Identification of plasmids in avian-associated Escherichia coli using nanopore and illumina sequencing.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project. RESULTS: Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected. CONCLUSIONS: Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.

Antimicrobial susceptibility of western Canadian Brachyspira isolates: Development and standardization of an agar dilution susceptibility test method.

The re-emergence of Brachyspira-associated disease in pigs since the late 2000s has illuminated some of the diagnostic challenges associated with this genus; notably, the lack of standardized antimicrobial susceptibility testing (AST) methods and interpretive criteria. Consequently, laboratories have relied heavily on highly variable in-house developed methods. There are currently no published investigations describing the antimicrobial susceptibility of Brachyspira isolates collected from pigs in Canada. The first objective of this study was therefore to develop a standardized protocol for conducting agar dilution susceptibility testing of Brachyspira spp., including determining the optimal standardized inoculum density, a key test variable that impacts test performance. The second objective was to determine the susceptibility of a collection of western Canadian Brachyspira isolates using the standardized methodology. After assessing multiple media, an agar dilution test was standardized in terms of starting inoculum (1-2 × 108 CFU/ml), incubation temperature and time, and assessed for repeatability. The antimicrobial susceptibility of a collection of clinical porcine Brachyspira isolates (n = 87) collected between 2009-2016 was then determined. This method was highly reproducible; repeat susceptibility testing yielded identical results 92% of the time. Although most of the isolates had very low MICs to the commonly used antimicrobials to treat Brachyspira-associated infections, several isolates with elevated MICs (>32 µg/ml) for tiamulin, valnemulin, tylosin, tylvalosin, and lincomycin were identified. Overall, this study underscores the importance of establishing CLSI approved clinical breakpoints for Brachyspira to facilitate the interpretation of test results and support the evidence-based selection of antimicrobials in swine industry.

Hybrid Genome Assemblies of 245 Avian and Broiler Barn Environment-Associated Escherichia coli Strains Isolated from Saskatchewan Broiler Farms.

Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. In a 3-year period, we collected and sequenced the whole genomes of E. coli disease isolates (n = 91), isolates from presumed healthy birds (n = 61), and isolates from 8 barn sites (n = 93) on broiler farms in Saskatchewan.

Computational Modeling and Design of New Inhibitors of Carbapenemases: A Discussion from the EPIC Alliance Network.

The EPIC consortium brings together experts from a wide range of fields that include clinical, molecular and basic microbiology, infectious diseases, computational biology and chemistry, drug discovery and design, bioinformatics, biochemistry, biophysics, pharmacology, toxicology, veterinary sciences, environmental sciences, and epidemiology. The main question to be answered by the EPIC alliance is the following: "What is the best approach for data mining on carbapenemase inhibitors and how to translate this data into experiments?" From this forum, we propose that the scientific community think up new strategies to be followed for the discovery of new carbapenemase inhibitors, so that this process is efficient and capable of providing results in the shortest possible time and within acceptable time and economic costs.

Biofilm Formation and Antimicrobial Susceptibility of E. coli Associated With Colibacillosis Outbreaks in Broiler Chickens From Saskatchewan.

The global poultry industry has grown to the extent that the number of chickens now well exceeds the number of humans on Earth. Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. We obtained 94 E. coli isolates from 12 colibacillosis outbreaks on Saskatchewan farms and screened them for antimicrobial resistance and biofilm formation. Fifty-six isolates were from broilers with confirmed colibacillosis, and 38 isolates were from healthy broilers in the same flocks (cecal E. coli). Resistance to penicillins, tetracyclines, and aminoglycosides was common in isolates from all 12 outbreaks, while cephalosporin resistance varied by outbreak. Most E. coli were able to form biofilms in at least one of three growth media (1/2 TSB, M63, and BHI broth). There was an overall trend that disease-causing E. coli had more antibiotic resistance and were more likely to form biofilms in nutrient-rich media (BHI) as compared to cecal strains. However, on an individual strain basis, there was no correlation between antimicrobial resistance and biofilm formation. The 21 strongest biofilm forming strains consisted of both disease-causing and cecal isolates that were either drug resistant or susceptible. Draft whole genome sequencing indicated that many known antimicrobial resistance genes were present on plasmids, with disease-causing E. coli having more plasmids on average than their cecal counterparts. We tested four common disinfectants for their ability to kill 12 of the best biofilm forming strains. All disinfectants killed single cells effectively, but biofilm cells were more resistant, although the difference was less pronounced for the disinfectants that have multiple modes of action. Our results indicate that there is significant diversity and complexity in E. coli poultry isolates, with different lifestyle pressures affecting disease-causing and cecal isolates.

Writing for Wikipedia: An Exercise in Microbiology, Writing, Research, Communication, and Public Service.

The COVID-19 pandemic has had a tremendous impact on how postsecondary education was and is delivered. Educators were forced to rapidly move their instruction online and find new and innovative ways to convey content, stimulate student engagement, and satisfy curricular requirements. The purpose of this article is to describe the use of a Wikipedia writing assignment in Veterinary Bacteriology and Mycology, a second-year course in the Doctor of Veterinary Medicine (DVM) curriculum at the University of Saskatchewan. Students had the opportunity to write and edit incomplete articles on topics related to course content. In addition to deep exploration of a topic, this fostered in students the development of research and communication skills, which have been articulated as core competencies in veterinary programs.

A review of antimicrobial resistance in imported foods.

Antimicrobial resistance is one of the most serious threats to medical science. Food supply is recognized as a potential source of resistant bacteria, leading to the development of surveillance programs targeting primarily poultry, pork, and beef. These programs are limited in scope, not only in the commodities tested, but also in the organisms targeted (Escherichia coli, Salmonella, and Campylobacter); consequently, neither the breadth of food products available nor the organisms that may harbour clinically relevant and (or) mobile resistance genes are identified. Furthermore, there is an inadequate understanding of how international trade in food products contributes to the global dissemination of resistance. This is despite the recognized role of international travel in disseminating antimicrobial-resistant organisms, notably New Delhi metallo-beta-lactamase. An increasing number of studies describing antimicrobial-resistant organisms in a variety of imported foods are summarized in this review.

Evaluation of selective media in antimicrobial surveillance programs capturing broad-spectrum ß-lactamase producing Escherichia coli from chickens at slaughter.

Antimicrobial resistance surveillance targeting agricultural animals is practiced in many countries but does not often include media selective for cephalosporin resistance. Here, we compared the frequency of recovery of resistant Escherichia coli using selective and non-selective media from the cecal contents of 116 chickens collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). Third generation cephalosporin resistance was detected in 24 samples including 12, 10, and 2 on selective, non-selective, and both media, respectively. Isolates producing the CTX-M-1 ESBL were grown from 11 samples, 10 on selective medium only. Our results suggest that current surveillance approaches underestimate the true prevalence of resistance to critically important antimicrobials.

Évaluation de milieux sélectifs dans des programmes de surveillance antimicrobienne isolant Escherichia coli produisant des ß-lactamases à large spectre provenant de poulets à l'abattage. La surveillance de la résistance aux antimicrobiens ciblant les animaux d'élevage est pratiquée dans de nombreux pays mais n'inclut pas souvent les milieux sélectifs pour la résistance aux céphalosporines. Ici, nous avons comparé la fréquence d'isolement d'Escherichia coli résistants à l'aide de milieux sélectifs et non sélectifs à partir du contenu caecal de 116 poulets collectés dans le cadre du Programme intégré canadien de surveillance de la résistance aux antimicrobiens (PICRA). Une résistance aux céphalosporines de troisième génération a été détectée dans 24 échantillons dont 12, 10 et 2 sur des milieux sélectifs, non sélectifs et les deux, respectivement. Les isolats produisant les BLSE CTX-M-1 ont été cultivés à partir de 11 échantillons, 10 sur un milieu sélectif uniquement. Nos résultats suggèrent que les approches de surveillance actuelles sous-estiment la prévalence réelle de la résistance aux antimicrobiens d'importance critique.(Traduit par Dr Serge Messier).

Characterization of antimicrobial-resistant Escherichia coli causing urinary tract infections in dogs: Passive surveillance in Saskatchewan, Canada 2014 to 2018.

BACKGROUND: Urinary tract infections (UTIs) are common in dogs and can be caused by multidrug-resistant Escherichia coli (E coli). OBJECTIVE: To describe the frequency and mechanisms of antimicrobial resistance (AMR) among E coli causing UTIs in dogs in Western Canada during a 4-year surveillance period. ANIMALS: Urine from 516 dogs. METHODS: From November 2014 to 2018, 516 nonduplicate E coli isolates from the urine of dogs were collected from a diagnostic laboratory. Susceptibility testing was determined for a panel of 14 antimicrobials belonging to 7 drug classes. Resistant isolates were screened for the presence of extended-spectrum beta-lactamases (ESBLs), AmpC ß-lactamases, and plasmid-mediated quinolone resistance (PMQR) genes. Epidemiological relationships were assessed by MLST. RESULTS: 80.2% (414/516) of isolates were susceptible to all antimicrobials tested. There was no significant increase in the proportion of isolates resistant to any of the tested antimicrobials during the study period. Resistance to ampicillin was the most common (14.9%, 77/516). Overall, 12 isolates had blaCMY-2 -type AmpC ß-lactamases, and 7 produced CTX-M-type ESBLs. A single isolate had the aac(6')-Ib-cr PMQR gene. The qnr and qepA determinants were not detected. A single isolate belonging to the pandemic lineage ST131 was identified. CONCLUSION: Escherichia coli isolated from the urine of dogs in our region remain susceptible to first-line therapies, though resistance, particularly to the aminopenicillins, warrants monitoring. This is the first description of E coli ST131 from a companion animal in Canada.

Recovery of borderline oxacillin-resistant Staphylococcus pseudintermedius (BORSP) from bone and soft tissue of a rheumatoid arthritis patient with severe osteoporosis: transmission from the family dog.

We report a case of borderline oxacillin-resistant S. pseudintermedius (BORSP) in a rheumatoid arthritis patient with severe osteoporosis. The organism is also resistant to erythromycin and clindamycin. We also present clear evidence on transmission from the family dog.

Bacteremia with Staphylococcus pseudintermedius in a 4 month old pediatric oncology patient.

Staphylococcus pseudintermedius colonizes and is a pathogen of dogs and is being increasingly recognized from specimens from humans with various infections. We describe a case of S. pseudintermedius bacteremia in a 4 month old pediatric oncology patient with clear evidence of transmission from the family pet.

Methicillin-resistant Staphylococcus aureus replication in the presence of high (≥32 µg/ml) drug concentration of vancomycin as seen by electron microscopy.

Methicillin-resistant Staphylococcus aureus (MRSA) has unfortunately become a common pathogen in many healthcare facilities. In many institutions, vancomycin remains the preferred agent for treating serious MRSA infections including bacteraemia with or without endocarditis. The mutant prevention concentration (MPC) testing ≥109 colony forming units of bacteria, describes the antimicrobial drug concentration blocking the growth of the least susceptible cell from high density bacterial populations. With blood culture isolates of MRSA, we discovered strains with MPC values ≥32 µg/ml and viable cells could be readily recovered from agar plates containing 32 µg/ml of vancomycin. To investigate MRSA strains surviving in high concentrations of vancomycin on drug containing agar plates, we utilized electron microscopy to measure cell wall thickness as this has been previously reported as a potential mechanism of resistance1 along with septum thickening. Our data shows MRSA replication from high density bacterial populations in the presence of ≥32 µg/ml of vancomycin. Such observations may explain vancomycin failure in some patients and/or persistent bacteraemia and could potentially question the use of this drug in some critically ill patients in favour of an alternative agent.

Antimicrobial Resistance in Clostridium and Brachyspira spp. and Other Anaerobes.

This article describes the antimicrobial resistance to date of the most frequently encountered anaerobic bacterial pathogens of animals. The different sections show that antimicrobial resistance can vary depending on the antimicrobial, the anaerobe, and the resistance mechanism. The variability in antimicrobial resistance patterns is also associated with other factors such as geographic region and local antimicrobial usage. On occasion, the same resistance gene was observed in many anaerobes, whereas some were limited to certain anaerobes. This article focuses on antimicrobial resistance data of veterinary origin.

Identification of antimicrobial resistant bacteria from plant-based food products imported into Canada.

The role of plant-based foods in the epidemiology of antimicrobial resistance has been inadequately studied. In this investigation, resistant organisms from vegetables, fruits and spices imported into Canada were identified and characterized. A total of 143 products imported from primarily Asian and African countries were purchased from international markets in Saskatoon, Saskatchewan. Samples were selectively cultured for bacterial species where resistance is known to be emerging. The proportions of samples positive for each organism were as follows: E. coli (n = 13, 9.1%), Salmonella spp. (n = 2, 1.4%), ESBL producing Enterobacter spp. (n = 2, 1.4%) and K. pneumoniae (n = 2, 1.4%), S. aureus (n = 7, 4.9%) and Enterococcus spp. (n = 66, 46.2%). Antimicrobial minimum inhibitory concentrations were determined by broth micro-dilution and agar-dilution. Based on the susceptibility of each organism, isolates were screened for resistance genes (ß-lactamases and plasmid mediated quinolones resistance determinants) by PCR. Extended-spectrum ß-lactamase producing Enterobacteriaceae and methicillin resistant S. aureus (MRSA) were identified from 6/143 (4.2%) and 2/143 (1.4%) of samples respectively. The qnrB, qnrS and aac(6')-Ib-cr plasmid mediated quinolone resistance determinants were identified in 2/143 (1.4%) of samples tested. None of the Enterobacteriaceae isolates were resistant to meropenem or colistin. Similarly, all Enterococcus isolates remained susceptible to ampicillin, penicillin and vancomycin. Finding multi-drug resistant bacteria which are frequently isolated from human infections is concerning, although the contribution of the global food trade to the dissemination of resistance remains cryptic. These results suggest that imported plant-based foods may be an underappreciated source of clinically relevant resistant organisms. Further study is required to address these gaps in our understanding of the epidemiology of resistance, and the magnitude of the risk posed to human health by these organisms.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

Extended-Spectrum ß-Lactamase and AmpC ß-Lactamase-Producing Escherichia coli Isolates from Chickens Raised in Small Flocks in Ontario, Canada.

Food-producing animals are recognized to play a role in the epidemiology of antimicrobial resistance in Canada. However, the presence of resistant organisms in particular groups of animals, such as chickens raised in small-holder flocks, has not been studied. The purpose of this study was, therefore, to identify and characterize Escherichia coli possessing broad-spectrum ß-lactamase genes among a collection of third-generation cephalosporin-resistant isolates recovered from 205 small flocks in southern Ontario. Extended-spectrum ß-lactamase (ESBL; CTX-M-1) positive strains were isolated from 26 out of 205 flocks (12.7%), whereas 39 strains possessing AmpC (CMY-2) were grown from 31 out of 205 flocks (15.1%). Pulsed-field gel electrophoresis (PFGE) revealed that the isolates were genetically heterogeneous. Further testing by multi-locus sequence typing confirmed that none of the PFGE-defined clusters belonged to ST131. Our results suggest that the dissemination of this resistance in bacteria isolated from chickens in small-holder flocks may be associated with the spread of plasmids rather than particular E. coli clones and that these isolates do not possess the ESBL types most commonly associated with human infections (CTX-M-15).