195 isolates examined. CRISPR-Cas was detected in 52 (46/88) and 42 % (45/107) E. faecalis and E. hirae isolates, respectively. The prevalence of Orphan CRISPR arrays was higher in E. faecalis isolates (95 %, 84/88) compared with E. hirae isolates (49 %, 53/107). Species-specific repeat sequences were identified in Orphan CRISPR arrays, and 42 unique spacer sequences were identified. Only two spacers matched previously characterized pig virome sequences, and many were apparently derived from chromosomal sequences of enterococci. Surprisingly, 17 (40 %) of the spacers were detected in both species. Shared spacer sequences are evidence of a lack of species specificity in the agents and mechanisms responsible for integration of spacers, and the abundance of spacer sequences corresponding to bacterial chromosomal sequences reflects interspecific interactions within the intestinal microbiota.

mPUMA: a computational approach to microbiota analysis by de novo assembly of operational taxonomic units based on protein-coding barcode sequences.

BACKGROUND: Formation of operational taxonomic units (OTU) is a common approach to data aggregation in microbial ecology studies based on amplification and sequencing of individual gene targets. The de novo assembly of OTU sequences has been recently demonstrated as an alternative to widely used clustering methods, providing robust information from experimental data alone, without any reliance on an external reference database. RESULTS: Here we introduce mPUMA (microbial Profiling Using Metagenomic Assembly, http://mpuma.sourceforge.net), a software package for identification and analysis of protein-coding barcode sequence data. It was developed originally for Cpn60 universal target sequences (also known as GroEL or Hsp60). Using an unattended process that is independent of external reference sequences, mPUMA forms OTUs by DNA sequence assembly and is capable of tracking OTU abundance. mPUMA processes microbial profiles both in terms of the direct DNA sequence as well as in the translated amino acid sequence for protein coding barcodes. By forming OTUs and calculating abundance through an assembly approach, mPUMA is capable of generating inputs for several popular microbiota analysis tools. Using SFF data from sequencing of a synthetic community of Cpn60 sequences derived from the human vaginal microbiome, we demonstrate that mPUMA can faithfully reconstruct all expected OTU sequences and produce compositional profiles consistent with actual community structure. CONCLUSIONS: mPUMA enables analysis of microbial communities while empowering the discovery of novel organisms through OTU assembly.

A 'universal' type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities.

Bacteria and Archaea are evolutionarily and biochemically distinct domains found together in many environments. Robust 'universal' PCR primer sets targeting both the bacterial 16S rRNA gene and the type I chaperonin gene have been established. However, 'universal' PCR primers for Archaea are currently limited to the 16S rRNA gene. We investigated the type II chaperonin (known as the thermosome, TF55, CCT or TCP-1) as a potential universal target (UT) for Archaea. Reproducible amplification of thermosome gene sequences from all major phyla tested was achieved through the application of a mixture or 'cocktail' of two forward and two reverse primers. Phylogenies based on the ∼750-bp thermosome UT were congruent with 16S rRNA gene phylogenies while exhibiting longer branch lengths, improving resolution of closely related taxa. 'Universal' thermosome primers were applied to profiling the archaeal community of dairy cow rumen and results compared with profiles based on the 16S rRNA gene and methyl co-enzyme M reductase (methanogen-specific) gene. Clone libraries generated from each target gene, as well as a pyrosequencing profile of one thermosome rumen library, revealed that all three targets consistently detected Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanosphaera stadtmanae as the dominant constituents; however, thermosome gene sequences were more diverse than either of the other targets providing a higher resolution description of the archaeal community. These findings demonstrate that a 'universal' thermosome PCR protocol is a powerful metagenomic tool for detecting and characterizing Archaea and archaeal communities.