Multiple processes contribute to methane emission in a riparian cottonwood forest ecosystem.

Methane emission from trees may partially or completely offset the methane sink in upland soils, the only process that has been regularly included in methane budgets for forest ecosystems. Our objective was to analyze multiple biogeochemical processes that influence the production, oxidation and transport of methane in a riparian cottonwood ecosystem and its adjacent river. We combined chamber flux measurements on tree stems, forest soil and the river surface with eddy covariance measurements of methane net ecosystem exchange. In addition, we tested whether methanogens were present in cottonwood stems, shallow soil layers and alluvial groundwater. Average midday peak in net methane emission measured by eddy covariance was c. 12 nmol m-2  s-1 . The average uptake of methane by soils (0.87 nmol m-2  s-1 ) was largely offset by tree stem methane emission (0.75 nmol m-2  s-1 ). There was evidence of methanogens in tree stems but not in shallow soil. Growing season (May-September) cumulative net methane emission (17.4 mmol CH4  m-2 ) included methane produced in cottonwood stems and methane input to the nocturnal boundary layer from the forest and the adjacent river. The multiple processes contributing to methane emission illustrated the linked nature of these adjacent terrestrial and aquatic ecosystems.

Structural analysis of broiler chicken small intestinal mucin O-glycan modification by Clostridium perfringens.

Clostridium perfringens is a Gram-positive opportunistic pathogen that is the principal etiological agent of necrotic enteritis (NE) in poultry. The ability of C. perfringens to incite NE depends upon its ability to penetrate the protective mucus barrier within the small intestine, which is largely composed of heavily glycosylated proteins called mucins. Mucins are decorated by N- and O-linked glycans that serve both as a formidable gel-like barrier against invading pathogens and as a rich carbon source for mucolytic bacteria. The composition of avian O-linked glycans is markedly different from mucins in other vertebrates, being enriched in sulfated monosaccharides and N-acetyl-d-neuraminic acid (Neu5Ac, sialic acid). These modifications increase the overall negative charge of mucins and are believed to impede colonization by enteric pathogens. The mechanism by which C. perfringens penetrates the poultry intestinal mucus layer during NE is still unknown. However, the CAZome (i.e., the total collection of proteins encoded within a genome active on carbohydrates) of C. perfringens strain CP1 encodes several putative and known enzymes with activities consistent with the modification of mucin. To further investigate this relationship, O-glycans from Gallus gallus domesticus mucus were extracted from the small intestine and characterized using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Chicken mucin monosaccharides included l-fucose (Fuc), d-mannose (Man), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), N-acetyl-d-glucosamine (GlcNAc), and Neu5Ac (sialic acid). Using these monosaccharides as sole carbon sources, we showed that C. perfringens CP1 grew on Neu5Ac, Man, Gal, and GlcNAc but not on Fuc and GalNAc. We also demonstrated C. perfringens grew on different native-state preparations of intestinal mucins and mucus including porcine mucins, chicken mucus, and chicken mucins. Finally, anaerobic incubation of chicken mucin O-glycans with C. perfringens and subsequent analysis of the glycans revealed that there was preferential removal of Neu5Ac. These observations are discussed in the context of the predicted metabolic potential of C. perfringens CP1 and the mucolytic enzymes encoded within its CAZome.

Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni.

Enrofloxacin is registered for therapeutic use in beef cattle to treat bovine respiratory disease in Canada. A murine model was used to experimentally examine the impact of therapeutic administration of enrofloxacin on fluoroquinolone resistance development in Campylobacter jejuni. Administration of enrofloxacin to mice via subcutaneous injection or per os routes resulted in equivalent levels of bioactive enrofloxacin within the intestine, but bioactivity was short-lived (<48 h after cessation). Enrofloxacin administration did not affect densities of total bacteria, Firmicutes, or Bacteroidetes in digesta and had modest impacts on densities of Enterobacteriaceae. All mice inoculated with C. jejuni NCTC 11168 became persistently colonized by the bacterium. Enrofloxacin reduced C. jejuni cell densities within the cecal and colonic digesta for all treatments, and densities shed in feces as a function of antibiotic duration. None of the C. jejuni isolates recovered from mice after administration of enrofloxacin (n = 260) developed resistance to ciprofloxacin regardless of method or duration of administration. Furthermore, only modest shifts in the minimum inhibitory concentration of the isolates by treatment were noted. The study findings indicate that the risk posed by short-term subcutaneous administration of enrofloxacin for the development of fluoroquinolone resistance in mammals is low.

Subtype-Specific Selection for Resistance to Fluoroquinolones but Not to Tetracyclines Is Evident in Campylobacter jejuni Isolates from Beef Cattle in Confined Feeding Operations in Southern Alberta, Canada.

Campylobacter jejuni was longitudinally isolated from beef cattle housed in four confined feeding operations (CFOs) in Southern Alberta, Canada, over 18 months. All of the cattle were administered a variety of antimicrobial agents (AMAs) nontherapeutically and metaphylactically during their time in the CFOs. In total, 7,966 C. jejuni isolates were recovered from cattle. More animals were colonized by the bacterium after >60 days in the CFO (interim) than were individuals upon entry at the CFO (arrival). Subtyping and resistance to seven AMAs were determined for 1,832 (23.0%) and 1,648 (20.7%) isolates, respectively. Increases in the proportion of isolates resistant to tetracycline were observed at all four CFOs between sample times and to ciprofloxacin and nalidixic acid at one or more CFOs. The vast majority of isolates resistant to tetracycline carried tetO, whereas ciprofloxacin resistance was predominantly attributed to mutations in the gyrA gene. Although considerable diversity was observed, a majority of C. jejuni isolates belonged to one of five predominant subtype clusters. There was no difference in subtype diversity by CFO, but the population structure differed between sample times. Selection for resistance to ciprofloxacin and nalidixic acid was subtype dependent, whereas selection for resistance to tetracycline was not. The findings indicate that a proportion of cattle entering CFOs carry resistant C. jejuni subtypes, and the characteristics of beef cattle CFOs facilitate transmission/proliferation of diverse subtypes, including those resistant to AMAs, which coupled with the densities of CFOs likely contribute to the high rates of cattle-associated campylobacteriosis in Southern Alberta.IMPORTANCE A small proportion of cattle entering a CFO carry Campylobacter jejuni, including subtypes resistant to AMAs. The large numbers of cattle arriving from diverse locations at the CFOs and intermingling within the CFOs over time, coupled with the high-density housing of animals, the high rates of transmission of C. jejuni subtypes among animals, and the extensive use of AMAs merge to create an ideal situation where the proliferation of diverse antimicrobial-resistant C. jejuni subtypes is facilitated. Considering that Southern Alberta reports high rates of campylobacteriosis in the human population and that many of these clinical cases are due to C. jejuni subtypes associated with cattle, it is likely that the characteristics of beef cattle CFOs favor the propagation of clinically relevant C. jejuni subtypes, including those resistant to medically important AMAs, which constitute a risk to human health.

Streamlined purification of fluorescently labeled Escherichia coli phosphate-binding protein (PhoS) suitable for rapid-kinetics applications.

Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (Pi) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of Pi binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni2+-affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the Pi release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a Pi detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates.

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, ß1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and ß1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both ß-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

Changes in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine.

Escherichia coli O157:H7 is a foodborne pathogen that colonizes ruminants. Cattle are considered the primary reservoir of E. coli O157:H7 with super-shedders, defined as individuals excreting > 104 E. coli O157:H7 CFU g-1 feces. The mechanisms leading to the super-shedding condition are largely unknown. Here, we used 16S rRNA gene pyrosequencing to examine the composition of the fecal bacterial community in order to investigate changes in the bacterial microbiota at several locations along the digestive tract (from the duodenum to the rectal-anal junction) in 5 steers previously identified as super-shedders and 5 non-shedders. The overall bacterial community structure did not differ by E. coli O157:H7 shedding status; but several differences in the relative abundance of taxa and OTUs were noted between the two groups. The genus Prevotella was most enriched in the non-shedders while the genus Ruminococcus and the Bacteroidetes phylum were notably enriched in the super-shedders. There was greater bacterial diversity and richness in samples collected from the lower- as compared to the upper gastrointestinal tract (GI). The spiral colon was the only GI location that differed in terms of bacterial diversity between super-shedders and non-shedders. These findings reinforced linkages between E. coli O157:H7 colonization in cattle and the nature of the microbial community inhabiting the digestive tract of super-shedders.

Prevalence and diversity of waterborne Arcobacter butzleri in southwestern Alberta, Canada.

Arcobacter butzleri is a potential enteric pathogen to human beings, but its reservoirs and modes of transmission are largely unverified. Microbiological and molecular detection and subtyping techniques can facilitate surveillance of A. butzleri in hosts and environmental reservoirs. We isolated A. butzleri from 173 surface water samples (25.6%) and 81 treated wastewater samples (77.9%) collected in southwestern Alberta over a 1-year period. Arcobacter butzleri isolates (n = 500) were genotyped and compared to determine diversity of A. butzleri in southwestern Alberta. Culture methods affected the frequency of detection and genotype diversity of A. butzleri, and isolation comprehensiveness was different for surface waters and treated wastewaters. Detection of A. butzleri in the Oldman River Watershed corresponded with season, river flow rates, and fecal coliform densities. Arcobacter butzleri was detected most frequently in treated wastewater, in the Oldman River downstream from treated wastewater outfalls, and in tributaries near areas of intensive confined feeding operations. All sample sources possessed high genotype diversity, and A. butzleri isolates from treated wastewaters were genetically similar to isolates from the Oldman River downriver from treated wastewater outfall sites. In southwestern Alberta, municipal and agricultural activities contribute to the density and genotype diversity of A. butzleri in surface waters.

Correction: Changes in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine.

[This corrects the article DOI: 10.1371/journal.pone.0170050.].

Efficacy of wastewater treatment on Arcobacter butzleri density and strain diversity.

Arcobacter butzleri is a suspected waterborne enteric pathogen that is ubiquitous in the environment, but the degree to which wastewater treatment prevents entry of A. butzleri into environmental waters and the risks posed are not well established. Untreated and treated wastewater samples (n = 260) were collected weekly from the Lethbridge and Fort Macleod wastewater treatment facilities (the two major municipal inputs in southwestern Alberta, Canada) from May 2008 to April 2009. Untreated wastewaters contained high densities of A. butzleri and fecal coliform indicators, and densities at Lethbridge were typically higher than at Fort Macleod. Data indicated that A. butzleri and fecal coliform densities in wastewater were greatest in autumn and lowest in winter. Mechanical and biological treatment of wastewaters reduced but did not eliminate fecal coliform indicators or A. butzleri. At Lethbridge, UVB irradiation of mechanically and biologically treated wastewater further reduced densities of fecal coliform indicators. There was high A. butzleri genotype diversity in all sample sources, and survival during treatment was not strain-dependent. No genotype was dominant in any sample source, but 8.9% of genotypes were recurrent over time, and 4.4% of genotypes were detected at both wastewater treatment facilities. The current study demonstrates that viable A. butzleri are able to survive wastewater treatment, including UVB irradiation, which may lead to increased density and genetic diversity of this suspected pathogen in environmental waters via wastewater effluent discharge.

Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle.

Cattle are the primary reservoir of the foodborne pathogen Escherichia coli O157:H7, with the concentration and frequency of E. coli O157:H7 shedding varying substantially among individual hosts. The term ''super-shedder" has been applied to cattle that shed ≥10(4) cfu E. coli O157:H7/g of feces. Super-shedders have been reported to be responsible for the majority of E. coli O157:H7 shed into the environment. The objective of this study was to determine if there are phenotypic and/or genotypic differences between E. coli O157:H7 isolates obtained from super-shedder compared to low-shedder cattle. From a total of 784 isolates, four were selected from low-shedder steers and six isolates from super-shedder steers (4.01-8.45 log cfu/g feces) for whole genome sequencing. Isolates were phage and clade typed, screened for substrate utilization, pH sensitivity, virulence gene profiles and Stx bacteriophage insertion (SBI) sites. A range of 89-2473 total single nucleotide polymorphisms (SNPs) were identified when sequenced strains were compared to E. coli O157:H7 strain Sakai. More non-synonymous SNP mutations were observed in low-shedder isolates. Pan-genomic and SNPs comparisons did not identify genetic segregation between super-shedder or low-shedder isolates. All super-shedder isolates and 3 of 4 of low-shedder isolates were typed as phage type 14a, SBI cluster 3 and SNP clade 2. Super-shedder isolates displayed increased utilization of galactitol, thymidine and 3-O-ß-D-galactopyranosyl-D-arabinose when compared to low-shedder isolates, but no differences in SNPs were observed in genes encoding for proteins involved in the metabolism of these substrates. While genetic traits specific to super-shedder isolates were not identified in this study, differences in the level of gene expression or genes of unknown function may still contribute to some strains of E. coli O157:H7 reaching high densities within bovine feces.

ß2-1 Fructan supplementation alters host immune responses in a manner consistent with increased exposure to microbial components: results from a double-blinded, randomised, cross-over study in healthy adults.

ß2-1 Fructans are purported to improve health by stimulating growth of colonic bifidobacteria, increasing host resistance to pathogens and stimulating the immune system. However, in healthy adults, the benefits of supplementation remain undefined. Adults (thirteen men, seventeen women) participated in a double-blinded, placebo-controlled, randomised, cross-over study consisting of two 28-d treatments separated by a 14-d washout period. Subjects' regular diets were supplemented with ß2-1 fructan or placebo (maltodextrin) at 3×5 g/d. Fasting blood and 1-d faecal collections were obtained at the beginning and at the end of each phase. Blood was analysed for clinical, biochemical and immunological variables. Determinations of well-being and general health, gastrointestinal (GI) symptoms, regularity, faecal SCFA content, residual faecal ß2-1 fructans and faecal bifidobacteria content were undertaken. ß2-1 Fructan supplementation had no effect on blood lipid or cholesterol concentrations or on circulating lymphocyte and macrophage numbers, but significantly increased serum lipopolysaccharide, faecal SCFA, faecal bifidobacteria and indigestion. With respect to immune function, ß2-1 fructan supplementation increased serum IL-4, circulating percentages of CD282+/TLR2+ myeloid dendritic cells and ex vivo responsiveness to a toll-like receptor 2 agonist. ß2-1 Fructans also decreased serum IL-10, but did not affect C-reactive protein or serum/faecal Ig concentrations. No differences in host well-being were associated with either treatment, although the self-reported incidence of GI symptoms and headaches increased during the ß2-1 fructan phase. Although ß2-1 fructan supplementation increased faecal bifidobacteria, this change was not directly related to any of the determined host parameters.

Comparative Detection and Quantification of Arcobacter butzleri in Stools from Diarrheic and Nondiarrheic People in Southwestern Alberta, Canada.

Arcobacter butzleri has been linked to enteric disease in humans, but its pathogenicity and epidemiology remain poorly understood. The lack of suitable detection methods is a major limitation. Using comparative genome analysis, we developed PCR primers for direct detection and quantification ofA. butzleri DNA in microbiologically complex matrices. These primers, along with existing molecular and culture-based methods, were used to detectA. butzleri and enteric pathogens in stools of diarrheic and nondiarrheic people (n= 1,596) living in southwestern Alberta, Canada, from May to November 2008. In addition, quantitative PCR was used to compare A. butzleridensities in diarrheic and nondiarrheic stools.Arcobacter butzleriwas detected more often by PCR (59.6%) than by isolation methods (0.8%). Comparison by PCR-based detection found no difference in the prevalence ofA. butzleri between diarrheic (56.7%) and nondiarrheic (45.5%) individuals. Rates of detection in diarrheic stools peaked in June (71.1%) and October (68.7%), but there was no statistically significant correlation between the presence ofA. butzleri and patient age, sex, or place of habitation. Densities ofA. butzleriDNA in diarrheic stools (1.6 ± 0.59 log10 copies mg(-1)) were higher (P= 0.007) than in nondiarrheic stools (1.3 ± 0.63 log10copies mg(-1)). Of the 892 diarrheic samples that were positive for A. butzleri, 74.1% were not positive for other bacterial and/or viral pathogens. The current study supports previous work suggesting that A. butzleri pathogenicity is strain specific and/or dependent on other factors, such as the level of host resistance.

Development of a comparative genomic fingerprinting assay for rapid and high resolution genotyping of Arcobacter butzleri.

BACKGROUND: Molecular typing methods are critical for epidemiological investigations, facilitating disease outbreak detection and source identification. Study of the epidemiology of the emerging human pathogen Arcobacter butzleri is currently hampered by the lack of a subtyping method that is easily deployable in the context of routine epidemiological surveillance. In this study we describe a comparative genomic fingerprinting (CGF) method for high-resolution and high-throughput subtyping of A. butzleri. Comparative analysis of the genome sequences of eleven A. butzleri strains, including eight strains newly sequenced as part of this project, was employed to identify accessory genes suitable for generating unique genetic fingerprints for high-resolution subtyping based on gene presence or absence within a strain. RESULTS: A set of eighty-three accessory genes was used to examine the population structure of a dataset comprised of isolates from various sources, including human and non-human animals, sewage, and river water (n=156). A streamlined assay (CGF40) based on a subset of 40 genes was subsequently developed through marker optimization. High levels of profile diversity (121 distinct profiles) were observed among the 156 isolates in the dataset, and a high Simpson's Index of Diversity (ID) observed (ID > 0.969) indicate that the CGF40 assay possesses high discriminatory power. At the same time, our observation that 115 isolates in this dataset could be assigned to 29 clades with a profile similarity of 90% or greater indicates that the method can be used to identify clades comprised of genetically similar isolates. CONCLUSIONS: The CGF40 assay described herein combines high resolution and repeatability with high throughput for the rapid characterization of A. butzleri strains. This assay will facilitate the study of the population structure and epidemiology of A. butzleri.

Perspectives on super-shedding of Escherichia coli O157:H7 by cattle.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 104 colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

Escherichia coli O157:H7 super-shedder and non-shedder feedlot steers harbour distinct fecal bacterial communities.

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed "super-shedders". A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a "normal" microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.

Comparative variation within the genome of Campylobacter jejuni NCTC 11168 in human and murine hosts.

Campylobacteriosis incited by C. jejuni is a significant enteric disease of human beings. A person working with two reference strains of C. jejuni National Collection of Type Cultures (NCTC) 11168 developed symptoms of severe enteritis including bloody diarrhea. The worker was determined to be infected by C. jejuni. In excess of 50 isolates were recovered from the worker's stool. All of the recovered isolates and the two reference strains were indistinguishable from each other based on comparative genomic fingerprint subtyping. Whole genome sequence analysis indicated that the worker was infected with a C. jejuni NCTC 11168 obtained from the American Type Culture Collection; this strain (NCTC 11168-GSv) is the genome sequence reference. After passage through the human host, major genetic changes including indel mutations within twelve contingency loci conferring phase variations were detected in the genome of C. jejuni. Specific and robust single nucleotide polymorphism (SNP) changes in the human host were also observed in two loci (Cj0144c, Cj1564). In mice inoculated with an isolate of C. jejuni NCTC 11168-GSv from the infected person, the isolate underwent further genetic variation. At nine loci, mutations specific to inoculated mice including five SNP changes were observed. The two predominant SNPs observed in the human host reverted in mice. Genetic variations occurring in the genome of C. jejuni in mice corresponded to increased densities of C. jejuni cells associated with cecal mucosa. In conclusion, C. jejuni NCTC 11168-GSv was found to be highly virulent in a human being inciting severe enteritis. Host-specific mutations in the person with enteritis occurred/were selected for in the genome of C. jejuni, and many were not maintained in mice. Information obtained in the current study provides new information on host-specific genetic adaptation by C. jejuni.

Are super-shedder feedlot cattle really super?

The objective of this study was to determine the frequency and duration of super-shedding in cattle by enumerating Escherichia coli O157:H7 in feces and to compare lineage and pulsed-field gel electrophoresis (PFGE) subtypes from super- and low-shedders. E. coli O157:H7 was enumerated from fecal samples obtained from the rectums of 400 feedlot cattle. Super-shedding steers (N=11) were identified, transported, and penned individually. Freshly voided fecal pats were sampled 2 h before and 6 h after feeding for 7 d, then once daily for an additional 19 d. Isolates (N=126) were subtyped using PFGE, and lineage was typed using a lineage-specific polymorphism assay. Of the 11 super-shedders identified at the commercial feedlot, only five were confirmed as super-shedders at the research feedlot, with no super-shedders identified 6 d after sampling at the commercial feedlot. Super-shedding was not consistent in fecal pats collected from the same individual at different times of the day. Isolates exhibited three distinct PFGE subtypes, with most isolates (97.6%) displaying the same subtype, including those obtained from steers that transitioned from super- to low-shedding. The short duration of super-shedding and its lack of continuance suggest that these individuals may not play as great a role in the dissemination of E. coli O157:H7 within the feedlot as previously proposed.

Campylobacter jejuni colonization is associated with a dysbiosis in the cecal microbiota of mice in the absence of prominent inflammation.

BACKGROUND: Campylobacter jejuni causes enterocolitis in humans, but does not incite disease in asymptomatic carrier animals. To survive in the intestine, C. jejuni must successfully compete with the microbiota and overcome the host immune defense. Campylobacter jejuni colonization success varies considerably amongst individual mice, and we examined the degree to which the intestinal microbiota was affected in mice (i.e. a model carrier animal) colonized by C. jejuni at high relative to low densities. METHODS: Mice were inoculated with C. jejuni or buffer, and pathogen shedding and intestinal colonization were measured. Histopathologic scoring and quantification of mRNA expression for α-defensins, toll-like receptors, and cytokine genes were conducted. Mucosa-associated bacterial communities were characterized by two approaches: multiplexed barcoded pyrosequencing and terminal restriction fragment length polymorphism analysis. RESULTS: Two C. jejuni treatments were established based on the degree of cecal and colonic colonization; C. jejuni Group A animals were colonized at high cell densities, and C. jejuni Group B animals were colonized at lower cell densities. Histological examination of cecal and colonic tissues indicated that C. jejuni did not incite visible pathologic changes. Although there was no significant difference among treatments in expression of mRNA for α-defensins, toll-like receptors, or cytokine genes, a trend for increased expression of toll-like receptors and cytokine genes was observed for C. jejuni Group A. The results of the two methods to characterize bacterial communities indicated that the composition of the cecal microbiota of C. jejuni Group A mice differed significantly from C. jejuni Group B and Control mice. This difference was due to a reduction in load, diversity and richness of bacteria associated with the cecal mucosa of C. jejuni Group A mice. CONCLUSIONS: High density colonization by C. jejuni is associated with a dysbiosis in the cecal microbiota independent of prominent inflammation.

Effect of antimicrobial growth promoter administration on the intestinal microbiota of beef cattle.

BACKGROUND: Antimicrobial growth promoters (AGPs) are antimicrobial agents administered to livestock in feed for prolonged periods to enhance feed efficiency. Beef cattle are primarily finished in confined feeding operations in Canada and the USA, and the administration of AGPs such as chlortetracycline and sulfamethazine (Aureo S-700 G) is the standard. The impacts of AGPs on the intestinal microbiota of beef cattle are currently uncertain; it is documented that AGPs administered to beef cattle pass through the rumen and enter the intestine. To ascertain the impacts of Aureo S-700 G on the small and large intestinal microbiota of beef cattle (mucosa-associated and within digesta), terminal restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for total bacteria were applied. Beef cattle were maintained in an experimental feedlot (five replicate pens per treatment), and AGP treatment cattle were administered Aureo S-700 G in feed, whereas control cattle were administered no antimicrobials. As the intestinal microbiota of beef cattle has not been extensively examined, clone library analysis was applied to ascertain the primary bacterial constituents of the intestinal microbiota. RESULTS: Comparative T-RFLP and qPCR analysis (n = 122 samples) revealed that bacterial community fingerprints and bacterial load within digesta differed from those associated with mucosa. However, the administration of Aureo S-700 G did not affect bacterial community fingerprints or bacterial load within the small and large intestine relative to control cattle. Analysis of >1500 near full length 16S rDNA clones revealed considerably greater bacterial diversity in the large relative to the small intestine of beef cattle. Mucosa-associated bacterial communities in the jejunum were dominated by Proteobacteria, and differed conspicuously from those in the ileum and large intestine. Although the ileum contained bacterial clones that were common to the jejunum as well as the cecum, Firmicutes clones associated with mucosa dominated in the ileum, cecum, and descending colon. In the descending colon, clone library analysis did not reveal a difference in the richness or diversity of bacterial communities within digesta relative to those associated with mucosa. However, T-RFLP analysis indicated a significant difference in T-RF relative abundance (i.e. difference in relative taxon abundance) between mucosa-associated and digesta communities attributed in part to the differential abundance of Bacteriodes, Alistipes, Oscillibacter, and unclassified Clostridiales. CONCLUSIONS: These data demonstrate that there was no significant difference in the composition of the predominant intestinal bacteria constituents within animals administered Aureo S-700 G and those not administered AGPs after a 28 day withdrawal period.