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Background: We aimed to investigate the prevalence, molecular epidemiology and prevalence factors for Extended Spectrum β-Lactamase-producing Enterobacteriaceae (ESBL-E) shedding by race horses. A cross-sectional study was performed involving fecal samples collected from 169 Thoroughbred horses that were housed at a large racing facility in Ontario, Canada. Samples were enriched, plated on selective plates, sub-cultured to obtain pure cultures and ESBL production was confirmed. Bacterial species were identified and antibiotic susceptibility profiles were assessed. E. coli sequence types (ST) and ESBL genes were determined using multilocus sequence type (MLST) and sequencing. Whole genome sequencing was performed to isolates harboring CTX-M-1 gene. Medical records were reviewed and associations were investigated., Results: Adult horses (n = 169), originating from 16 different barns, were sampled. ESBL-E shedding rate was 12% (n = 21/169, 95% CI 8-18%); 22 ESBL-E isolates were molecularly studied (one horse had two isolates). The main species was E. coli (91%) and the major ESBL gene was CTX-M-1 (54.5%). Ten different E. coli STs were identified. Sixty-four percent of total isolates were defined as multi-drug resistant. ESBL-E shedding horses originated from 8/16 different barns; whereas 48% (10/21) of them originated from one specific barn. Overall, antibiotic treatment in the previous month was found as a prevalence factor for ESBL-E shedding (p = 0.016, prevalence OR = 27.72, 95% CI 1.845-416.555)., Conclusions: Our findings demonstrate the potential diverse reservoir of ESBL-E in Thoroughbred race horses. Multi-drug resistant bacteria should be further investigated to improve antibiotic treatment regimens and equine welfare.

Clostridium perfringens is an important cause of foal necrotizing enteritis and canine acute hemorrhagic diarrhea. A major virulence determinant of the strains associated with these diseases appears to be a beta-sheet pore-forming toxin, NetF, encoded within a pathogenicity locus (NetF locus) on a large tcp-conjugative plasmid. Strains producing NetF also produce the putative toxin NetE, encoded within the same pathogenicity locus, as well as CPE enterotoxin and CPB2 on a second plasmid, and sometimes the putative toxin NetG within a pathogenicity locus (NetG locus) on another separate large conjugative plasmid. Previous genome sequences of two netF-positive C. perfringens showed that they both shared three similar plasmids, including the NetF/NetE and CPE/CPB2 toxins-encoding plasmids mentioned above and a putative bacteriocin-encoding plasmid. The main purpose of this study was to determine whether all NetF-producing strains share this common plasmid profile and whether their distinct NetF and CPE pathogenicity loci are conserved. To answer this question, 15 equine and 15 canine netF-positive isolates of C. perfringens were sequenced using Illumina Hiseq2000 technology. In addition, the clonal relationships among the NetF-producing strains were evaluated by core genome multilocus sequence typing (cgMLST). The data obtained showed that all NetF-producing strains have a common plasmid profile and that the defined pathogenicity loci on the plasmids are conserved in all these strains. cgMLST analysis showed that the NetF-producing C. perfringens strains belong to two distinct clonal complexes. The pNetG plasmid was absent from isolates of one of the clonal complexes, and there were minor but consistent differences in the NetF/NetE and CPE/CPB2 plasmids between the two clonal complexes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Clostridium difficile is a significant pathogen with over 300 000 cases reported in North America annually. Previously, it was thought that C. difficile was primarily a clinically associated infection. However, through the use of whole genome sequencing it has been revealed that the majority of cases are community acquired. The source of community-acquired C. difficile infections (CDI) is open to debate with foodborne being one route considered. Clostridium difficile fits the criteria of a foodborne pathogen with respect to being commonly encountered in a diverse range of foods that includes meat, seafood and fresh produce. However, no foodborne illness outbreaks have been directly linked to C. difficile there is also no conclusive evidence that its spores can germinate in food matrices. This does not exclude food as a potential vehicle but it is likely that the pathogen is also acquired through zoonosis and the environment. The most significant factor that defines susceptibility to CDI is the host microbiome and functioning immune system. In this respect, effective control can be exercised by reducing the environmental burden of C. difficile along with boosting the host defences against the virulent enteric pathogen., (© 2016 The Society for Applied Microbiology.)

Aims: This study investigated the prevalence of Methicillin-resistant Staphylococcus aureus (MRSA), their spa-types, and antimicrobial resistance profiles at various steps during commercial pork production from three plants designated as A, B and C., Methods and Results: Over a period of 1 year 2640 samples from three commercial pork plants were obtained on a rotating basis. Sample sources were: nasal swabs after bleeding (NSAB), nasal swab after scalding (NSASs, plant C) or skinning (NSASk, plants A, B), carcass swabs after pasteurization (CSAP, plant C) or washing (CSAW, plants A, B) and retail pork (RP). Overall MRSA prevalence at each sampling point in the pork plants after adjusting for clustering was: 61·93, 28·38 7·58 and 1·21% for NSAB, NSASc/Sk, CSAP/CSAW and RP respectively. The majority of MRSA isolates from the three pork plants belonged to livestock-associated MRSA spa-types t034 and t011 (3·8%; ST398). The mainly human spa-type t002 (15%) was also recovered. All MRSA isolates were resistant to β-lactam and tetracycline antibiotics. Overall resistance to tigecycline was found in about 10% of MRSA isolates while <3% isolates were resistant to daptomycin, gentamicin, quinupristin/dalfopristin and trimethoprim/sulfamethoxazole., Conclusion: A higher prevalence of MRSA in the nasal cavity of incoming pigs was observed at all three plants, but a notable reduction in MRSA along the pork processing steps occurred., Significance and Impact of the Study: The highest prevalence of MRSA was found in the nasal cavity of incoming pigs in three commercial pig slaughter and pork processing plants. A reduction in MRSA prevalence occurred along the processing chain, and pork products from these plants showed significantly lower MRSA than the initial steps of slaughter and processing, suggesting a reduction in MRSA during the slaughter process with minimal cross-contamination., (© 2015 Her Majesty the Queen in Right of Canada © 2015 The Society for Applied Microbiology.)

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

Aims: To determine the persistence of Clostridium difficile spores in biosolids during composting or when amended into soil and held under natural environmental climatic conditions., Methods and Results: Five log CFU g(-1) Cl. difficile spores (ribotypes 027 or 078) were inoculated into agricultural soils (sandy loam or loam) amended with 10% w/w anaerobically digested biosolids. The inoculated soil : biosolids mixture was then placed into sentinel vials which were introduced at a depth of 15 cm within the field plot consisting of the corresponding soil type. Two trials were performed, the first of which started in late spring (May 2013 through to August 2014) and second from November 2013 through to October 2014 (fall trial). Ribotype 078 endospores in loam or sandy loam soil decreased during the summer but then increased in numbers towards the fall. At the end of the trial, levels of ribotype 078 spores had decreased by 1·5 log CFU g(-1) , with 027 spores decreasing by <1 log CFU g(-1) over the same time period. Windrow composting of biosolids decreased Cl. difficile levels from 3·7 log CFU g(-1) down to 0·3 log CFU g(-1) with the greater reduction occurring during the curing phase. In comparison, Cl. perfringens decreased from 6·3 log CFU g(-1) down to 2·4 log CFU g(-1) but mainly in the thermal phase of the composting process., Conclusions: Composting of biosolids is a more effective means of inactivating Cl. difficile compared to land application., Significance and Impact of the Study: Windrow composting represents an effective method to reduce the environmental burden of Cl. difficile associated with biosolids., (© 2015 The Society for Applied Microbiology.)

The study described in this article aimed at establishing a baseline assessment of the sanitary status of ice and guest rooms within Canadian hotels. Collectively, 54 hotel rooms belonging to six different national chains were sampled. High-contact surfaces (comforter, alarm clock, bedside lamp, TV remote, bathroom countertop, faucet, and toilet seat) were sampled using adenosine triphosphate (ATP) swabs and replicate organism detection and counting plates. ATP swab readings ranged from 2.12 to 4.42 log relative light units. Coliforms were recovered from 36% of surfaces with high prevalence being recovered from the comforter, TV remote, bathroom countertop, faucet, and toilet seat. Oxacillin-resistant bacteria were recovered from 19% of surfaces with 46% of isolates confirmed as methicillin-resistant Staphylococcus aureus. Two toxigenic Clostridium difficile isolates were recovered in the course of the study. Collectively, 24% of the ice samples harbored coliforms with a single sample testing positive for E. coli. The authors' study demonstrates that hotel rooms represent a potential source of community-acquired infections and the need for enhanced sanitation practices.

The oral and conjunctival microbiotas likely play important roles in protection from opportunistic infections, while also being the source of potential pathogens. Yet, there has been limited investigation in cats, and the impact of comorbidities such as feline immunodeficiency virus (FIV) infection has not been reported. Oral and conjunctival swabs were collected from cats with FIV infection and FIV-uninfected controls, and subjected to 16S rRNA gene (V4) PCR and next generation sequencing. 9,249 OTUs were identified from conjunctival swabs, yet the most common 20 (0.22%) OTUs accounted for 76% of sequences. The two most abundant OTUs both belonged to Staphylococcus, and accounted for 37% of sequences. Cats with FIV infection had significantly lower relative abundances of Verrucomicrobia, Fibrobacteres, Spirochaetes, Bacteroidetes and Tenericutes, and a higher relative abundance of Deinococcus-Thermus. There were significant differences in both community membership (P = 0.006) and community structure (P = 0.02) between FIV-infected and FIV-uninfected cats. FIV-infected cats had significantly higher relative abundances of Fusobacteria and Actinobacteria in the oral cavity, and significantly higher relative abundances of several bacterial classes including Fusobacteria (0.022 vs 0.007, P = 0.006), Actinobacteria (0.017 vs 0.003, P = 0.003), Sphingobacteria (0.00015 vs 0.00003, P = 0.0013) and Flavobacteria (0.0073 vs 0.0034, P = 0.030). The feline conjunctival and oral microbiotas are complex polymicrobial communities but dominated by a limited number of genera. There is an apparent impact of FIV infection on various components of the microbiota, and assessment of the clinical relevance of these alterations in required.

Background: Quantitative PCR is rapidly becoming the standard method for analyzing gene expression in a wide variety of biological samples however it can suffer from significant error if stably expressed reference genes are not identified on which to base the analysis. Suitable reference genes for qPCR experiments on Staphylococcus pseudintermedius have yet to be identified., Results: Three reference genes in S. pseudintermedius were identified and validated from a set of eight potential genes (proC, gyrB, rplD, rho, rpoA, ftsZ, recA, sodA). Two strains of S. pseudintermedius were used, and primer specificity and efficiency were confirmed and measured. Ranking of the genes with respect to expression stability revealed gyrB, rho and recA as the best reference genes. This combination was used to quantify expression of a single biofilm associated gene, icaA, in logarithmic, stationary and biofilm growth phases, revealing that expression was significantly upregulated in the biofilm growth phase in both strains., Conclusion: Three reference genes, gyrB, rho and recA, were identified and validated for use as reference genes for quantitative PCR experiments in S. pseudintermedius. Also, the biofilm associated gene icaA was shown to be significantly upregulated in biofilm samples, consistent with its role in biofilm production.

Background: Fecal bacteriotherapy ('stool transplant') can be effective in treating recurrent Clostridium difficile infection, but concerns of donor infection transmission and patient acceptance limit its use. Here we describe the use of a stool substitute preparation, made from purified intestinal bacterial cultures derived from a single healthy donor, to treat recurrent C. difficile infection that had failed repeated standard antibiotics. Thirty-three isolates were recovered from a healthy donor stool sample. Two patients who had failed at least three courses of metronidazole or vancomycin underwent colonoscopy and the mixture was infused throughout the right and mid colon. Pre-treatment and post-treatment stool samples were analyzed by 16 S rRNA gene sequencing using the Ion Torrent platform., Results: Both patients were infected with the hyper virulent C. difficile strain, ribotype 078. Following stool substitute treatment, each patient reverted to their normal bowel pattern within 2 to 3 days and remained symptom-free at 6 months. The analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples but constituted over 25% of the sequences up to 6 months after treatment., Conclusion: This proof-of-principle study demonstrates that a stool substitute mixture comprising a multi-species community of bacteria is capable of curing antibiotic-resistant C. difficile colitis. This benefit correlates with major changes in stool microbial profile and these changes reflect isolates from the synthetic mixture., Clinical Trial Registration Number: CinicalTrials.gov NCT01372943.

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.

Treatment of galactosemia requires a galactose-restricted diet. Although meats are not traditionally thought of as a dietary carbohydrate source, small amounts may be present in free form and/or bound to proteins or lipids. The purpose of this study was to determine the free and bound galactose contents of baby food meats. Galactose was assayed using high-performance liquid chromatography. The free galactose content of baby food meats ranged from 0 to 0.031 mg/100 g. No statistically significant differences in free galactose content were found among the meats. Bound galactose was found in all analyzed baby food meats, ranging from 0.065 to 0.148 mg/100 g. The mean galactose content of BeechNut chicken (St. Louis, MO) was significantly less than that found in Gerber (Fremont, MI) and Heinz (Pittsburgh, PA) brands of chicken, beef, and turkey, and Gerber lamb and veal. Based on current recommendations, all examined baby food meats would be acceptable for infants with galactosemia.

Objective: To determine the effect of differences in plasma phenylalanine (Phe) concentrations (< 363 umol/L, 363 to 605 umol/L, and > 605 umol/L) on hematological and immunological parameters in 22 children with phenylketonuria (PKU)., Methods: Children with PKU were divided into one of three groups based on fasting plasma Phe levels. Hematologic and immunologic parameters of the children with PKU were compared between the groups and also compared with published values from age-matched children without PKU., Results: Hematologic and immunologic parameters did not differ among children with different plasma Phe concentrations. Specifically, no significant differences between groups of PKU children with differing plasma Phe levels were found for plasma levels of albumin, hemoglobin, amino acids, IgM, complement C3, interleukins 1 and 2, erythrocyte, leukocyte and differential cell counts, hematocrit, percentages and numbers of CD4+, CD8+, CD3+ and total lymphocytes, or CD4 to CD8 ratio. Mean plasma IgG and IgA concentrations of the PKU children were, however, significantly lower than values from similar aged children. Moreover, positive correlations were obtained between plasma albumin and percentages and numbers of CD3+ and CD4+, between plasma IgG and interleukins 1 and 2, and between intakes of energy, protein, iron and plasma IgG levels. No correlations were found between plasma Phe and immunological parameters., Conclusion: While differences in plasma Phe concentrations up to concentrations of 866 umol/L do not appear to affect selected immune system parameters, further studies are needed to investigate the relationship between dietary nutrient intake, nutritional status, antibody biosynthesis and cytokine production. Assessment of plasma and cell membrane lipids and trace mineral status of PKU children would be helpful to determine if relationships exist between these nutrients and antibody production.