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- ItemNon-therapeutic administration of a model antimicrobial growth promoter modulates intestinal immune responses(BioMed Central, 2011) Costa, Estela; Uwiera, Richard R. E.; Kastelic, John P.; Selinger, L. Brent; Inglis, G. DouglasBackground: The development of efficacious alternatives to antimicrobial growth promoters (AGP) in livestock production is an urgent issue, but is hampered by a lack of knowledge regarding the mode of action of AGP. The belief that AGP modulate the intestinal microbiota has become prominent in the literature; however, there is a lack of experimental evidence to support this hypothesis. Using a chlortetracycline-murine-Citrobacter rodentium model, the ability of AGP to modulate the intestinal immune system in mammals was investigated. Results: C. rodentium was transformed with the tetracycline resistance gene, tetO, and continuous oral administration of a non-therapeutic dose of chlortetracycline to mice did not affect densities of C. rodentium CFU in feces throughout the experiment or associated with mucosal surfaces in the colon (i.e. at peak and late infection). However, chlortetracycline regulated transcription levels of Th1 and Th17 inflammatory cytokines in a temporal manner in C. rodentium-inoculated mice, and ameliorated weight loss associated with infection. In mice inoculated with C. rodentium, those that received chlortetracycline had less pathologic changes in the distal colon than mice not administered CTC (i.e. relative to untreated mice). Furthermore, chlortetracycline administration at a non-therapeutic dose did not impart either prominent or consistent effects on the colonic microbiota. Conclusion: Data support the hypothesis that AGP function by modulating the intestinal immune system in mammals. This finding may facilitate the development of biorationale-based and efficacious alternatives to AGP.
- ItemLongitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics(Biomed Central Ltd, 2011) Alexander, Trevor W.; Yanke, L. Jay; Reuter, Tim; Topp, Ed; Read, Ronald R.; Selinger, L. Brent; McAllister, Tim A.Background: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA. Results: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments. Conclusion: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.
- ItemEffect of antimicrobial growth promoter administration on the intestinal microbiota of beef cattle(BioMed Central, 2013) Reti, Kristen L.; Thomas, Matthew C.; Yanke, L. Jay; Selinger, L. Brent; Inglis, G. DouglasBackground: 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.
- ItemCampylobacter jejuni colonization is associated with a dysbiosis in the cecal microbiota of mice in the absence of prominent inflammation(Public Library of Science, 2013) Lone, Abdul G.; Selinger, L. Brent; Uwiera, Richard R. E.; Xu, Yong; Inglis, G. DouglasBackground: 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, tolllike 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.
- ItemStructural and biochemical analysis of a unique phosphatase from Bdellovibrio bacteriovorus reveals its structural and functional relationship with the protein tyrosine phosphatase class of phytase(Public Library of Science, 2014) Gruninger, Robert J.; Thibault, John; Capeness, Michael J.; Till, Robert; Mosimann, Steven C.; Sockett, R. Elizabeth; Selinger, L. Brent; Lovering, Andrew L.Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.
- ItemComparative variation within the genome of Campylobacter jejuni NCTC 11168 in human and murine hosts(Public LIbrary of Science, 2014) Thomas, Dallas K.; Lone, Abdul G.; Selinger, L. Brent; Taboada, Eduardo N.; Uwiera, Richard R.E.; Abbott, D. Wade; Inglis, G. DouglasCampylobacteriosis 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 .
- ItemDevelopment of a comparative genomic fingerprinting assay for rapid and high resolution genotyping of Arcobacter butzleri(BioMed Central, 2015) Webb, Andrew L.; Kruczkiewicz, Peter; Selinger, L. Brent; Inglis, G. Douglas; Taboada, Eduardo N.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.
- ItemComparative genomic analysis of Escherichia coli 0157:H7 isolated from super-shedder and low-shedder cattle(Public Library of Science, 2016) Munns, Krysty D.; Zaheer, Rahat; Xu, Yong; Stanford, Kim; Laing, Chad R.; Gannon, Victor P. J.; Selinger, L. Brent; McAllister, Tim A.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 104 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 supershedder 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-β-D-galactopyranosyl-Darabinose 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 0157:H7 reaching high densities within bovine feces.
- ItemComparative transcriptomic analysis of rectal tissue from beef steers revealed reduced host immunity in Escherichia coli 0157:H7 super-shedders(Public LIbrary of Science, 2016) Wang, Ou; Liang, Guanxiang; McAllister, Tim A.; Plastow, Graham; Stanford, Kim; Selinger, L. Brent; Guan, Le LuoSuper-shedder cattle are a major disseminator of E . coli O157:H7 into the environment, and the terminal rectum has been proposed as the primary E . coli O157:H7 colonization site. This study aimed to identify host factors that are associated with the super-shedding pro- cess by comparing transcriptomic profiles in rectal tissue collected from 5 super-shedder cattle and 4 non-shedder cattle using RNA-Seq. In total, 17,859 ± 354 genes and 399 ± 16 miRNAs were detected, and 11,773 genes were expressed in all animals. Fifty-eight differ- entially expressed (DE) genes (false discovery rate < 0.05) including 11 up-regulated and 47 down-regulated (log 2 (fold change) ranged from -5.5 to 4.2), and 2 up-regulated DE miRNAs (log 2 (fold change) = 2.1 and 2.5, respectively) were identified in super-shedders compared to non-shedders. Functional analysis of DE genes revealed that 31 down-regu- lated genes were potentially associated with reduced innate and adaptive immune functions in super-shedders, including 13 lymphocytes membrane receptors, 3 transcription factors and 5 cytokines, suggesting the decreased key host immune functions in the rectal tissue of super-shedders, including decreased quantity and migration of immune cells such as lym- phocytes, neutrophils and dendritic cells. The up-regulation of bta-miR-29d-3p and the down regulation of its predicted target gene, regulator of G-protein signaling 13 , suggested a potential regulatory role of this miRNA in decreased migration of lymphocytes in super- shedders. Based on these findings, the rectal tissue of super-shedders may inherently exhibit less effective innate and adaptive immune protection. Further study is required to confirm if such effect on host immunity is due to the nature of the host itself or due to actions mediated by E . coli O157:H7.
- ItemChanges in bacterial community composition of Escherichia coli O157:H7 super-shedder cattle occur in the lower intestine(Public Library of Science, 2017) Zaheer, Rahat; Bony-Dugat, Eric; Holman, Devon; Cousteix, Elodie; Xu, Yong; Munns, Krysty D.; Selinger, Lorna J.; Barbieri, Rutn; Alexander, Trevor W.; McAllister, Tim A.; Selinger, L. BrentEscherichia 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 supershedders.