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Background The heightened prevalence of Salmonella Typhimurium remains a public health and food safety concern. Studies have reported antibiotic, flavophospholipol, may have the ability to reduce Salmonella in swine, as well as alter the gut microbiota in favour of beneficial bacteria by inhibiting pathogenic bacteria. Thus, the objective of this study was to investigate the fecal microbiota of weaned pigs receiving in-feed flavophospholipol and challenged with Salmonella Typhimurium. Results Twenty-one weaned pigs were fed either a diet containing 4 ppm of flavophospholipol (treatment group) or a non-medicated feed (control group) for 36 days post-weaning (Day 1 to Day 36). The pigs were orally challenged with a 2 mL dose of 108 CFU/mL of S. Typhimurium at Day 7 and Day 8. Community bacterial DNA extracted from fecal samples collected at Day 6 (before challenge) and Day 36 (28 days after challenge) were used to assess the fecal microbiota using the V4 region of the 16S rRNA gene with Illumina MiSeq next-generation sequencing. Sequencing data were visualized using mothur and analyzed in JMP and R software. The fecal microbiota of pigs in the treatment group had differences in abundance of phyla (Firmicutes, Proteobacteria) and genera (Lactobacillus, Roseburia, Treponema, unclassified Ruminococcaceae, Blautia, Streptococcus, Megasphaera, Dorea, Sporobacter, Peptococcus, unclassified Firmicutes, Clostridium IV and Campylobacter) when compared to pigs that were controls, 28 days after challenge with Salmonella (P P = 0.001) and decrease in Firmicutes (P = 0.012) and genus Roseburia (P = 0.003) in the treated pigs suggestive of possible microbial dysbiosis. An increased abundance of genera Lactobacillus (P = 0.012) was also noted in the treated group in comparison to the control. Conclusion Based on these findings, it is difficult to conclude whether treatment with 4 ppm of flavophospholipol is promoting favorable indigenous bacteria in the pig microbiota as previous literature has suggested.

Objective To evaluate the presence of quaternary ammonium compound (QAC) (resistance genes, qac A/B, smr, qacG, and qacJ, in clinical isolates of methicillin-susceptible Staphylococcus pseudintermedius (MSSP) and methicillin-resistant S pseudintermedius (MRSP) from dogs and the impact on in vitro chlorhexidine susceptibility. Study design Experimental in vitro study. Sample population Seventy isolates from dogs colonized or infected with MRSP (n = 50) or MSSP (n = 20). Methods Agar dilution was used to determine the minimum inhibitory concentration (MIC) of chlorhexidine digluconate. Real-time polymerase chain reaction was used to detect the presence of QAC resistance genes, qacA/B, smr, qacG, and qacJ genes. Results One or more qac genes were identified in 52 of 70 (74%) isolates. Overall, there was no association between chlorhexidine MIC and the presence of one or more qac genes (P = .85) or the presence of qacA/B (P = .31), smr (P = .72) or qacJ (P = .93) individually. There was an association between qacG and MIC (P = .012), with a median MIC of 1.5 μg/mL for isolates possessing this gene and 1 μg/mL for those not possessing it. Conclusion Quaternary ammonium compound resistance genes were present in MRSP and MSSP isolates. With the exception of qacG, the presence of these genes was not associated with increased MIC. All isolates exhibited MIC 5000 to 80 000 times lower than the concentration recommended for use. Clinical significance Despite the presence of QAC genes, chlorhexidine digluconate should be effective against MRSP and MSSP if used correctly.

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal’s improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model., Author summary Simian Immunodeficiency virus in macaques remains the most translational model for HIV. Macaques are also key models for other infectious diseases and colitis, where background colon health and inflammation could confound experimental results. To address these issues SPF breeding colonies were established to exclude specific viruses detrimental to mucosal and systemic health. To further improve macaque models, we established a gastrointestinal pathogen free (GPF) colony by administration of a multimodal therapeutic regimen for common endemic pathogens (EPs) and established expanded operational practices for continued exclusion. Through extensive longitudinal microbiota, parasitology, microbiology, and tissue sampling we demonstrated that our treatment and exclusion practices successfully eliminated common EPs, improved mucosal barriers, and reduced mucosal and systemic inflammation resulting in less inter-animal variation without overt evidence of microbiota disruption. Finally, compared to treatment-naïve controls, GPF animals challenged with SIV intrarectally demonstrated a more controlled and consistent rate of SIV acquisition, further indicating underlying EPs even at subclinical levels may cause deleterious variations between study subjects. Collectively the GPF macaque represents a model that eliminates a significant source of morbidity, reduces inter-animal variability, and thus has the potential to improve both animal welfare and research outcomes.

Bats are associated with the emergence of several mammalian diseases. Their sessional migration, and tendency to form large colonies in close proximity to human habitats enables effective intra- and inter-species transmission of pathogens. Clostridioides difficile is an important enteric pathogen in humans and animals; however, the source of its dissemination in the population is unknown. The purpose of this study was to determine the prevalence of C. difficile in bats, and to characterize C. difficile isolates. Feces (n = 93) was sampled from bats during their migration across Europe. Eighteen samples (19.4%) were positive for C. difficile; ribotypes 078, 056, and a new ribotype CDB3 were identified. Clostridioides difficile ribotypes 078 and 056 are associated with human and animal diseases. The C. difficile prevalence and ribotypes in this study do not necessarily identify bats as a significant source, but more likely as an indicator of C. difficile perpetuation in the environment.

Clostridium (Clostridioides) difficile has been identified in humans and a wide range of animal species, but there has been little study of remote animal populations with limited human contact. The objective of this study was to determine the prevalence and types of C. difficile in wild and captive polar bears (Ursus maritimus). Fecal samples were collected from two populations of wild polar bears in Nunavut Canada; M’Clintock Channel and Hudson Strait (Davis Strait or Foxe Basin), as well as from a facility (PBJ) in Churchill, Manitoba that temporarily houses nuisance polar bears and from captive bears in a zoological park. Enrichment culture was performed and isolates were characterized by ribotyping and toxinotyping. Clostridium difficile was isolated from 24/143 (16.8%) of samples; 18/120 (15%) wild bear samples, 4/7 (57%) from the PBJ and 2/16 (13%) samples from three zoo bears. The prevalence of C. difficile was significantly higher in bears that were housed at the PBJ vs wild bears (P = 0.0042), but there was no difference between wild bears from M’Clintock Channel (14/100, 14%) and those from Hudson Strait (4/20, 20%) (P = 0.50). Fourteen of the 24 (58%) isolates were toxigenic; 13/18 (72%) wild bear isolates, 0/4 PBJ isolate and 1/2 zoo isolates. Four toxigenic ribotypes were identified, with one that possessed tcdB and cdtA predominating. None of the toxigenic isolates were ribotypes that have been identified previously by the authors. There was no overlap in toxigenic ribotypes between the different populations. Clostridium difficile was not uncommonly identified in polar bears, with differences in type distribution amongst the different regions. The presence of strains that have not been identified in humans or domestic animals suggests that polar bears may be a natural reservoir of unique strains of this important bacterium.

Historically, extended-spectrum cephalosporin resistance in bacteria from animals in Canada has been attributed to the SHV and CMY β-lactamase families. This pattern is beginning to change with the emergence of the blaCTX-M gene family among Escherichia coli recovered from various animal species. Here we analyze and compare whole genome sequences of blaCTX-M-positive E. coli isolates (n = 173) from dogs, chicken, swine, horses and beef cattle in Canada. Ten blaCTX-M variants were identified with blaCTX-M-1,-14, -15, -27 and blaCTX-M-55 being identified in most animal species. These variants occurred across many sequence types, suggesting that mobile genetic elements mediate the spread of blaCTX-M. The variants blaCTX-M-14, -15, -27 and blaCTX-M-55 are associated with the global spread of blaCTX-M in human clinical isolates and their presence could be indicative of transfer between humans and animals. These variants were also the principal variants identified among sequence type 131 isolates, which were not associated with any other species than dogs. These isolates carried the same blaCTX-M variants as E. coli isolates found in humans. Close contact may promote the transmission of these isolates between humans and companion animals.

The goal of this study was to determine the frequency of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and other Enterobacterales from turkeys in Canada and characterize the associated resistance determinants. Pooled fecal samples were collected in 77 turkey farms across British Columbia, Québec, and Ontario. Isolates were obtained with and without selective enrichment cultures and compared to isolates from diagnostic submissions of suspected colibacillosis cases in Ontario. Isolates were identified using MALDI-TOF and susceptibility to ESCs was assessed by disk diffusion. The presence of blaCMY, blaCTX-M, blaTEM, and blaSHV was tested by PCR. Transformation experiments were used to characterize blaCMY plasmids. Genome sequencing with short and long reads was performed on a representative sample of blaCTX-M-positive isolates to assess isolates relatedness and characterize blaCTX-M plasmids. For the positive enrichment cultures (67% of total samples), 93% (587/610) were identified as E. coli, with only a few other Enterobacterales species identified. The frequency of ESC resistance was low in E. coli isolates from diagnostic submission (4%) and fecal samples without selective enrichment (5%). Of the ESC-resistant Enterobacterales isolates from selective enrichments, 71%, 18%, 14%, and 8% were positive for blaCMY, blaTEM, blaCTX-M, and blaSHV, respectively. IncI1 followed by IncK were the main incompatibility groups identified for blaCMY plasmids. The blaCTX-M-1 gene was found repeatedly on IncI1 plasmids of the pMLST type 3, while blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were associated with a variety of IncF plasmids. Clonal spread of strains carrying blaCTX-M genes between turkey farms was observed, as well as the presence of an epidemic blaCTX-M-1 plasmid in unrelated E. coli strains. In conclusion, Enterobacterales resistant to ESCs were still widespread at low concentration in turkey feces two years after the cessation of ceftiofur use. Although blaCMY-2 is the main ESC resistance determinant in E. coli from Canadian turkeys, blaCTX-M genes also occur which are often carried by multidrug resistance plasmids. Both clonal spread and horizontal gene transfer are involved in parallel in the spread of blaCTX-M genes in Enterobacterales from Canadian turkeys.

Significance Skin forms a critical protective barrier between a mammal and its external environment. Baseline data on the mammalian skin microbiome elucidates which microorganisms are found on healthy skin and provides insight into mammalian evolutionary history. To our knowledge, this study represents the largest existing mammalian skin microbiome survey. Our findings demonstrate that human skin is distinct, not only from other Primates, but from all 10 mammalian orders sampled. Identifying significant similarities between branching of mammalian phylogenetic trees and relatedness trees for their corresponding microbial communities raises the possibility that mammals have experienced coevolution between skin microbiota and their corresponding host species., Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment, yet the factors that govern skin microbial community composition among mammals are poorly understood. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. Skin from the back, torso, and inner thighs of 177 nonhuman mammals was sampled, representing individuals from 38 species and 10 mammalian orders. Animals were sampled from farms, zoos, households, and the wild. The DNA extracts from all skin swabs were amplified by PCR and sequenced, targeting the V3-V4 regions of bacterial and archaeal 16S rRNA genes. Previously published skin microbiome data from 20 human participants, sampled and sequenced using an identical protocol to the nonhuman mammals, were included to make this a comprehensive analysis. Human skin microbial communities were distinct and significantly less diverse than all other sampled mammalian orders. The factor most strongly associated with microbial community data for all samples was whether the host was a human. Within nonhuman samples, host taxonomic order was the most significant factor influencing skin microbiota, followed by the geographic location of the habitat. By comparing the congruence between host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing evidence of phylosymbiosis between skin microbial communities and their hosts.

Several factors are known to affect the intestinal microbiota of cattle. However, how these changes occur over time is poorly understood. This study aimed to investigate the consequences of entrance into a feedlot and the effects of virginiamycin used as a growth promoter on the bovine fecal microbiota. Two batches of beef cattle (B1, n = 50 and B2, n = 36) entering a feedlot operation were randomly divided into two pens: one receiving virginiamycin and one group not receiving antibiotic (control group). Fecal samples were collected at arrival, mid feedlot and at exit to slaughter. The V4 region of 16S rRNA gene was amplified and sequenced. Escherichia coli strains isolated in samples from arrival and exit of B2 were also isolated and used as indicators of antimicrobial susceptibility. Marked changes in membership and structure of fecal microbiota occurred following entrance into the feedlot. At mid feedlot, virginiamycin affected bacterial community membership, but not structure, suggesting that the antibiotic had a stronger effect on the rare, but not on the most abundant species. The use of virginiamycin had no demonstrable effect on antibiotic resistance in E. coli. The differences found between batches are suggestive that variations in study conditions are important and can strongly affect the intestinal microbiota.

Many cases of Clostridioides difficile infection (CDI) are poorly responsive to standard antibiotic treatment strategies, and often patients suffer from recurrent infections characterized by severe diarrhea. Our group previously reported the successful cure of two patients with recurrent CDI using a standardized stool-derived microbial ecosystem therapeutic (MET-1). Using an in vitro model of the distal gut to support bacterial communities, we characterized the metabolite profiles of two defined microbial ecosystems derived from healthy donor stool (DEC58, and a subset community, MET-1), as well as an ecosystem representative of a dysbiotic state (ciprofloxacin-treated DEC58). The growth and virulence determinants of two C. difficile strains were then assessed in response to components derived from the ecosystems. CD186 (ribotype 027) and CD973 (ribotype 078) growth was decreased upon treatment with DEC58 metabolites compared to ciprofloxacin-treated DEC58 metabolites. Furthermore, CD186 TcdA and TcdB secretion was increased following treatment with ciprofloxacin-treated DEC58 spent medium compared to DEC58 spent medium alone. The net metabolic output of C. difficile was also modulated in response to spent media from defined microbial ecosystems, although several metabolite levels were divergent across the two strains examined. Further investigation of these antagonistic properties will guide the development of microbiota-based therapeutics for CDI.

Objective To compare the minimum inhibitory concentration (MIC) of four antimicrobials in planktonic vs. biofilm-associated Staphylococcus pseudintermedius. Study Design In vitro study. Sample Population 78 isolates from dogs colonized or infected with methicillin-resistant S. pseudintermedius (MRSP, n=39) or methicillin-susceptible S. pseudintermedius (MSSP, n=39). Methods Agar dilution was used to determine the MIC of amikacin, cefazolin, enrofloxacin, and gentamicin for planktonic bacteria. A modified broth microdilution assay was used to assess the MIC of biofilm-associated bacteria. Results MIC were significantly higher in biofilm-associated vs. planktonic bacteria for all antimicrobials; amikacin (median MIC: biofilm >2,000 μg/mL vs. planktonic 3 μg/mL, P 1,000 vs. 0.5 μg/mL, P 1,000 vs. 0.25 μg/mL, P 1,000 vs. 0.3 μg/mL, P

Objective To evaluate the expression of biofilm-associated genes in Staphylococcus pseudintermedius on multiple clinically relevant surfaces. Study Design In vitro experimental study. Sample Population Two strains of methicillin-resistant S. pseudintermedius isolated from clinical infections representing the most common international isolates. Methods A quantitative polymerase chain reaction (qPCR) assay for expression of genes related to biofilm initial adhesion, formation/maturation, antimicrobial resistance, and intracellular communication was developed and validated. S. pseudintermedius biofilms were grown on 8 clinically relevant surfaces (polymethylmethacrylate, stainless steel, titanium, latex, silicone, polydioxanone, polystyrene, and glass) and samples of logarithmic and stationary growth phases were collected. Gene expression in samples was measured by qPCR. Results Significant differences in gene expression were identified between surfaces and between bacterial strains for most gene/strain/surface combinations studied. Expression of genes responsible for production of extracellular matrix were increased in biofilms. Expression of genes responsible for initial adhesion and intracellular communication was markedly variable. Antimicrobial resistance gene expression was increased on multiple surfaces, including stainless steel and titanium. Conclusion A method for evaluation of expression of multiple biofilm-associated genes in S. pseudintermedius was successfully developed and applied to the study of biofilms on multiple surfaces. Variations in expression of these genes have a bearing on understanding the development and treatment of implant-associated biofilm infections and will inform future clinical research.

Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-β and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. Reducing Campylobacter numbers in the intestinal tract of chickens will minimize transmission to humans, thereby reducing the incidence of infection. We have previously shown that oral pre-treatment of chickens with C. jejuni lysate and Poly D, L-lactide-co-glycolide polymer nanoparticles (PLGA NPs) containing CpG oligodeoxynucleotide (ODN) can reduce the number of C. jejuni in infected chickens. In the current study, the effects of these pre-treatments on the composition and functional diversity of the cecal microbiota, in chickens experimentally infected with C. jejuni, were investigated using next-generation sequencing. The taxonomic composition analysis revealed a reduction in cecal microbial diversity and considerable changes in the taxonomic profiles of the microbial communities of C. jejuni-challenged chickens. On the other hand, irrespective of the dose, the microbiota of PLGA-encapsulated CpG ODN- and C. jejuni lysate-treated chickens exhibited higher microbial diversity associated with high abundance of members of Firmicutes and Bacteroidetes and lower numbers of Campylobacter than untreated-chickens. These findings suggest that oral administration of encapsulated CpG ODN and C. jejuni lysate can reduce colonization by C. jejuni by enhancing the proliferation of specific microbial groups. The mechanisms that mediate these changes remain, however, to be elucidated.

The lone star tick, Amblyomma americanum, is spreading northward from its historical stronghold in the southeastern United States. As a vector and biting pest, public and veterinary health officials must remain vigilant of the lone star tick's expanding range. We use ticks submitted to Public Health Ontario Laboratory (1999-2016) to describe the spatial and temporal dynamics of A. americanum in Ontario, Canada, as well as submitter demographics. We identified 847 A. americanum submissions during the surveillance period, with 773 (91.3%) non-travel-related and 74 (8.7%) travel-related submissions. Annual A. americanum submissions increased over the surveillance period. Approximately 91% of non-travel-related submissions were adult ticks and 9% were nymphs. The highest submission rates were from individuals living in the Eastern and South West regions of the province. Adult specimens were primarily submitted from May through July and nymphs from March through September. Higher numbers of submissions were from young children (

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile. We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile.

Objective—To evaluate the efficacy of various endotracheal tube disinfection strategies for elimination of Streptococcus zooepidemicus and Bordetella bronchiseptica. Design—Experimental in vitro study. Sample—12 sterile endotracheal tubes. Procedures—Endotracheal tubes were inoculated with S zooepidemicus or B bronchiseptica and subjected to 1 of 5 treatments (spraying with accelerated hydrogen peroxide solution [AHP] or soaking in one of the following: AHP, 0.5% chlorhexidine gluconate solution [CHG], 0.3% triclosan-containing soap solution, or tap water) or left untreated (controls). After 5 minutes, tubes were rinsed with water and swabbed for direct and enrichment culture. Culture results were scored semiquantitatively. Each isolate was tested separately (10 endotracheal tubes/isolate/treatment). Results—No growth was identified by direct culture of any samples collected from CHG-treated endotracheal tubes, whereas S zooepidemicus and B bronchiseptica were each identified from 1 of 10 tubes sprayed or soaked with AHP and from all tubes (10/10 each) treated by other methods or used as controls. The CHG and AHP treatments resulted in significantly lower median growth scores after direct culture than did other treatments. After enrichment culture, samples from CHG-treated tubes had significantly lower growth scores than samples from AHP-treated tubes, which had significantly lower scores than samples from other treatment groups. Conclusions and Clinical Relevance—High-level disinfection (ie, elimination of all vegetative bacterial growth) was not achieved with any treatment tested. Although optimal approaches are not known, processing of endotracheal tubes with CHG or AHP appears to be the best approach when sterilization is not feasible.

Case Description—A 9-year-old castrated male domestic shorthair cat was evaluated because of hematuria and weight loss after an 8-year history of intermittent signs of feline lower urinary tract disease (FLUTD). A complete diet history revealed that the cat was eating a commercial diet that does not undergo the same processing procedures as most pet foods and so might be at increased risk for bacterial contamination owing to a nonstandard industry cooking procedure. Clinical Findings—The cat had a history consistent with FLUTD, but bacteriologic culture of the urine revealed Salmonella organisms. Additional analysis revealed Salmonella enterica serotype I:ROUGH-O:g,m,s:- in samples of urine and feces as well as Salmonella enterica serotype Johannesburg and Salmonella enterica serotype Senftenberg in the diet. Treatment and Outcome—The cat responded positively to antimicrobial treatment for the Salmonella bacteriuria as well as to dietary and environmental management for the clinical signs associated with FLUTD. Clinical Relevance—Findings in this case highlighted an additional health consequence associated with ingestion of Salmonella-contaminated food. Such contamination is of particular concern with raw meat–based diets or diets that have not undergone standard industry cooking practices. Veterinarians should obtain a diet history for every companion animal during every evaluation to help with diagnosis and optimal treatment. (J Am Vet Med Assoc 2015;247:525–530)

OBJECTIVE To evaluate the impact of gentamicin, silver, or both additives in polymethylmethacrylate (PMMA) beads on methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilm formation in vitro. SAMPLE 4 preparations of PMMA beads (formed with no additive [control], gentamicin, silver, and gentamicin and silver). PROCEDURES Beads from each group were exposed to 10 MRSP isolates known to be strong biofilm formers. Following incubation, the beads were rinsed to remove planktonic bacteria, then sonicated to dislodge biofilm-associated bacteria. Resulting suspensions were serially diluted, plated on blood agar, and incubated overnight; CFUs were counted. Variance of mean CFU counts following log10 transformation was analyzed among PMMA groups. RESULTS None of the PMMA additives tested completely inhibited MRSP biofilm formation. There was a significant effect of gentamicin and gentamicin plus silver on this variable, compared with controls, but not of silver alone. There was no difference between gentamicin and gentamicin plus silver. When only isolates not susceptible to gentamicin were evaluated, there were no significant differences among PMMA additive groups. Within gentamicin-susceptible isolates, there was an impact of gentamicin and gentamicin plus silver, but no impact of silver alone and no difference between gentamicin and gentamicin plus silver. CONCLUSIONS AND CLINICAL RELEVANCE Gentamicin-impregnated PMMA was effective at reducing biofilm formation of gentamicin-susceptible MRSP isolates but had no effect on isolates not susceptible to gentamicin. Silver-impregnated PMMA had no effect on MRSP biofilm formation. Results suggested that gentamicin-impregnated PMMA may not be effective in vivo against MRSP isolates not susceptible to gentamicin. Antibacterial efficacy of silver should not be assumed without proper testing of the target bacteria and specific silver compound.

Recent viral metagenomic studies have demonstrated the diversity of eukaryotic viruses and bacteriophage shed in the feces of domestic species. Although enteric disease is a major concern in the commercial mink farming industry, few etiologic agents have been well characterized. This study aimed to identify viruses shed in the fecal matter of clinically healthy commercial mink from 40 southern Ontario farms. Viral RNA was extracted from 67 pooled fecal samples (30 adult female mink and 37 kit) and amplified for Illumina sequencing on the NextSeq platform, and the resulting contigs were trimmed and assembled using Trimmomatic 0.36.0 and Spades 3.8.0 in iVirus (CyVerse, AZ, USA) and SeqMan NGen 12 (DNAStar, WI, USA). Identification of assembled sequences >100 bp (Geneious 10.1.3) showed an abundance of bacteriophage sequences, mainly from families Siphoviridae (53%), Podoviridae (22%), Myoviridae (20%), Inoviridae (1%), Leviviridae (0.04%), Tectiviridae (0.01%), and Microviridae (0.01%). A diverse range of vertebrate viruses were detected, of which posavirus 3, mink bocavirus, gyroviruses, and avian‐associated viruses were most abundant. Additionally, sequences from nonvertebrate viruses with water and soil‐associated amebal and algal hosts were also highly prevalent. The results of this study show that viruses shed in the fecal matter of healthy commercial mink are highly diverse and could be closely associated with diet, and that more research is necessary to determine how the detected viruses may impact mink health.

The gastrointestinal microbiome is known to play a critical role in animal health but has been relatively poorly characterized in commercial mink, an obligate carnivore. Whether the microbiota can be manipulated in mink to improve pelt quality, health, and well-being is unknown. The objectives of this study were to characterize the fecal microbiota of commercial mink, and to evaluate potential changes due to year (2014 vs 2015), life stage (adult female vs weaned kit), season (summer vs winter), and between Canadian farms. Pooled fecal samples were collected from adult females and weaned kits in the summers of 2014 (n = 173) and 2015 (n = 168), and from females in the winter of 2016 (n = 39), a time when females undergo marked calorie restriction, from 49 mink farms in Ontario. Bacterial DNA was extracted and the V4 region of the 16S rRNA gene was amplified. Approximately 22 million sequences were identified following quality control filtering. A total of 31 bacterial phyla were identified; however, only 3 comprised >1% of the total sequences identified, with Firmicutes and Proteobacteria together comprising 95% of the total sequences. Comparisons were made by life stage, season and year; no differences were found in the relative abundance of any taxa between samples collected from adult females and weaned kits from the same year and the greatest number of differences at each taxonomic level were noted between 2014 and 2015. Significantly more operational taxonomic units (OTUs) were found in 2014 than 2015 or 2016 (p

Background The incomplete iron salt of polyacrylic acid (IIS-PAA) has been used for its haemostatic, wound healing and antibacterial properties in human patients. IIS-PAA is a water-soluble, acidic, hydrophilic polymer that is active at pH 2.9–4.0. Hypotheses/Objectives To evaluate the antibacterial effect of IIS-PAA against Pseudomonas aeruginosa, meticillin-resistant Staphylococcus pseudintermedius (MRSP) and meticillin-resistant S. aureus (MRSA). Methods This was a prospective, nonblinded in vitro study. Sixty canine isolates of P. aeruginosa, MRSP and MRSA were included. Minimum inhibitory concentrations (MICs) of IIS-PAA were determined by agar dilution on Mueller–Hinton agar using testing concentrations ranging from 0.0625–0.4% (raw material 100%) and based on Clinical and Laboratory Standards Institute guidelines. Results The median MIC for P. aeruginosa was 0.2% (range 0.2–0.3%), mean 0.2% and standard deviation (SD) of 0.018%. The median MIC for MRSP was 0.05% (0.025–0.1%), mean 0.058% and SD 0.022%, and the median MIC for MRSA was 0.1% (0.00625–0.4%), mean 0.126% and SD 0.080%. There were significant differences in MICs between the three bacteria types with MICs highest for Pseudomonas and lowest for MRSP (all P < 0.0001). Conclusions IIS-PAA had in vitro antibacterial activity against three important bacterial pathogens. Further studies of IIS-PAA are indicated to assess the potential clinical benefit and in vitro studies to investigate the spectrum of anti-bacterial activity and mode of action.

The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP. Infection of chickens with H9N2 resulted in an increase in phylum Proteobacteria, and differential enrichment with the genera Vampirovibrio, Pseudoflavonifractor, Ruminococcus, Clostridium cluster XIVb and Isobaculum while control chickens were differentially enriched with genera Novosphingobium, Sphingomonas, Bradyrhizobium and Bifidobacterium. Analysis of pre- and post-H9N2 infection of the same chickens showed that, before infection, the fecal microbiota was characterized by Lachnospiracea and Ruminococcaceae family and the genera Clostridium sensu stricto, Roseburia and Lachnospiraceae incertae sedis. However, post-H9N2 infection, class Deltaproteobacteria, orders Clostridiales and Bacteroidiales and the genus Alistipes were differentially enriched. Findings from the current study show that influenza virus infection in chickens results in the shift of the gut microbiota, and the disruption of the host-microbial homeostasis in the gut might be one of the mechanisms by which influenza virus infection is established in chickens.

The microbiota of hard ticks has been an area of growing interest due to the potential role that the broader microbial community may play in pathogen carriage and transmission. In the last two decades, Ontario, Canada has experienced rapid changes in the risk of tick-borne disease, primarily due to the range expansion of Ixodes scapularis . Another human-biter, Dermacentor variabilis , is a longstanding resident of the province, but currently poses minimal risk of pathogen transmission. To examine the microbiota of these two species, we collected adult and nymphal I. scapularis and D. variabilis from the eastern and southern regions of the province via tick dragging, and conducted next generation sequencing of 19 samples (composed of 45 ticks) via Illumina MiSeq. A total of 1400469 sequences were detected (median 69118/sample; range 23350–155227). The most abundant families of bacteria were unclassified Clostridiales and Ruminococcaceae for both I. scapularis and D. variabilis . No significant differences in the relative abundances of any phylum, class, order, family or genus were detected between locations (east vs south), sex, life stage or tick species. There were no differences in community membership or structure based on unifrac and AMOVA analyses. Female and male ticks had lower microbial diversity when compared to nymphs, based on the Simpson’s index and Shannon evenness index. The findings of our study differ from previous studies of these tick species conducted in other geographic areas, and highlight the potential role geography and related ecological factors may have in shaping the tick microbiota.

Antimicrobials are sometimes given to food animals at low doses in order to promote faster growth. However, the mechanisms by which those drugs improve performance are not fully understood. This study aimed to investigate the impact of zinc bacitracin (55g/ton), enramycin (10g/ton); halquinol® (30g/ton); virginiamycin (16,5g/ton) and avilamycin (10g/ton) on the cecal microbiota of broiler chicken, compared to a control group. Six hundred and twenty four chicks (Cobb 500) arriving to an experimental unit were randomly assigned into each treatment with four repetitions per treatment. The cecal content of 16 animals per treatment (n = 96) was used for DNA extraction and sequencing of the V4 region of the 16S rRNA gene using Illumina technology. The use of antimicrobials induced significant changes in membership but not in structure of the cecal microbiota compared to the control group, suggesting a greater impact on the less abundant species of bacteria present in that environment. Halquinol was the only drug that did not affect microbial membership. Firmicutes comprised the major bacterial phylum present in the cecum of all groups. There was no statistical difference in relative abundances of the main phyla between treated animals and the control group (all P>0.05). Treatment with enramycin was associated with decreased richness and with lower relative abundance of unclassified Firmicutes, Clostridium XI, unclassified Peptostreptococcaceae (all P

To elucidate the ecological effect of high oral doses of halotolerant (resistant to table salt) indigenous-gut bacteria on other commensals early in life, we conducted a culture-based study to quantify the effect of intestinalLactobacillus plantarumstrain of bovine origin (with remarkable aerobic growth capabilities and inhibitory activity againstEscherichia coliO157:H7 and F5) on clinical health and gut lactobacilli/coliforms in newborn calves. In a double-blind placebo-randomized trial twelve colostrum-fed calves, consecutively born at a farm, were fedL. plantarumwithin 12 hours from birth at low (107-8 CFU/day) or high concentrations (1010-11) or placebo (q24 h, 5 d; 10 d follow-up). We developed a 2.5% NaCl-selective culture strategy to facilitate the enumeration ofL. plantarum-strain-B80, and tested 384 samples (>1,152 cultures).L. plantarum-B80-like colonies were detected in a large proportion of calves (58%) even before their first 24 hours of life indicating endemic presence of the strain in the farm. In contrast to studies where human-derivedLactobacillusLGG orrhamnosushad notoriously high, but short-lived, colonization, we found thatL. plantarumcolonized stably with fecal shedding of6±1 log10·g−1(irrespective of dose,P>0.2). High doses significantly reduced other fecal lactic acid bacteria (e.g., lactobacilli,P<0.01) and slightly reduced body weight gain in calves after treatment. For the first time, a halotolerant strain ofL. plantarumwith inhibitory activity against a human pathogen has the ability to inhibit other lactobacilliin vivowithout changing its species abundance, causing transintestinal translocation, or inducing clinical disease. The future selection of probiotics based on halotolerance may expand therapeutic product applicability.

Clostridium difficile is an important cause of enteric infections in humans. Recently, concerns have been raised regarding whether animals could be a source of C. difficile spores. Although colonization has been identified in a number of domestic species, the ability of commensal pests to serve as a reservoir for C. difficile has not been well investigated. The objective of this study was to determine whether urban rats ( Rattus spp.) from Vancouver, Canada, carry C. difficile . Clostridium difficile was isolated from the colon contents of trapped rats and was characterized using ribotyping, toxinotyping, and toxin gene identification. Generalized linear mixed models and spatial analysis were used to characterize the ecology of C. difficile in rats. Clostridium difficile was isolated from 95 of 724 (13.1%) rats, although prevalence differed from 0% to 46.7% among city blocks. The odds of being C. difficile positive decreased with increasing weight (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.87), suggesting that carriage is more common in younger animals. The strains isolated included 9 ribotypes that matched recognized international designations, 5 identified by our laboratory in previous studies, and 21 “novel” ribotypes. Some strains were clustered geographically; however, the majority were dispersed throughout the study area, supporting environmental sources of exposure and widespread environmental contamination with a variety of C. difficile strains. Given that urban rats are the source of a number of other pathogens responsible for human morbidity and mortality, the potential for rats to be a source of C. difficile for humans deserves further consideration.

Made available in DSpace on 2018-12-11T17:08:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 Recent studies performed in humans and rats have reported that exercise can alter the intestinal microbiota. Athletic horses perform intense exercise regularly, but studies characterizing horse microbiome during aerobic conditioning programs are still limited. Evidence has indicated that this microbial community is involved in the metabolic homeostasis of the host. Research on ergogenic substances using new sequencing technologies have been limited to the intestinal microbiota and there is a considerable demand for scientific studies that verify the effectiveness of these supplements in horses. L-carnitine and chromium are potentially ergogenic substances for athletic humans and horses since they are possibly able to modify the metabolism of carbohydrates and lipids. This study aimed to assess the impact of acute exercise and aerobic conditioning, associated either with L-carnitine or chromium supplementation, on the intestinal microbiota of fillies. Twelve Mangalarga Marchador fillies in the incipient fitness stage were distributed into four groups: control (no exercise), exercise, L-carnitine (10g/day) and chelated chromium (10mg/day). In order to investigate the impact of acute exercise or aerobic conditioning on fecal microbiota all fillies undergoing the conditioning program were analyzed as a separate treatment. The fillies underwent two incremental exercise tests before and after training on a treadmill for 42 days at 70-80% of the lactate threshold intensity. Fecal samples were obtained before and 48 h after acute exercise (incremental exercise test). Bacterial populations were characterized by sequencing the V4 region of the 16S rRNA gene using the MiSeq Illumina platform, and 5,224,389 sequences were obtained from 48 samples. The results showed that, overall, the two most abundant phyla were Firmicutes (50.22%) followed by Verrucomicrobia (15.13%). The taxa with the highest relative abundances were unclassified Clostridiales (17.06%) and 5 genus incertae sedis from the phylum Verrucomicrobia (12.98%). There was a decrease in the phylum Chlamydiae and in the genus Mycobacterium after the second incremental exercise test. Intense exercise changed the community's structure and aerobic conditioning was associated with changes in the composition and structure of the intestinal bacterial population of fillies. The intra-group comparison showed that chromium or L-carnitine induced moderate changes in the fecal microbiota of fillies, but the microbiota did not differ from the control group, which was exercised with no supplementation. Fecal pH correlated positively with Simpson's index, while plasma pH correlated negatively. Our results show that exercise and aerobic conditioning can change in the microbiota and provide a basis for further studies enrolling a larger number of horses at different fitness levels to better understand the effects of exercise and training on the intestinal microbiota of horses. Department of Technology Faculdades de Ciencias Agrárias e Veterinárias UNESP Univástadual Paulista Laboratório de Bioquómica de Microrganismos e Plantas Department of Animal Morphology and Physiology Faculdades de Ciencias Agrárias e Veterinárias UNESP Univástadual Paulista Laboratório de Farmacologia e Fisiologia do Exercócio Equino (LAFEQ) Department of Animal Sciences Escola de Veterinária Universidade Federal de Minas Gerais Department of Pathobiology Ontario Veterinary College University of Guelph Department of Animal Morphology and Physiology Faculdades de Ciencias Agrárias e Veteriná rias UNESP Univástadual Paulista Laboratório de Farmacologia e Fisiologia do Exercócioáquino (LAFEQ) Department of Technology Faculdades de Ciencias Agrárias e Veterinárias UNESP Univástadual Paulista Laboratório de Bioquómica de Microrganismos e Plantas Department of Animal Morphology and Physiology Faculdades de Ciencias Agrárias e Veterinárias UNESP Univástadual Paulista Laboratório de Farmacologia e Fisiologia do Exercócio Equino (LAFEQ) Department of Animal Morphology and Physiology Faculdades de Ciencias Agrárias e Veteriná rias UNESP Univástadual Paulista Laboratório de Farmacologia e Fisiologia do Exercócioáquino (LAFEQ)

Otitis externa is a common multifactorial disease in dogs. The diversity of the cutaneous microbiota in dogs appears to decrease in diseased states. However, little is known about the microbiota of the canine ear and how it is altered by disease.To describe the otic bacterial microbiota in dogs with otitis externa compared to healthy dogs.Samples were collected from 18 dogs with clinical and cytological evidence of otitis externa, and eight clinically normal dogs without cytological evidence of otitis externa.DNA from each sample was isolated and IlluminaBacteria from 27 different phyla were identified. Affected ears had significantly decreased alpha diversity when compared to healthy ears. Community structure and membership also differed between the two groups. Linear discriminant analysis effect size analysis identified 153 operational taxonomic units (OTUs) that were differentially abundant. Eleven OTUs were over-represented in the affected ears, including Staphylococcus, Pseudomonas and Parvimonas.The otic bacterial microbiota is much more complex than has been identified with previous culture-based studies; otitis externa is accompanied by broad and complex differences in the microbiota.L'otite externe est une maladie multifactorielle fréquente chez le chien. La diversité du microbiote cutané chez le chien semble diminuer dans les états pathologiques. Cependant, on sait peu de chose sur le microbiote de l'oreille canine et comment il est altéré en fonction des maladies. HYPOTHÈSES/OBJECTIFS: Décrire le microbiote bactérien auriculaire des chiens avec otite externe comparé aux chiens sains.Des échantillons ont été prélevés de 18 chiens avec des signes cliniques et cytologiques d'otite externe et huit chiens cliniquement sains sans signe cytologique d'otite externe. MATÉRIEL ET MÉTHODE: L’ADN de chaque échantillon a été isolé et un séquençage a été réalisé par IlluminaLe microbiote bactérien auriculaire est bien plus complexe que ce qui avait été mis en évidence par les études antérieures basées sur les cultures; l'otite externe est accompagnée par des différences larges et complexes du microbiote.INTRODUCCIÓN: la otitis externa es una enfermedad multifactorial común en los perros. La diversidad de la microbiota cutánea en perros parece disminuir en estados de enfermedad. Sin embargo, poco se sabe sobre la microbiota del oído canino y cómo se altera por enfermedad. HIPÓTESIS/OBJETIVOS: describir la microbiota bacteriana ótica en perros con otitis externa en comparación con perros sanos. ANIMALES: se recogieron muestras de 18 perros con pruebas clínicas y citológicas indicativas de otitis externa y ocho perros clínicamente normales sin pruebas citológicas de otitis externa. MÉTODOS Y MATERIALES: se aisló el DNA de cada muestra y se realizó la secuenciación con Illumina® de la región hipervariable V4 de los amplicones del gen 16S rRNA. Las secuencias fueron procesadas utilizando el software de bioinformática MOTHUR. RESULTADOS: Se identificaron bacterias de 27 filos diferentes. Las orejas afectadas tuvieron una disminución significativa de la diversidad alfa en comparación con las orejas sanas. La estructura de la comunidad y las especies también diferían entre los dos grupos. El análisis discriminante y el análisis del efecto en el tamaño identificaron 153 unidades taxonómicas operativas (OTU) que eran diferencialmente abundantes. Once OTUs estaban sobre representadas en los oídos afectados, incluyendo Staphylococcus, Pseudomonas y Parvimonas. CONCLUSIONES: la microbiota bacteriana ótica es mucho más compleja de lo que se ha identificado en estudios previos basados ​​en cultivos; la otitis externa se acompaña de amplias y complejas diferencias en la microbiota.Die Otitis externa ist eine häufige multifaktorielle Erkrankung von Hunden. Die Diversität der kutanen Mikrobioma bei Hunden scheint in Krankheitszuständen abzunehmen. Es ist jedoch wenig bekannt über das Mikrobiom des Hundeohres und wie es sich im Krankheitszustand verändert.Eine Beschreibung der otischen bakteriellen Mikrobioma bei Hunden mit Otitis externa im Vergleich zu gesunden Hunden. Tiere - Es wurden Proben von 18 Hunden mit klinischer und zytologischer Evidenz einer Otitis externa genommen sowie von acht klinisch normalen Hunden ohne zytologischer Evidenz einer Otitis externa.Es wurde DNA von jeder Probe isoliert und Illumina® Sequenzierung der V4 hypervariablen Region des 16S rRNA Gen Amplikons durchgeführt. Die Sequenzen wurden mittels Bioinformatics Software MOTHUR verarbeitet.Bakterien von 27 unterschiedlichen Phyla wurden identifiziert. Die betroffenen Ohren zeigten eine signifikant verminderte Alpha Diversität im Vergleich zu gesunden Ohren. Die Struktur der Gemeinschaft und Zugehörigkeit unterschied sich ebenfalls zwischen den zwei Gruppen. Die Größenanalyse der linearen Diskriminanzanalyse identifizierte 153 funktionierende taxonomische Einheiten (OTUs), die unterschiedlich reichlich vorhanden waren. Elf OTUs waren überrepräsentiert in den betroffenen Ohren, inklusive Staphylococcus, Pseudomonas und Parvimonas.Das otische bakterielle Mikrobiom ist wesentlich komplexer als bisher mittels Kultur-basierter Studien herausgefunden werden konnte; die Otitis externa wird von breiten und komplexen Unterschieden im Mikrobiom begleitet.背景: 犬外耳炎是常见的多因素疾病。犬皮肤微生物群的多样性似乎在患病状态下降低。然而,人们对犬耳道微生物群及其如何被疾病改变知之甚少。 假设/目标: 描述与健康犬相比,外耳炎患犬的耳道细菌菌群。 动物: 从18只临床和细胞学都证明患有外耳炎的犬,以及8只临床和细胞学证明无外耳炎正常犬的耳道采集样品。 方法和材料: 分离每个样品的DNA,进行16S rRNA基因V4高变区的扩增,并送Illumina®公司测序。使用生物信息学软件MOTHUR处理序列。 结果: 共鉴定出27种不同门的细菌。与健康耳道相比,患病耳道的α多样性明显降低。两组之间的细菌种群结构和种类也不同。线性判别分析的效应量分析确定了丰度差异的153个可操作分类单位(OTU)。患病耳道中有11个OTU过多,包括葡萄球菌、假单胞菌和细单胞菌等。 结论: 耳道细菌菌群比以往培养研究所鉴定的要复杂得多; 外耳炎常伴随广泛而复杂的微生物群差异。.背景: 外耳炎は犬の一般的な多因子疾患である。犬の皮膚細菌叢の多様性は病的状態で減少するようである。しかし、犬の耳細菌叢および耳細菌叢が病気によってどのように変化するかについてはほとんどわかっていない。 仮説/目的: 本研究の目的は、健常犬と比較した外耳炎犬の耳細菌叢を記述することである。 被験動物: 外耳炎の臨床的および細胞学的証拠のある18頭の犬、および外耳炎の細胞学的証拠のない8頭の臨床的に健常な犬からサンプルを採取した。 材料と方法: 各サンプルからDNAを分離し、16S rRNA遺伝子アンプリコンのV4超可変領域のIllumina®シーケンスを実施した。バイオインフォマティクスソフトウェアMOTHURを用いて塩基配列を処理した。 結果: 27の異なる系統の細菌を同定した。罹患耳は、健常耳と比較した場合、α多様性を有意に減少させた。群集構造およびメンバーシップも2グループ間で異なった。線形判別分析効果量解析は、差別的に豊富であった153の運用分類単位(OTU)を識別した。 Staphylococcus、Pseudomonas、Parvimonasなど、11OTUが罹患耳に過剰発現していた。 結論: 耳細菌叢は、既存の培養ベースの研究で確認されたものよりもはるかに複雑であ理、さらに、外耳炎は細菌叢において広く複雑な差異を伴っている。.A otite externa é uma doença multifatorial comum em cães. A diversidade da microbiota cutânea parece sofrer redução durante estados patológicos. Entretanto, pouco se sabe sobre a microbiota da orelha canina e as suas alterações causadas por enfermidades. HIPÓTESE/OBJETIVOS: Descrever a microbiota bacteriana ótica em cães com otite externa comparada a cães saudáveis.Coletou-se amostras de 18 cães com evidência clínica e citológica de otite externa, e oito cães clinicamente normais sem evidência citológica de otite externa. MÉTODOS E MATERIAIS: O DNA de cada amostra foi isolado e realizou-se o sequenciamento da região hipervariável V4 dos amplicons do gene 16S rRNA. As sequências foram processadas utilizando o software de bioinformática MOTHUR.Bactérias de 27 filos diferentes foram identificadas. As orelhas afetadas apresentaram alfa diversidade significativamente reduzida comparado a orelhas normais. Houve também diferença entre os grupos na estrutura da comunidade e no parentesco. A análise do efeito da análise discriminante linear identificou 153 unidades taxonômicas operacionais (operational taxonomic units, OTUs) que foram diferentemente abundantes. Onze OTUs estavam super-representadas nas orelhas afetadas, incluindo Staphylococcus, Pseudomonas e Parvimonas. CONCLUSÕES: A microbiota bacteriana ótica é muito mais complexa do que havia sido identificado anteriormente nos estudos baseados em cultura; a otite externa está acompanhada de diferenças amplas e complexas na microbiota.

Certain 3D printed metals and surface finishes may be better suited for canine patient specific orthopedic implants on the basis of minimizing potential bacterial biofilm growth. Thirty disks each of titanium alloy, stainless steel, and cobalt chromium alloy were 3D printed via laser powder bed fusion. Fifteen disks of each metal were subsequently polished. After incubation with a robust biofilm-forming methicillin-resistant Staphylococcus pseudintermedius isolate, disks were rinsed and sonicated to collect biofilm bacteria. Serial dilutions were plated on blood agar, and colony forming units were counted log (ln) transformed for analysis of variance. Interference microscopy quantified surface roughness for comparison to biofilm growth. Scanning electron microscopy on both pre- and post-sonicated disks confirmed biofilm presence and subsequent removal, and visualized surface features on cleaned disks. Significantly more bacteria grew on rough versus polished metal preparations (p < 0.0001). Titanium alloy had more bacterial biofilm growth compared to cobalt chromium alloy (p = 0.0001) and stainless steel (p < 0.0001). There were no significant growth differences between cobalt chromium alloy and stainless steel (p = 0.4737). Relationships between biofilm growth and surface roughness varied: positive with the rough preparations and negative with the smooth. Polished preparations had increased variance in surface roughness compared to rough preparations, and within disk variance predominated over between disk variance for all preparations with the exception of rough cobalt chromium alloy and rough stainless steel. Using scanning electron microscopy, bacterial biofilms tended to form in crevices. Overall, manual polishing of 3D printed surfaces significantly reduced biofilm growth, with preparation-specific relationships between surface roughness and biofilm growth. These results suggest that metallic implants produced by laser powder bed fusion should be polished. Further research will elucidate the optimal surface roughness per preparation to reduce potential biofilm formation and implant associated infection.

Community associated Clostridium difficile and MRSA are among the most common infection causing pathogens encountered in developed nations. It has been proposed that both pathogens may be foodborne with supporting evidence being based on the carriage in animals and recovery from meat at retail. The following reports on the incidence and dissemination routes of C. difficile and MRSA within a high capacity pork processing facility. Sampling of carcasses and processing environment was performed over two visits to the facility. C. difficile was isolated 80% (16/20) of manure samples taken within the holding area, with 45% (9/20) of holding area floor and wall samples testing positive for the pathogen. Three of the twenty carcasses (15%) sampled at post-bleed and a further 3 at post-evisceration also tested positive for C. difficile . No C. difficile were recovered from scald water samples, polisher/scraper units or on carcasses in the cold room at the end of the line. Ribotype 078 was the predominant C. difficile recovered with the remaining belonging to other ribotypes. MRSA was recovered from a single carcass at post-bleed and within the cold room, in addition to one sample taken from the holding area (overall prevalence of 2.7%). The MRSA isolates recovered from carcasses was spa t034 which is a commonly associated with pigs. The results of the study confirm that the holding area of slaughterhouse acts as a reservoir for C. difficile and MRSA although there was no evidence to suggest that the pathogens were disseminated into the processing lines. Consequently, it can be concluded that pork processing does not represent a significant risk of disseminating MRSA and C. difficile between carcasses.

Three previously described methods for culture of Clostridium difficile from meats were evaluated by microbiologists with experience in C. difficile culture and identification. A consensus protocol using BHI broth enrichment followed by ethanol shock and plating to selective and non-selective media was selected for use, and all participating laboratories received hands-on training in the use of this method prior to study initiation. Retail meat products (N = 1755) were cultured for C. difficile over 12 months during 2010–2011 at 9 U.S. FoodNet sites. No C. difficile was recovered, although other clostridia were isolated.

Antimicrobial resistance (AMR) in zoonotic (e.g. Salmonella spp.), pathogenic, and opportunistic (e.g. E. coli) bacteria in animals represents a potential reservoir of antimicrobial resistant bacteria and resistance genes to bacteria infecting humans and other animals. This study evaluated the prevalence of E. coli and Salmonella enterica, and the presence of associated AMR in commercial meat, companion, research, and shelter rabbits in Canada. Associations between antimicrobial usage and prevalence of AMR in bacterial isolates were also examined in commercial meat rabbits. Culture and susceptibility testing was conducted on pooled fecal samples from weanling and adult commercial meat rabbits taken during both summer and winter months (n=100, 27 farms), and from pooled laboratory (n=14, 8 laboratory facilities), companion (n=53), and shelter (n=15, 4 shelters) rabbit fecal samples. At the facility level, E. coli was identified in samples from each commercial rabbit farm, laboratory facility, and 3 of 4 shelters, and in 6 of 53 companion rabbit fecal samples. Seventy-nine of 314 (25.2%; CI: 20.7-30.2%) E. coli isolates demonstrated resistance to >1 antimicrobial agent. At least one E. coli isolate resistant to at least one antimicrobial agent was present in samples from 55.6% of commercial farms, and from 25% of each laboratory and shelter facilities, with resistance to tetracycline being most common; no resistance was identified in companion animal samples. Salmonella enterica subsp. was identified exclusively in pooled fecal samples from commercial rabbit farms; Salmonella enterica serovar London from one farm and Salmonella enterica serovar Kentucky from another. The S. Kentucky isolate was resistant to amoxicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, streptomycin, and tetracycline, whereas the S. London isolate was pansusceptible. Routine use of antimicrobials on commercial meat rabbit farms was not significantly associated with the presence of antimicrobial resistant E. coli or S. enterica on farms; trends towards resistance were present when resistance to specific antimicrobial classes was examined. E. coli was widely prevalent in many Canadian domestic rabbit populations, while S. enterica was rare. The prevalence of AMR in isolated bacteria was variable and most common in isolates from commercial meat rabbits (96% of the AMR isolates were from commercial meat rabbit fecal samples). Our results highlight that domestic rabbits, and particularly meat rabbits, may be carriers of phenotypically antimicrobial-resistant bacteria and AMR genes, possibly contributing to transmission of these bacteria and their genes to bacteria in humans through food or direct contact, as well as to other co-housed animal species.

Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a d -glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester–phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin.

Fecal samples were collected to establish the apparent prevalence of Clostridium difficile shedding in Standardbred and Thoroughbred racehorses housed at 4 racetracks and 2 breeding facilities, and in horses admitted to a referral large animal clinic. Forty-one (7.59%) of 540 racetrack horses, seven (5.83%) of 120 breeding farm horses, and four (4.88%) out of 82 horses admitted to the referral clinic were culture-positive for C. difficile. An overall fecal culture prevalence of 7.01% for C. difficile was identified in 742 fecal samples. PCR-ribotyping and toxin gene identification was performed and seventeen 17 PCR-ribotypes were identified among the 52 C. difficile isolates.

The objective of this study was to compare full binary toxin loci (CDTloc) sequences from a collection of Clostridium difficile isolates in an effort to further understand the regulation of the binary toxin (CdtAB) and its putative regulator (CdtR). Sequences from different ribotypes and toxinotypes were analysed phylogenetically and for polymorphisms, non-sense mutations, promoter features and signal sequences. Expression of cdtA, which was also representative of cdtB expression, was measured by quantitative PCR (qPCR). Several consensus promoter features and various polymorphisms were identified including a non-sense mutation identified in a ribotype 078 cdtR gene that is predicted to result in a severely truncated protein. Despite this mutation, cdtA expression was still detected by qPCR. Dendrograms based on total sequences indicated that isolates belonging to the same ribotype shared the greatest similarity within the binary toxin locus. Although cdtR is thought to be involved in regulation of cdtA expression, a cdtR non-sense mutation did not inhibit expression of cdtA, suggesting that either the truncated protein is functional or another regulator of the binary toxin exists.

A variety of bacteria are known or suspected of being able to cause enteritis in dogs and cats. Campylobacter spp, Clostridium difficile, Clostridium perfringens, and Salmonella spp are most commonly implicated, but many other organisms are likely involved. Poor understanding of the intestinal microflora and the fact that many, if not all, of these microorganisms can also be found in healthy individuals complicates testing, thereby affecting the use of specific treatments and assessment of potential infection control and zoonotic disease risks. An understanding of the strengths and limitations of various diagnostic options is important for the management of canine and feline enteritis.

This study was conducted to determine the occurrence of zinc and copper resistances in methicillin-resistant Staphylococcus aureus (MRSA) from swine and veal calves in a global strain collection. The test population consisted of 476 porcine MRSA isolates from ten European countries, 18 porcine MRSA isolates from Canada and seven MRSA from China, 92 MRSA and 60 methicillin-susceptible S. aureus (MSSA) isolates from veal calves in the Netherlands and 88 porcine MSSA isolates from four European countries. Most porcine MRSA (n=454) and all bovine MRSA belonged to clonal complex (CC) 398 whereas 37 of the pig MRSA from Europe and the seven Chinese isolates belonged to other CCs and 3 isolates were not classified into a CC. All isolates were tested for susceptibility to zinc chloride and copper sulphate using agar dilution and tested by PCR for the czrC gene encoding zinc resistance. Phenotypic zinc resistance (MIC>2mM) was observed in 74% (n=324) and 42% (n=39) of European MRSA CC398 from pigs and veal calves, respectively, and in 44% of the Canadian isolates (n=8), but not among the Chinese isolates. Almost all (99%) zinc-resistant MRSA carried czrC. Of the 37 European non-CC398 MRSA, 62% were resistant to zinc, but only 46% of them carried czrC. The MICs of the MSSA isolates to zinc chloride ranged from 1 to 4mM and none carried czrC. The MICs of copper sulphate were associated neither with methicillin resistance nor with the detection of czrC. This study showed that zinc resistance and the czrC gene are widespread among CC398 MRSA isolates. This suggests that the use of zinc in feed might have contributed to the emergence of MRSA.

To the Editor: Staphylococcus aureus is a well-known pathogen of horses (1), but the role of other coagulase-positive staphylococcal species in these animals is unclear. S. pseudintermedius and S. delphini, members of the S. intermedius group (SIG), cause infections in some companion animals and equids (2), can be multidrug resistant, and could be a concern in horses. Members of SIG are difficult to differentiate by using biochemical methods and require molecular techniques for accurate species-level identification (3); therefore, misidentification of these pathogens could occur. Methicillin-resistant or unusual staphylococci that are isolated at the Ontario Veterinary College Health Sciences Centre by the University of Guelph Animal Health Laboratory (AHL) routinely undergo further characterization. During 2011, the laboratory tested 5 isolates from different horses that were coagulase-positive staphylococci other than methicillin-resistant S. aureus (MRSA). Isolates were identified by using matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry, S. pseudintermedius or S. delphini PCR (4), and sodA sequence analysis (3). Isolates were further characterized, as indicated, by direct repeat unit typing (5), pulsed-field gel electrophoresis (PFGE) (6), mecA PCR (7), penicillin-binding protein 2a latex agglutination test, and antimicrobial drug susceptibility testing by broth microdilution and/or disk diffusion. A search of AHL’s database was performed to identify other S. pseudintermedius and S. delphini isolates for all submissions of samples from equids during January 2011–August 2012. Of the 5 isolates from the horses, 1 was identified as methicillin-resistant S. pseudintermedius (MRSP) and 4 as methicillin-susceptible S. delphini (Table). The MRSP isolate was classified by direct repeat unit typing as dt11a, a predominant MRSP clone in dogs in North America (8). In addition to β-lactams, the MRSP isolate was resistant to chloramphenicol, clindamycin, erythromycin, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole and susceptible to nitrofurantoin, rifampin, streptomycin, and vancomycin. Table Results of investigation of Staphylococcus delphini and S. pseudintermedius infection in horses, Canada* The 4 S. delphini isolates were initially identified biochemically as S. pseudintermedius but subsequently classified as group A (n = 1) and group B (n = 3) S. delphini by molecular methods (Table). One isolate (SD-4) was resistant to only erythromycin; the remaining isolates were susceptible to all tested antimicrobial drugs. PFGE showed that 2 of the S. delphini isolates (SD-1 and SD-2) were possibly related, with a 4-band difference. The remaining isolates were unrelated to each other and the 2 related isolates. Two of the horses (sources of isolates SD-2 and SD-3) had been recently acquired at the same auction and were sampled on the same day; however, PFGE showed that these samples were not related and came from different groups (A, B). No common epidemiologic links were identified for any of the horses. The AHL database search identified 8 additional horses from which S. pseudintermedius was biochemically identified; on the basis of drug-resistance patterns, 6 (75%) of these isolates were determined to be MRSP (Table). One additional S. delphini isolate was identified by using MALDI-TOF. No common epidemiologic links were identified for these infections. MRSP is an emerging pathogen in dogs and cats (1) but has been rarely identified in horses (2). The role of these bacteria in disease in horses is unclear, but given their ability to cause opportunistic infections in other species, these pathogens should not be dismissed. S. pseudintermedius rarely causes disease in humans (9), and transmission normally occurs from infected or colonized animals. Although rarely reported, infection with MRSP might be overlooked in horses; misidentification as S. aureus is possible if laboratories assume that coagulase-positive staphylococci from horses are S. aureus, and misidentification as methicillin susceptible is possible because the use of cefoxitin susceptibility and S. aureus breakpoints is ineffective for determination of methicillin resistance in S. pseudintermedius (10). Additionally, S. pseudintermedius generates coagulase-positive results by tube testing but coagulase-negative results by slide testing, which creates the potential for misidentification as coagulase-negative staphylococci. Given the rapid expansion of S. pseudintermedius infections among dogs, the potential for zoonotic transmission, and the highly resistant nature of this pathogen, ongoing surveillance is indicated in the equine population. Recently, S. delphini has been divided into groups A and B (3). The typical hosts for group A are believed to be mustelidae (i.e., mink, ferret, badger), whereas hosts for group B remain unknown. S. delphini has rarely been identified in horses, but, as we observed, it may be misidentified by conventional methods. Although colonization or contamination appeared most likely in the instances we describe, these findings suggest that this opportunistic pathogen can be found in horses and might be pathogenic in certain situations. Our findings highlight the importance of using additional identification methods (e.g., MALDI-TOF, Staphylococcus species–specific PCR) for differentiation of SIG members (notably S. delphini and S. pseudintermedius) to effectively document the emergence of these species in horses. In addition, these findings indicate the need to ensure proper differentiation of S. aureus from SIG in equine isolates, despite the historical predominance of S. aureus, because of the differences in methods for determination of methicillin resistance. Future studies are needed to determine prevalence trends and disease roles for these species in equids.

A longitudinal study of Clostridium difficile colonization in piglets was performed on a conventional swine farm in Ontario, Canada. Fecal samples were collected from 10 sows prior to their expected farrowing date, and then from all their piglets on days 2, 7, 30, 44 and 62 of life. C. difficile was isolated from 4/10 (40%) of sows prior to farrowing, 90/121 (74%) piglets on day 2, 66/117 (56%) on day 7, 45/113 (40%) on day 30, 23/101 (23%) on day 44 and 2/54 (3.7%) on day 62. There was a significant decrease in colonization over time (P 0.0001). Overall, C. difficile was isolated from one or more samples from 116/121 (96%) piglets. There was an inverse association between sow colonization and piglet colonization on day 2 (P 0.0001) and a positive association on day 7 (P = 0.001). Ribotype 078/toxinotype V predominated, accounting for 213/234 (91%) isolates. A toxinotype XIV strain that has been previously found in humans in the province was the 2nd most common, but was mainly found in sows, not piglets. Overall, 227/234 (97%) of isolates were from types that have been isolated from humans in the province. Intermittent colonization was detected in 11 (9.6%) piglets. The decline in C. difficile colonization over the first 2 months of life was remarkable. The variation in colonization over a relatively short period of time has important implications for the design and interpretation of studies evaluating C. difficile colonization in pigs, since relatively small differences in age may have a major confounding effect on the prevalence of colonization. The decline in prevalence over time may also have implications on public health concerns, since colonization rates of animals at the time of slaughter are presumably more relevant than those earlier in life.

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive, efficient and reproducible technique for studying gene expression. Identification of stably expressed reference genes is required to avoid bias in these studies yet mostly unvalidated reference genes are used in studying gene expression in Clostridium difficile. Here, we sought to identify a set of stable reference genes used to normalize C. difficile expression data comparing exponential versus stationary phases of growth. Eight candidate reference genes (rpoA, rrs, gyrA, gluD, adk, rpsJ, tpi, and rho) were assessed in 3 C. difficile genotypes (ribotypes 027, 078, and 001). The primers were analyzed for efficiency and the 8 genes were ranked according to their stability. Overall, the genes rrs, adk, and rpsJ ranked among the most stable. Identification of the most stable genes was, however, strain dependent and suggests that selection of reference genes in a heterogeneous species, such as C. difficile, requires multiple genes to be assessed to confirm their stability within the strains being studied.

Methods Fecal samples were collected from 92 bats in Slovenia, consisting of 12 different species, and the bacterial microbiota was assessed via next generation sequencing of the 16S rRNA gene V4 region. Results Sequences were assigned to 28 different phyla, but only Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria accounted for ≥1% of sequences. One phylum (Proteobacteria), one class (Gammaproteobacteria), three orders (Pseudomonadales, Lactobacillales, Bacillales), four families (Enterobacteriaceae, Pseudomonadaceae, Staphylococcaceae, Carnobacteriaceae), and five genera (Pseudomonas, Staphylococcus, Carnobacterium, an unclassified Enterobacteriaceae, Acinetobacter) accounted for 50% of sequences. There were no significant differences in the relative abundances of any phyla between bat species, but various differences were noted at lower taxonomic levels, such as Enterobacteriaceae (P = 0.007, most abundant in M. blythii), Pseudomonadaceae (P = 0.007, most abundant in Rhinolophus hipposideros) and Chlamydiaceae (P = 0.04, most abundant in Myotis myotis). There were significant differences in richness between species in both adults and juveniles/subadults, but there was no impact of sex on any alpha diversity index. When only adults are considered, there were significant differences in community membership between M. blythii and M. emarginatus (P = 0.011), and M. blythii and R. hipposideros (P = 0.004). There were also significant differences in community structure between M. blythii and M. emarginatus (P = 0.025), and M. blythii and R. hipposideros (P = 0.026). When adults of the four main species were compared, 14 OTUs were identified as differentially abundant using LEfSe. Only one difference was identified when comparing R. hipposideros adults and juvenile/subadults, with Klebsiella over-represented in the younger bats. Conclusions Bats have a complex and diverse microbiota with a high relative abundance of Proteobacteria. The relevance of this difference is unclear and requires further study. Differences in the microbiota were observed between bat species, perhaps reflecting different diets and environmental exposures.

To determine the incidence rate of infections with North American pulsed-field types 7 and 8 (NAP7/NAP8) strains of Clostrodium difficile, ribotype 078, and toxinotype V strains, we examined data collected for the Canadian Nosocomial Infections Surveillance Program (CNISP) CDI surveillance project during 2004-2008. Incidence of human infections increased from 0.5% in 2004/2005 to 1.6% in 2008.