Extended-spectrum cephalosporin (ESC)-resistant Klebsiella pneumoniae are a problem in human patients and have been studied extensively. However, there is a paucity of information regarding ESC-resistant K. pneumoniae from livestock in general, and in Canada in particular. This study characterized ESC-resistant K. pneumoniae recovered from dairy manure in Ontario, Canada, and their ESC-resistance plasmids. ESC-resistant K. pneumoniae (n = 73) and K. quasipneumoniae (n = 11) isolates were screened by PCR for blaCTX-M, blaCMY and blaSHV prior to undergoing antimicrobial susceptibility testing using disk diffusion. Isolates from dairy manure carrying blaCTX-M (n = 74), and additional isolates from turkeys (n = 8) and dogs (n = 2), underwent short-read whole genome sequencing (WGS) and a subset of these (n = 35) had additional long-read sequencing and hybrid assembly for confirmation. Isolates were characterized using multi-locus sequence typing (MLST) and antimicrobial resistance (AMR) gene profiles. Thirty known sequence types (STs) and four novel STs were identified for K. pneumoniae, and two STs among K. quasipneumoniae. The isolates were found at various treatment stages of the manure on one farm but were only sporadically found on any of the other farms investigated. The majority of isolates (86%) were multi-drug resistant. Variants of CTX-M were identified in diverse STs and included blaCTX-M-15 (n = 81), blaCTX-M-1 (n = 2) and blaCTX-M-32 (n = 1). The blaCTX-M-15 gene was located on diverse IncF, IncHI1 or IncY replicons as well as on the chromosome, whereas blaCTX-M-1 was harboured on the epidemic IncI1/ST3 plasmid, and blaCTX-M-32 on an IncN plasmid. Plasmids were characterized based on core gene SNPs, replicon types and AMR genes, and compared to plasmids from Escherichia coli from a parallel study. Transfer of blaCTX-M plasmids between bacterial species by conjugation was also assessed. Conjugation of IncI1 and IncFII plasmids occurred from K. pneumoniae to E. coli strains but not vice versa. Notably, IncY replicons were identified as conjugative plasmids and transfer was demonstrated between E. coli strains as well as from K. pneumoniae to E. coli. No strain overlap was observed between dairy manure isolates and those from turkey and dogs, however we did identify similarities between K. pneumoniae MLST and resistance profiles from publicly available human clinical isolates to our isolates found in dairy manure (ST37 and 405), turkeys (ST45) and dogs (ST711).

The Canadian Genomics Research and Development Initiative for Antimicrobial Resistance (GRDI-AMR) uses a genomics-based approach to understand how health care, food production and the environment contribute to the development of antimicrobial resistance. Integrating genomics contextual data streams across the One Health continuum is challenging because of the diversity in data scope, content and structure. To better enable data harmonization for analyses, a contextual data standard was developed. However, development of standards does not guarantee their use. Implementation strategies are critical for putting standards into practice. This work focuses on the development of implementation strategies to better operationalize data standards across the Canadian federal genomics ecosystem. Results include improved understanding of complex data models that can create challenges for existing systems. Technical implementation strategies included spreadsheet-based solutions, new exchange formats, and direct standards integration into new databases. Data curation exercises highlighted common data collection and sharing issues, which informed improved practices and evaluation procedures. These new practices are contributing to improved data quality and sharing within the GRDI-AMR consortium as evidenced by publicly available datasets. The implementation strategies and lessons learned described in this work are generalizable for other standards and can be applied more broadly within other initiatives.

The avermectin family of anthelmintics is largely considered to lack antibacterial activity against gram-positive and gram-negative bacteria. Here, we screened six avermectins (ivermectin, eprinomectin, doramectin, abamectin, selamectin, and emamectin benzoate) and a structurally related milbemycin (moxidectin) for antibacterial activity against a panel of representative gram-positive and gram-negative bacteria. We report that emamectin benzoate exhibited activity against several species of gram-positive bacteria, whereas selamectin was active against Staphylococcus aureus and Staphylococcus epidermidis. Emamectin benzoate was the only avermectin with activity against hyper-permeabilized Escherichia coli cells. Using the model bacterium, Bacillus subtilis, we demonstrated that emamectin benzoate is bactericidal, causing rapid lysis and extensive cell envelope damage. To gather insight into its molecular target, several mutant selection methods were employed but failed to yield emamectin benzoate-resistant mutants. Macromolecular synthesis assays indicated that emamectin benzoate inhibited peptidoglycan synthesis. Transcriptome sequencing showed that emamectin benzoate activates the Lia and SigM cell envelope stress-responsive systems and induces the expression of peptidoglycan biosynthetic genes. Although the inactivation or activation of the Lia system did not alter the susceptibility of B. subtilis to emamectin benzoate, CRISPRi-mediated repression of individual essential peptidoglycan biosynthetic genes sensitized B. subtilis to emamectin benzoate. Emamectin benzoate synergized with cell wall-targeting antibiotics (nisin, fosfomycin, daptomycin, and tunicamycin) against B. subtilis. Phosphatidylglycerol antagonized the antibacterial activity of emamectin benzoate, suggesting that EMB likely interacts with membrane phospholipids to exert its effect. Altogether, avermectins exhibit anti-gram-positive activity, albeit with differing levels of potency and selectivity, and, mechanistically, emamectin benzoate targets the cell envelope.IMPORTANCEAvermectins and the structurally related milbemycins are thought to lack antibacterial activity against gram-positive and gram-negative bacteria. Using antimicrobial susceptibility testing, we showed that avermectins and the milbemycin, moxidectin, possess anti-gram-positive activity, with the avermectin, emamectin benzoate, exhibiting the greatest spectrum of activity. Using B. subtilis as a model organism, we showed that emamectin benzoate is bactericidal, causes extensive cell envelope damage, and inhibits peptidoglycan synthesis. Transcriptome analysis of B. subtilis cells treated with emamectin benzoate showed that this avermectin activates two envelope stress-responsive systems and induces the expression of peptidoglycan biosynthetic genes, likely to counteract emamectin benzoate-mediated cell envelope damage. As some avermectins and milbemycins are approved for human and animal use, these drugs may be repurposed for the treatment of gram-positive bacterial infections. Furthermore, given the extensive use of these agents in agriculture, aquaculture, and medicine, examination of their off-target effects on various bacterial communities is warranted.

Some antibiotics are used for the treatment of various bacterial crop diseases, and there is a concern that this practice may represent a selection pressure that increases the reservoir of antibiotic resistance carried by bacteria in crop production systems. Since the 1950s the aminoglycoside antibiotic streptomycin has been widely used for the treatment of some bacterial crop diseases such as fire blight in apples and pears. Following application, the time that bacteria will be exposed to the antibiotic, and therefore the pressure for selection of resistance, will vary according to the environmental persistence of the antibiotic. In the present study, the dissipation of streptomycin was examined in soils supplemented with 5 mg streptomycin/kg soil and incubated for 21 days under laboratory conditions. The impact of two key rate-controlling variables, soil texture (sandy loam, loam, clay loam) and temperature (4, 20, 30 degrees C) on streptomycin persistence were explored. -Robust methods for streptomycin extraction and analysis by LC-MS/MS were developed. Streptomycin dissipation followed first order kinetics, with the time to dissipate 50 % of the parent compound (DT50) in soils of varying texture incubated at 20 degrees C ranging from about seven to 15 days. In contrast, the DT50 of streptomycin in autoclaved loam soil incubated at 20 degrees C was about 111 days. At 4 degrees C the DT50 ranged from 49 to 137 days. Under no incubation conditions were any extractable transformation products obtained. Streptomycin was dissipated significantly more rapidly in field soil that had a prior history of exposure to the antibiotic than in soil that did not. Taken together, these results indicate that streptomycin is amenable to biodegradation in agricultural soils with DT 50 s of several days when temperature is permissive.

Bradyrhizobium ottawaense MIAE 01942 is a symbiotic nitrogen-fixing bacterium isolated from the root nodules of soybeans grown in agricultural soils amended with veterinary antibiotics. The genome consists of a single 8.45 Mb circular chromosome that harbors genes involved in nitrogen fixation, denitrification, and antibiotic and metal resistance.

Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3′)-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade ‘A’ and ‘B’, with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.