Whole-genome sequencing of SARS-CoV-2 from the initial cases of domestic cat infections in Canada.

Two cat nasal swabs from Canada's earliest confirmed SARS-CoV-2 positive domestic cats were sequenced to over 99% SARS-CoV-2 genome coverage. One cat had lineage A.23.1 SARS-CoV-2 not reported before in animals. Both sequences have multiple spike gene mutations and clustered closely with human-derived sequences in the global SARS-CoV-2 phylogenetic tree.

mcr-1 colistin resistance gene sharing between Escherichia coli from cohabiting dogs and humans, Lisbon, Portugal, 2018 to 2020.

BackgroundThe emergence of colistin resistance is a One Health antimicrobial resistance challenge worldwide. The close contact between companion animals and humans creates opportunities for transmission and dissemination of colistin-resistant bacteria.AimTo detect potential animal reservoirs of colistin-resistant Escherichia coli and investigate the possible sharing of these bacteria between dogs, cats and their cohabiting humans in the community in Lisbon, Portugal.MethodsA prospective longitudinal study was performed from 2018 to 2020. Faecal samples from dogs and cats either healthy or diagnosed with a skin and soft tissue or urinary tract infection, and their cohabiting humans were screened for the presence of colistin-resistant E. coli. All isolates were tested by broth microdilution against colistin and 12 other antimicrobials. Colistin-resistant isolates were screened for 30 resistance genes, including plasmid-mediated colistin resistance genes (mcr-1 to mcr-9), and typed by multilocus sequence typing. Genetic relatedness between animal and human isolates was analysed by whole genome sequencing.ResultsColistin-resistant E. coli strains harbouring the mcr-1 gene were recovered from faecal samples of companion animals (8/102; 7.8%) and humans (4/125; 3.2%). No difference between control and infection group was detected. Indistinguishable multidrug-resistant E. coli ST744 strains harbouring the mcr-1 gene were found in humans and their dogs in two households.ConclusionsThe identification of identical E. coli strains containing the plasmid-mediated mcr-1 gene in companion animals and humans in daily close contact is of concern. These results demonstrate the importance of the animal-human unit as possible disseminators of clinically important resistance genes in the community setting.

Extended spectrum ß lactamase-producing Enterobacteriaceae shedding by race horses in Ontario, Canada.

BACKGROUND: We aimed to investigate the prevalence, molecular epidemiology and prevalence factors for Extended Spectrum ß-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.

Governing antimicrobial resistance: a narrative review of global governance mechanisms.

Antimicrobial resistance (AMR), a central health challenge of the twenty first century, poses substantial population health risks, with deaths currently estimated to be around 700,000 per year globally. The international community has signaled its commitment to exploring and implementing effective policy responses to AMR, with a Global Action Plan on AMR approved by the World Health Assembly in 2015. Major governance challenges could thwart collective efforts to address AMR, along with limited knowledge about how to design effective global governance mechanisms. To identify common ground for more coordinated global actions we conducted a narrative review to map dominant ideas and academic debates about AMR governance. We found two categories of global governance mechanisms: binding and non-binding and discuss advantages and drawbacks of each. We suggest that a combination of non-binding and binding governance mechanisms supported by leading antimicrobial use countries and important AMR stakeholders, and informed by One Health principles, may be best suited to tackle AMR.

Antimicrobial Sensitivity Testing of Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is particularly adept at evading the immune system, resulting in chronic infections of the lungs and joints of feedlot cattle. The chronicity of the lesions results in prolonged antimicrobial therapy, possibly exacerbating antimicrobial resistance. This cross-sectional study generated in vitro antimicrobial susceptibility testing (AST) data on 211 M. bovis isolates recovered from 159 healthy, diseased, and dead cattle, spanning the period of 2006-2018. Nine antimicrobials commonly administered to western Canadian feedlot cattle were assessed. The data were analyzed with non-parametric statistical tests with a level of significance of p < 0.05 (two-tailed). Minimum inhibitory concentration (MIC) values tended to increase between the isolates from healthy versus dead cattle and over time (2006-2018). Isolates from dead versus healthy cattle were more likely to be resistant to tulathromycin, gamithromycin, tylosin and enrofloxacin. There was no difference in the distributions of the MICs generated from the isolates recovered from the lungs and joints (p ≥ 0.124) and the lungs and deep nasal passages (p ≥ 0.157) of the same animals.

Optimization of Antimicrobial Treatment to Minimize Resistance Selection.

Optimization of antimicrobial treatment is a cornerstone in the fight against antimicrobial resistance. Various national and international authorities and professional veterinary and farming associations have released generic guidelines on prudent antimicrobial use in animals. However, these generic guidelines need to be translated into a set of animal species- and disease-specific practice recommendations. This article focuses on prevention of antimicrobial resistance and its complex relationship with treatment efficacy, highlighting key situations where the current antimicrobial drug products, treatment recommendations, and practices may be insufficient to minimize antimicrobial selection. The authors address this topic using a multidisciplinary approach involving microbiology, pharmacology, clinical medicine, and animal husbandry. In the first part of the article, we define four key targets for implementing the concept of optimal antimicrobial treatment in veterinary practice: (i) reduction of overall antimicrobial consumption, (ii) improved use of diagnostic testing, (iii) prudent use of second-line, critically important antimicrobials, and (iv) optimization of dosage regimens. In the second part, we provided practice recommendations for achieving these four targets, with reference to specific conditions that account for most antimicrobial use in pigs (intestinal and respiratory disease), cattle (respiratory disease and mastitis), dogs and cats (skin, intestinal, genitourinary, and respiratory disease), and horses (upper respiratory disease, neonatal foal care, and surgical infections). Lastly, we present perspectives on the education and research needs for improving antimicrobial use in the future.

Antibiotic prescriptions for outpatient acute rhinosinusitis in Canada, 2007-2013.

INTRODUCTION: Acute rhinosinusitis (ARS) is a respiratory disease commonly caused by viral infections. Physicians regularly prescribe antibiotics despite bacterial etiologies being uncommon. This is of concern, as this use adds to the selection pressure for resistance. Here we present the descriptive epidemiology of acute rhinosinusitis and corresponding antibiotic prescribing practices by Canadian outpatient physicians from 2007-2013. MATERIALS/METHODS: Diagnosis and antibiotic prescription data for ARS were extracted from the Canadian Disease and Therapeutic Index for 2007 to 2013, and population data were acquired from Statistics Canada. ARS diagnosis and antibiotic prescription rates and frequencies of antibiotic classes were calculated. RESULTS: Eighty-eight percent of patients diagnosed with ARS in 2013 were adults, with a greater rate of antibiotic prescriptions observed among the adults relative to the pediatric patients (1632.9 and 468.6 antibiotic prescriptions per 10,000 inhabitants). Between 2007 and 2013, the ARS diagnosis rate decreased from 596 to 464 diagnoses per 10,000 inhabitants, while the percentage of diagnoses with antibiotic prescriptions at the national level remained stable (87% to 84%). From 2007 to 2013, prescription rates for macrolides decreased from 203.5 to 105.4 prescriptions per 10,000 inhabitants. In 2013, penicillins with extended spectrum were more commonly prescribed compared to macrolides among adult patients (153.5 and 105.4 prescriptions per 10,000 inhabitants, respectively). CONCLUSION: This study is the first to describe physician antibiotic prescribing practices for treatment of ARS in Canada. Results show that antibiotic treatment for ARS represents an area for implementing antimicrobial stewardship, and through it, managing antibiotic resistance. Further work is required to better understand diagnosing practices and treatment criteria for ARS, and use this information to further assist physicians to limit unnecessary antibiotic prescribing practices.

NetF-producing Clostridium perfringens: Clonality and plasmid pathogenicity loci analysis.

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.

A human gut ecosystem protects against C. difficile disease by targeting TcdA.

BACKGROUND: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host. METHODS: A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed. RESULTS: MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis. CONCLUSIONS: MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.

A cross sectional study of animal and human colonization with Methicillin-Resistant Staphylococcus aureus (MRSA) in an Aboriginal community.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections are common among humans in Aboriginal communities in Canada, for unknown reasons. METHODS: Cross sectional study of humans and dogs in an Aboriginal community of approximately 1200 persons. Our objectives were to measure community-based prevalence of nasal MRSA colonization among humans, use multivariable logistic regression to analyze risk factors for MRSA colonization, and perform molecular typing of Staphylococci isolated to investigate interspecies transmission. RESULTS: 461 humans were approached for consent and 442 provided complete data. 109/442 (24.7 %, 95 % C.I. = 20.7-28.7 %) of humans were colonized with MRSA. 169/442 (38.2 %) of humans had received antibiotics in the last 12 months. Only number of rooms in the house (OR 0.86, p = 0.023) and recreational dog use (OR 7.7, p = 0.002) were significant risk factors for MRSA colonization. 95/109 (87.1 %) of MRSA strains from humans were of the same spa type (CMRSA10/USA300). 8/157 (5.1 %, 95 % C.I. = 1.7-8.5 %) of dogs were colonized with methicillin-susceptible S. aureus, and no dogs were colonized with MRSA. CONCLUSIONS: Human MRSA colonization in this community is very common, and a single clone is predominant, suggesting local transmission. Antibiotic use is also very common. Crowding may partially explain high colonization, but most considered risk factors including animal exposure were not predictive. Very few dogs carried human Staphylococcal strains.

Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

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.

Inactivation of Clostridium difficile in sewage sludge by anaerobic thermophilic digestion.

There has been an increase in community-associated Clostridium difficile infections with biosolids derived from wastewater treatment being identified as one potential source. The current study evaluated the efficacy of thermophilic digestion in decreasing levels of C. difficile ribotype 078 associated with sewage sludge. Five isolates of C. difficile 078 were introduced (final density of 5 log CFU/g) into digested sludge and subjected to anaerobic digestion at mesophilic (36 or 42 °C) or thermophilic (55 °C) temperatures for up to 60 days. It was found that mesophilic digestion at 36 °C did not result in a significant reduction in C. difficile spore levels. In contrast, thermophilic sludge digestion reduced endospore levels at a rate of 0.19-2.68 log CFU/day, depending on the strain tested. The mechanism of lethality was indirect - by stimulating germination then inactivating the resultant vegetative cells. Acidification of sludge by adding acetic acid (6 g/L) inhibited the germination of spores regardless of the sludge digestion temperature. In conclusion, thermophilic digestion can be applied to reduce C. difficile in biosolids, thereby reducing the environmental burden of the enteric pathogen.

The oral and conjunctival microbiotas in cats with and without feline immunodeficiency virus infection.

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.

Sanitary status and incidence of methicillin-resistant Staphylococcus aureus and Clostridium difficile within Canadian hotel rooms.

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.

Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012.

BACKGROUND: Data about molecular diversity of commonly circulating type A influenza viruses in Ontario swine are scarce. Yet, this information is essential for surveillance of animal and public health, vaccine updates, and for understanding virus evolution and its large-scale spread. METHODS: The study population consisted of 21 swine herds with clinical problems due to respiratory disease. Nasal swabs from individual pigs were collected and tested by virus isolation in MDCK cells and by rtRT-PCR. All eight segments of 10 H3N2 viruses were sequenced using high-throughput sequencing and molecularly characterized. RESULTS: Within-herd prevalence ranged between 2 and 100%. Structurally, Ontario H3N2 viruses could be classified into three different groups. Group 1 was the most similar to the original trH3N2 virus from 2005. Group 2 was the most similar to the Ontario turkey H3N2 isolates with PB1 and NS genes originating from trH3N2 virus and M, PB2, PA and NP genes originating from the A(H1N1)pdm09 virus. All Group 3 internal genes were genetically related to A(H1N1)pdm09. Analysis of antigenic sites of HA1 showed that Group 1 had 8 aa changes within 4 antigenic sites, A(1), B(3), C(2) and E(2). The Group 2 viruses had 8 aa changes within 3 antigenic sites A(3), B(3) and C(2), while Group 3 viruses had 4 aa changes within 3 antigenic sites, B(1), D(1) and E(2), when compared to the cluster IV H3N2 virus [A/swine/Ontario/33853/2005/(H3N2)]. CONCLUSIONS: The characterization of the Ontario H3N2 viruses clearly indicates reassortment of gene segments between the North American swine trH3N2 from cluster IV and the A(H1N1)pdm09 virus.

Occurrence of Clostridium difficile in seasoned hamburgers and seven processing plants in Iran.

BACKGROUND: The recent increment of the incidence of Community Associated Clostridium difficile Infection (CA)-CDI has led to speculation that this disease is associated to foodborne transmission. Therefore it is critical to establish the community sources of CDI in order to implement the appropriate interventions. The present study was conducted to evaluate the prevalence of C. difficile in seasoned hamburger and examine the sources of C. difficile dispersal in hamburger processing plants. A total of 211 samples including hamburger ingredients, the final product, processing equipment and food contact surfaces were collected from seven hamburger processing plants to evaluate the routes of dispersal of C. difficile. The samples were assessed for the occurrence of C. difficile using culture and polymerase chain reaction (PCR) methods. All isolates were screened for the existence of toxin A, B and binary toxin genes. In addition, isolates were subjected to PCR ribotyping. RESULTS: Overall, 9/211 (4.2%) samples were positive. Toxigenic C. difficile were detected from 2/7 (28.5%) hamburger processing plants, in (3/54) 5.6% of beef meat samples, (2/56) 3.5% of swabs taken from the environment and (4/56) 7.1% of hamburger samples after both molding and freezing. C. difficile was not found in 45 non-meat ingredients including 14 defrosted onions, 14 textured soy proteins and 17 seasonings. All isolates contained tcdB gene while 7 strains were positive for tcdA and two remaining strains were negative for tcdA. None of the isolates harbored binary toxin gene (cdtB). PCR ribotyping of 9 isolates categorized into four ribotypes (IR21, IR 22, IR 23 and IR24). Ribotype IR 22 was the most common type 6/9 (66.6%) found. This genotype was isolated from raw meat, environmental samples and hamburger after both forming and freezing in one processing plant, suggesting raw beef meat as a possible major source of contamination. CONCLUSIONS: Hyper-virulent strains of ribotype were not found in this study however, occurrence of other toxicgenic strains indicate the public health significance of contamination of this product.

Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria.

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.