Longitudinal study of the cutaneous microbiota of healthy horses.

BACKGROUND: Next-generation sequencing techniques have revealed that human and animal skin is colonised by a rich and diverse population of bacteria, and that microbial composition varies between different body sites and individuals. Very little is known about the normal microbiota of healthy equine skin. HYPOTHESIS/OBJECTIVES: To describe the taxonomic distributions of cutaneous bacterial microbiota in a population of healthy horses in Ontario, Canada, and to evaluate the effects of body site, individual and time of year on microbial diversity and community composition. ANIMALS: Samples were collected from four body sites (dorsum, ventral abdomen, pastern and groin) from 12 clinically healthy horses from the same farm. Samples were collected from all individuals at four time points (winter, spring, summer, autumn) within a calendar year. METHODS AND MATERIALS: Illumina sequencing of the V4 region of the 16S rRNA gene was performed following DNA extraction. Data were analysed using mothur software. RESULTS: Bacteria from 38 phyla and 1,665 genera were identified. Alpha diversity was higher in the winter and summer than spring and autumn although this was not statistically significant. Community membership and structure clustered more based on season than skin site. CONCLUSIONS AND CLINICAL IMPORTANCE: Healthy equine skin is inhabited by a marked diversity of microbiota. Individuals living in a similar environment share overlapping cutaneous microbial populations. These populations vary significantly over time and between body sites.

Feline conjunctival microbiota in a shelter: effects of time, upper respiratory disease and famciclovir administration.

OBJECTIVES: The aim of this study was to evaluate changes in the conjunctival microbiota of shelter-housed cats with time, upper respiratory disease (URD) and famciclovir administration. METHODS: Cats were assigned to treatment groups on shelter entry. Healthy cats or cats with URD received ~30 mg/kg or ~90 mg/kg of famciclovir or placebo PO q12h for 7 days, or were untreated. Swabs were collected from ventral conjunctival fornices prior to (day 1) and immediately after (day 8) the treatment period. Microbiota analysis was conducted on 124 randomly selected swabs from healthy (56 swabs) or URD-affected (68 swabs) cats. Following DNA extraction and amplification of the V4 region of the 16S rRNA gene, sequences were assembled into operational taxonomic units (OTUs). Over-represented OTUs (as determined by linear discriminate analysis effect size), alpha and beta diversity, and median relative abundance of known feline ocular surface pathogens were assessed for the entire population and in 10 clinically relevant subpopulations of cats. RESULTS: Bacteria from 33 phyla and 70 genera were identified. Considering all cats, median relative abundance of Mycoplasma increased from day 1 to day 8, while Proteobacteria decreased. Community membership and structure (beta diversity) differed between days 1 and 8 for all famciclovir-treated cats (regardless of health status or dose) and healthy or URD-affected cats (regardless of famciclovir dose). Differences in taxonomic diversity within a sample (alpha diversity) between day 1 and day 8 were not detected in any subpopulations. CONCLUSIONS AND RELEVANCE: Within 1 week of shelter entry, there were significant changes in community structure and membership of the feline conjunctival microbiota, with a shift towards over-representation of feline ocular surface pathogens. Although famciclovir may impact beta diversity of the feline conjunctival microbiota, absence of change in alpha diversity suggests minimal shift in individual cats.

Livestock-associated methicillin-resistant Staphylococcus aureus and Clostridium difficile in wild Norway rats (Rattus norvegicus) from Ontario swine farms.

Methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are important human pathogens that are also carried by animals. The role of wild mammals on farms in their maintenance and transmission, however, is poorly understood. To determine if Norway rats (Rattus norvegicus) are potential carriers of these bacteria on Canadian farms, we tested 21 rats from swine farms in Ontario. The MRSA spa type t034 was isolated from 1 (4.8%) rat. This livestock-associated strain often colonizes pigs and pig farmers, suggesting that transmission among rats and pigs or environmental transmission is possible on pig farms. Clostridium difficile ribotype 078 was isolated from 1 rat from a different farm. This strain is associated with infection in piglets, calves, and humans. The identification of MRSA and C. difficile in Norway rats on farms in Canada adds to the growing knowledge about the role of rats in the ecology of these pathogens. Further studies are required to determine if rats play a part in the epidemiology of these pathogens on farms.

Les bactéries Staphylococcus aureus résistants à la méthicilline (SARM) et Clostridium difficile sont des agents pathogènes importants chez l'humain et sont également retrouvées chez des animaux. Le rôle des animaux sauvages sur les fermes en lien avec leur maintenance et transmission est toutefois peu compris. Afin de déterminer si les rats (Rattus norvegicus) sont des porteurs potentiels de ces bactéries sur les fermes canadiennes, nous avons testé 21 rats provenant de fermes porcines en Ontario. Du SARM spa type t034 fut isolé à partir de un rat (4,8 %). Cette souche associée au bétail colonise souvent les porcs et les éleveurs de porcs, suggérant ainsi que la transmission entre les rats et les rats ou la transmission environnementale est possible sur les fermes porcines. Le ribotype 078 de Clostridium difficile fut isolé de un rat sur une ferme différente. Cette souche est associée à l'infection chez des porcelets, des veaux, et les humains. L'identification de SARM et de C. difficile chez des rats sur des fermes au Canada accroit les connaissances sur le rôle des rats dans l'écologie de ces agents pathogènes. Des études supplémentaires sont requises afin de déterminer si les rats jouent un rôle dans l'épidémiologie de ces agents pathogènes sur les fermes.(Traduit par Docteur Serge Messier).

Pilot study to evaluate 3 hygiene protocols on the reduction of bacterial load on the hands of veterinary staff performing routine equine physical examinations.

Reduction factors (RFs) for bacterial counts on examiners' hands were compared when performing a standardized equine physical examination, followed by the use of one of 3 hand-hygiene protocols (washing with soap, ethanol gel application, and chlorohexidine-ethanol application). The mean RFs were 1.29 log10 and 1.44 log10 at 2 study sites for the alcohol-gel (62% ethyl alcohol active ingredient) protocols and 1.47 log10 and 1.94 log10 at 2 study sites for the chlorhexidine-alcohol (61% ethyl alcohol plus 1% chlorhexidine active ingredients) protocols, respectively. The RFs were significantly different (P < 0.0001) between the hand-washing group and the other 2 treatment groups (the alcohol-gel and the chlorhexidine-alcohol lotion). The use of alcohol-based gels or chlorhexidine-alcohol hand hygiene protocols must still be proven effective in equine practice settings, but in this study, these protocols were equivalent or superior to hand washing for reduction in bacterial load on the hands of people after they perform routine physical examinations.

Suspected Clostridium difficile-associated hemorrhagic diarrhea in a 1-week-old elk calf.

Clostridium difficile-associated diarrhea was suspected in a 1-week-old elk (Cervus elaphus) calf. The isolation of a toxigenic strain of C. difficile from a diarrheic fecal sample, along with exclusion of other enteropathogens, formed the basis of this presumptive diagnosis. Further study is indicated to evaluate the role of C. difficile in neonatal diarrhea in elk.

Cellulosic fuel ethanol: alternative fermentation process designs with wild-type and recombinant Zymomonas mobilis.

Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The "Iogen Process" for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.

Steady-state measurements of lactic acid production in a wild-type and a putative D-lactic acid dehydrogenase-negative mutant of Zymomonas mobilis: influence of glycolytic flux.

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of D- and L-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of DL-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, D-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. D-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose.

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory's metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN's feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN's biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L x h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L x h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.