One Health Genomic Analysis of Extended-Spectrum ß-Lactamase‒Producing Salmonella enterica, Canada, 2012‒2016.

Extended-spectrum ß-lactamases (ESBLs) confer resistance to extended-spectrum cephalosporins, a major class of clinical antimicrobial drugs. We used genomic analysis to investigate whether domestic food animals, retail meat, and pets were reservoirs of ESBL-producing Salmonella for human infection in Canada. Of 30,303 Salmonella isolates tested during 2012-2016, we detected 95 ESBL producers. ESBL serotypes and alleles were mostly different between humans (n = 54) and animals/meat (n = 41). Two exceptions were blaSHV-2 and blaCTX-M-1 IncI1 plasmids, which were found in both sources. A subclade of S. enterica serovar Heidelberg isolates carrying the same IncI1-blaSHV-2 plasmid differed by only 1-7 single nucleotide variants. The most common ESBL producer in humans was Salmonella Infantis carrying blaCTX-M-65, which has since emerged in poultry in other countries. There were few instances of similar isolates and plasmids, suggesting that domestic animals and retail meat might have been minor reservoirs of ESBL-producing Salmonella for human infection.

A One Health Genomic Investigation of Gentamicin Resistance in Escherichia coli from Human and Chicken Sources in Canada, 2014 to 2017.

We investigated whether gentamicin resistance (Genr) in Escherichia coli isolates from human infections was related to Genr E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Genr E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Genr gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Genr E. coli contained a blaCTX-M extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Genr isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Genr and spectinomycin resistance (Specr) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Genr and Specr genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Genr isolates and plasmids were mostly different in chickens and humans.

A One-Health Genomic Investigation of Gentamicin Resistance in Salmonella from Human and Chicken Sources in Canada, 2014 to 2017.

We investigated whether the increased prevalence of gentamicin resistance in Salmonella from human infections was related to a similar increased prevalence in isolates from broiler chickens and whether this increase may have been due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on gentamicin-resistant (Genr) Salmonella isolates from human and chicken sources collected from 2014 to 2017 by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). We determined the genomic relatedness of strains and characterized resistance genes and plasmids. From 2014 to 2017, 247 isolates of Genr Salmonella were identified by CIPARS: 188 were from humans, and 59 were from chicken sources (26 from live animals on farm and 33 from retail meat). The five most common Genr serovars were Salmonella enterica serovars Heidelberg (n = 93; 31.5%), 4,[5],12:i:- (n = 42; 14.2%), Kentucky (n = 37; 12.5%), Infantis (n = 33; 11.2%), and Typhimurium (n = 23; 7.8%). Phylogenomic analysis revealed that for S. Heidelberg and S. Infantis, there were closely related isolates from human and chicken sources. In both sources, resistance to gentamicin and spectinomycin was most frequently conferred by aac(3)-VIa and ant(3'')-Ia, respectively. Plasmid closure confirmed linkages of gentamicin and spectinomycin resistance genes and revealed instances of similar plasmids from both sources. Gentamicin and spectinomycin resistance genes were linked on the same plasmids, and some plasmids and isolates from humans and chickens were genetically similar, suggesting that the use of lincomycin-spectinomycin in chickens may be selecting for gentamicin-resistant Salmonella in broiler chickens and that these resistant strains may be acquired by humans.

Genomic Investigation of the Emergence of Invasive Multidrug-Resistant Salmonella enterica Serovar Dublin in Humans and Animals in Canada.

Salmonella enterica subsp. enterica serovar Dublin is a zoonotic pathogen that often leads to invasive bloodstream infections in humans that are multidrug resistant. Described here are the results of Canadian national surveillance of S Dublin from 2003 to 2015 in humans and bovines, principally collected through the Canadian Integrated Program for Antibiotic Resistance Surveillance (CIPARS). An increase in human infections due to multidrug-resistant (MDR) S Dublin was observed in 2010, many of which were bloodstream infections. Phylogenomic analysis of human and bovine isolates revealed a closely related network that differed by only 0 to 17 single nucleotide variants (SNVs), suggesting some potential transmission between humans and bovines. Phylogenomic comparison of global publicly available sequences of S Dublin showed that Canadian isolates clustered closely with those from the United States. A high correlation between phenotypic and genotypic antimicrobial susceptibility was observed in Canadian isolates. IS26 replication was widespread among U.S. and Canadian isolates and caused the truncation and inactivation of the resistance genes strA and blaTEM-1B A hybrid virulence and MDR plasmid (pN13-01125) isolated from a Canadian S Dublin isolate was searched against NCBI SRA data of bacteria. The pN13-01125 coding sequences were found in 13 Salmonella serovars, but S Dublin appears to be a specific reservoir. In summary, we have observed the rise of invasive MDR S Dublin in humans in Canada and found that they are closely related to bovine isolates and to American isolates in their mobile and chromosomal contents.

A Novel Hybrid Plasmid Carrying Multiple Antimicrobial Resistance and Virulence Genes in Salmonella enterica Serovar Dublin.

Virulence plasmids and antibiotic resistance plasmids are usually maintained separately in Salmonella spp.; however, we report an instance of a hybrid plasmid (pN13-01125) in Salmonella enterica serovar Dublin. Review of the complete sequence of the 172,265-bp plasmid suggests that pN13-01125 is comprised of the previously described pSDVr and pSH696_135 plasmids and that the mechanism of hybridization likely involves IS6 (IS26) insertion sequence elements. The plasmid has a low conjugation frequency, confers resistance to six classes of antimicrobials, and contains a complete spv virulence operon.

Antimicrobial Use and Antimicrobial Resistance Indicators-Integration of Farm-Level Surveillance Data From Broiler Chickens and Turkeys in British Columbia, Canada.

Using data from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS), we aimed to describe trends in antimicrobial use (AMU) in broiler chickens and turkeys, to compare AMU across species, to compare with trends in antimicrobial resistance (AMR), and to assess the effects of various AMU/AMR units of measurement (metrics and indicators) on data integration. Data on AMU and AMR in enteric bacteria, collected from 2013 to 2017 from broiler chickens (n = 143 flocks) and turkeys (n = 145) were used. In broiler chickens, the total AMU in milligrams/population correction unit (mg/PCUBr) decreased by 6%, the number (n) of defined daily doses for animals using Canadian standards (nDDDvetCA) per 1,000 broiler chicken-days decreased by 12%, and nDDDvetCA/PCU decreased by 6%. In turkeys, the mg/PCUTk decreased by 1%, whereas the nDDDvetCA/1,000 turkey-days and the nDDDvetCA/PCU increased by 1 and 5%, respectively. The types of antimicrobial classes used in both species were similar. Using the frequency of flocks reporting use (i.e., number of flocks reporting use/number of flocks participating) as a measurement, the use of certain antimicrobials changed over time (e.g., Broilers, decreased cephalosporin use, virginiamycin use, emerging use of lincomycin-spectinomycin, and avilamycin; Turkeys: increased trimethoprim-sulfonamides and macrolide use). The trends in resistance to specific antimicrobials paralleled the frequency and quantity of use (e.g., ceftriaxone use decreased-ceftriaxone resistance decreased, and gentamicin use increased-gentamicin resistance increased) in some situations, but not others (decreased fluoroquinolone use-increased ciprofloxacin resistance). AMR data were summarized using the AMR indicator index (AMR Ix). The most notable AMR Ix trend was the decrease in ceftriaxone AMR Ix among Escherichia coli (0.19 to 0.07); indicative of the success of the poultry industry action to eliminate the preventive use of third generation cephalosporins. Other trends observed were the increase in ciprofloxacin AMR Ix among Campylobacter from 0.23 to 0.41 and gentamicin AMR Ix among E. coli from 0.11 to 0.22, suggestive of the persistence/emergence of resistance related to previous and current AMU not captured in our surveillance timeframe. These data highlight the necessity of multiple AMU and AMR indicators for monitoring the impact of stewardship activities and interventions.

Antimicrobial use on 24 beef farms in Ontario.

Understanding risk factors for antimicrobial resistance requires knowledge of antimicrobial selection pressure. The objectives of this research were to develop methodology for collecting quantitative antimicrobial use information from beef producers in Ontario, to document the types and quantities of antimicrobials reported (for a minimum of 12 mo), and to compare 2 metrics for injectable use reporting. Twenty-four volunteer beef producers were asked to complete a questionnaire, document drug use in a treatment diary, and retain empty medication containers. For injectable antimicrobials, producers recorded approximately 60% of the total use in the treatment diaries; oxytetracycline, penicillin, macrolides, florfenicol, and spectinomycin were used in the greatest quantities. Based on estimated weights of active ingredients (calculated according to number of animals exposed, duration, and average dose per day) the antimicrobials most commonly used in feed were monensin, tylosin, lasalocid, and tetracyclines. The antimicrobials most commonly used in water were lincomycin-spectinomycin, chlortetracycline, and oxytetracycline. Based on estimated weights and measured quantities, < 1% of antimicrobials used were in the Canadian category of highest importance to human medicine. A comparison of animal daily dosages to kilograms of active ingredient demonstrated that the relative ranking of use of antimicrobials varied with the chosen metric, and that further investigation into the best measure in relation to antimicrobial resistance is warranted.

Antimicrobial resistance in generic fecal Escherichia coli from 29 beef farms in Ontario.

The occurrence of antimicrobial resistance in generic Escherichia coli can serve as an indicator of the pool of resistance genes potentially available for transfer to pathogenic organisms. This study was conducted on 29 volunteer beef farms in Ontario to describe the prevalence and patterns of antimicrobial resistance in E. coli, and to describe changes in the prevalence of resistance during the feedlot stage of production. From the pooled fecal samples on 28 of the 29 farms, 31% of isolates from feedlots (n = 993) and 12% of isolates from cow-calf farms (n = 807) were resistant to one or more of 16 antimicrobials tested. No isolates were resistant to ceftriaxone, ciprofloxacin, gentamicin, or nalidixic acid, and < 1% of the pooled isolates were resistant to ceftiofur. Two percent of both feedlot and cow-calf isolates were resistant to > or = 5 antimicrobials. Cow-calf farms were at significantly lower risk than feedlots for having E. coli isolates that were resistant to streptomycin, sulfamethoxazole, and tetracycline. On average, the prevalence of sulfamethoxazole resistant E. coli isolates was significantly higher in calves than in cows. No resistance was observed to ceftriaxone or ciprofloxacin among isolates (n = 1265) obtained from individually sampled feedlot animals on 2 farms. Less than 1% of these isolates were resistant to gentamicin, nalidixic acid, and ceftiofur. From the individually sampled feedlot animals, resistance to streptomycin (on 1 farm), sulfamethoxazole, and tetracycline increased significantly from arrival to mid-point during the feeding period, and these levels persisted until market-readiness.

Antimicrobial use in the Alberta sheep industry.

Information regarding antimicrobial use in sheep is scarce. In 2001, a scrapie surveillance program was initiated in Alberta that also provided a mechanism for collecting other sheep health data including antimicrobial use information between April 2001 and April 2002. A major objective of this study was to describe antimicrobial use in the Alberta sheep industry. This was done by obtaining qualitative antimicrobial use information from all flocks (n = 212) providing cull ewes to the program using a brief, primarily flock-level, questionnaire. The respondents' flocks represented 13.6% of the total provincial flock in Alberta in 2001. By a substantial amount, the most frequent method of administering antimicrobials was through injection followed by in-feed, oral (liquids, pills, boluses), and in-water routes, respectively. Drug-specific use data were collected for injectable antimicrobials only, with the most commonly used antimicrobial classes being penicillins followed by tetracyclines. Producers rarely treated some or all of their flock with injectable antimicrobials after discovering an individual sick animal. Adult sheep were the most common age group treated with injectable antimicrobials and the most frequent reason for injectable antimicrobial use was mastitis followed by respiratory problems. This study provides some initial insight regarding antimicrobial use in Alberta sheep flocks. However, collection of more drug-specific data (drug type, dose/concentration, duration of treatment) for noninjectable routes of administration should be conducted in future studies. Assessing antimicrobial use in other sectors of the Alberta sheep industry (feedlots) and other provinces across Canada would also be beneficial.

Characterization of a colistin-resistant Salmonella enterica 4,[5],12:i:- harbouring mcr-3.2 on a variant IncHI-2 plasmid identified in Canada.

We have identified a Salmonella enterica serotype 4,[5],12:i:- containing a mcr-3.2 in a patient who travelled to Thailand 1 month prior to the identification of it in Canada. The isolate was multidrug resistant, but remained susceptible to the carbapenems, amikacin and piperacillin/tazobactam. The mcr-3.2 was carried on a 261 Kb variant of the IncHI2 pWJ1. This report provides further evidence of the emergence of a ST34 colistin-resistant clone.

Antimicrobial use surveillance in broiler chicken flocks in Canada, 2013-2015.

There is a paucity of data on the reason for and the quantity of antimicrobials used in broiler chickens in Canada. To address this, the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) implemented surveillance of antimicrobial use (AMU) and antimicrobial resistance (AMR) in broiler chicken flocks in 2013. Shortly after this (2014), the poultry industry banned the preventive use of ceftiofur in broiler chickens. The objectives of this analysis were to describe antimicrobial use (AMU) in Canadian broiler chickens between 2013 and 2015 (n = 378 flocks), compare these results to other animal species in Canada, to highlight the utility of farm surveillance data to evaluate the impact of a policy change, and to explore how different antimicrobial use metrics might affect data interpretation and communication. The surveillance data indicated that the poultry industry policy resulted in lower antimicrobial use and resistance, and they successfully captured information on when, where, why, and how much antimicrobials were being used. The majority of antimicrobials were administered via the feed (95%). The relative frequency of antimicrobial classes used in broiler chickens differed from those used in swine or in food animal production in general. Coccidiostats were the most frequently used antimicrobial classes (53% of total kg). Excluding coccidiostats, the top three most frequently used antimicrobial classes were bacitracin (53% of flocks), virginiamycin (25%) and avilamycin (21%), mainly used for the prevention of necrotic enteritis. Depending on the AMU metric utilized, the relative rankings of the top antimicrobials changed; hence the choice of the AMU metric is an important consideration for any AMU reporting. When using milligrams/Population Correction Unit (mg/PCU) the top three antimicrobial classes used were bacitracins (76 mg/PCU), trimethoprim-sulfonamides (24 mg/PCU), and penicillins (15 mg/PCU), whereas when using a number of Defined Daily Doses in animals using Canadian standards /1,000 chicken-days at risk (nDDDvetCA/1,000 CD) the ranking was bacitracins (223 nDDDvetCA/1,000 CD), streptogramins (118 nDDDvetCA/1,000 CD), and trimethoprim-sulfonamides (87 nDDDvetCA/1,000 CD). The median animal treatment days in feed for one cycle (ATD/cycle) during the three-year study were 34 ATD/cycle; this was equal to the mean age of the flocks at pre-harvest sampling day (days at risk), indicating that the studied flocks except those that were raised without antibiotics and organic, were fed with medicated rations throughout the observation period. Overall, more than half (59%) of antimicrobials used in broiler chickens were in classes not used in human medicine, such as ionophores and chemical coccidiostats aimed to prevent coccidiosis. Compared to grower-finisher pigs and in production animal species (national sales data), the mg/PCU of antimicrobials used in broiler chickens was relatively lower. The findings of this paper highlighted the importance of farm-level AMU surveillance in measuring the impact of interventions to reduce antimicrobials in poultry.

Contamination of Canadian private drinking water sources with antimicrobial resistant Escherichia coli.

BACKGROUND: Surface and ground water across the world, including North America, is contaminated with bacteria resistant to antibiotics. The consumption of water contaminated with antimicrobial resistant Escherichia coli (E. coli) has been associated with the carriage of resistant E. coli in people who drink it. OBJECTIVES: To describe the proportion of drinking water samples submitted from private sources for bacteriological testing that were contaminated with E. coli resistant to antibiotics and to determine risk factors for the contamination of these water sources with resistant and multi-class resistant E. coli. METHODS: Water samples submitted for bacteriological testing in Ontario and Alberta Canada were tested for E. coli contamination, with a portion of the positive isolates tested for antimicrobial resistance. Households were invited to complete questionnaires to determine putative risk factors for well contamination. RESULTS: Using multinomial logistic regression, the risk of contamination with E. coli resistant to one or two classes of antibiotics compared to susceptible E. coli was higher for shore wells than drilled wells (odds ratio [OR] 2.8) and higher for farms housing chickens or turkeys (OR 3.0) than properties without poultry. The risk of contamination with multi-class resistant E. coli (3 or more classes) was higher if the properties housed swine (OR 5.5) or cattle (OR 2.2) than properties without these livestock and higher if the wells were located in gravel (OR 2.4) or clay (OR 2.1) than in loam. CONCLUSIONS: Housing livestock on the property, using a shore well, and having a well located in gravel or clay soil increases the risk of having antimicrobial resistant E. coli in E. coli contaminated wells. To reduce the incidence of water borne disease and the transmission of antimicrobial resistant bacteria, owners of private wells need to take measures to prevent contamination of their drinking water, routinely test their wells for contamination, and use treatments that eliminate bacteria.

Antimicrobial resistance in Campylobacter, Salmonella, and Escherichia coli isolated from retail grain-fed veal meat from Southern Ontario, Canada.

This study estimated the prevalence of Salmonella, Campylobacter, and Escherichia coli isolates in fresh retail grain-fed veal obtained in Ontario, Canada. The prevalence and antimicrobial resistance patterns were examined for points of public health significance. Veal samples (n = 528) were collected from February 2003 through May 2004. Twenty-one Salmonella isolates were recovered from 18 (4%) of 438 samples and underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 6 (29%) of 21 Salmonella isolates; 5 (24%) of 21 isolates were resistant to five or more antimicrobials. No resistance to antimicrobials of very high human health importance was observed. Ampicillin-chloramphenicolstreptomycin-sulfamethoxazole-tetracycline resistance was found in 5 (3%) of 21 Salmonella isolates. Campylobacter isolates were recovered from 5 (1%) of 438 samples; 6 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was documented in 3 (50%) of 6 Campylobacter isolates. No Campylobacter isolates were resistant to five or more antimicrobials or category I antimicrobials. E. coli isolates were recovered from 387 (88%) of 438 samples; 1,258 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 678 (54%) of 1,258 E. coli isolates; 128 (10%) of 1,258 were resistant to five or more antimicrobials. Five (0.4%) and 7 (0.6%) of 1,258 E. coli isolates were resistant to ceftiofur and ceftriaxone, respectively, while 34 (3%) of 1,258 were resistant to nalidixic acid. Ciprofloxacin resistance was not detected. There were 101 different resistance patterns observed among E. coli isolates; resistance to tetracycline alone (12.7%, 161 of 1,258) was most frequently observed. This study provides baseline prevalence and antimicrobial resistance data and highlights potential public health concerns.

Antimicrobial resistance in Escherichia coli isolated from retail milk-fed veal meat from Southern Ontario, Canada.

This study estimated the prevalence of Escherichia coli isolates in fresh retail milk-fed veal scallopini pieces obtained from grocery stores in Ontario, Canada. In addition, the prevalence and antimicrobial resistance patterns were examined for points of public health significance. One hundred fifty-three milk-fed veal samples were collected over the course of two sampling phases, January to May 2004 and November 2004 to January 2005. E. coli isolates were recovered from 87% (95% confidence interval, 80.54 to 91.83%) of samples, and antimicrobial susceptibility testing was conducted on 392 isolates. The prevalence of resistance to one or more antimicrobials was 70% (274 of 392), while the resistance to five or more antimicrobials was 33% (128 of 392). Resistance to ceftiofur (2.8%), ceftriaxone (3.6%), nalidixic acid (12%), and ciprofloxacin (3.8%) alone or in combination was observed. Eighty-five resistance patterns were observed; resistance to tetracycline only (7.4%) was observed most frequently. Individual antimicrobial resistance prevalence levels were compared with grain-fed veal and retail beef data from samples collected in Ontario. In general, resistance to individual antimicrobials was observed more frequently in E. coli isolates from milk-fed veal than in isolates from grain-fed veal and beef. Resistance to one or more antimicrobials and to five or more antimicrobials in E. coli isolates was more frequent in isolates from milk-fed veal than in isolates from grain-fed veal and beef. This study provides baseline data on the occurrence of resistance in E. coli isolates from milk-fed veal that can be compared with data for other commodities. Additionally, E. coli resistance patterns may serve as an indicator of antimicrobial exposure.