First Canadian report of transmission of fluconazole-resistant Candida parapsilosis within two hospital networks confirmed by genomic analysis.

Candida parapsilosis is a common cause of non-albicans candidemia. It can be transmitted in healthcare settings resulting in serious healthcare-associated infections and can develop drug resistance to commonly used antifungal agents. Following a significant increase in the percentage of fluconazole (FLU)-nonsusceptible isolates from sterile site specimens of patients in two Ontario acute care hospital networks, we used whole genome sequence (WGS) analysis to retrospectively investigate the genetic relatedness of isolates and to assess potential in-hospital spread. Phylogenomic analysis was conducted on all 19 FLU-resistant and seven susceptible-dose dependent (SDD) isolates from the two hospital networks, as well as 13 FLU susceptible C. parapsilosis isolates from the same facilities and 20 isolates from patients not related to the investigation. Twenty-five of 26 FLU-nonsusceptible isolates (resistant or SDD) and two susceptible isolates from the two hospital networks formed a phylogenomic cluster that was highly similar genetically and distinct from other isolates. The results suggest the presence of a persistent strain of FLU-nonsusceptible C. parapsilosis causing infections over a 5.5-year period. Results from WGS were largely comparable to microsatellite typing. Twenty-seven of 28 cluster isolates had a K143R substitution in lanosterol 14-α-demethylase (ERG11) associated with azole resistance. As the first report of a healthcare-associated outbreak of FLU-nonsusceptible C. parapsilosis in Canada, this study underscores the importance of monitoring local antimicrobial resistance trends and demonstrates the value of WGS analysis to detect and characterize clusters and outbreaks. Timely access to genomic epidemiological information can inform targeted infection control measures.

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.

Four genomic clades of Candida auris identified in Canada, 2012-2019.

Candida auris is an emerging yeast that is associated with antifungal resistance and healthcare-associated outbreaks. From 2012 to 2019, there were 24 known cases of C. auris colonization or infection in Canada. Isolates were from axilla/groin (n = 6), ear (n = 5), blood (n = 4), toe (n = 2), and a variety of other sites (n = 7). Canadian isolates belonged to the four main genomic clades: Clade I (formerly called South Asian clade, n = 12), Clade II (East Asian, n = 3), Clade III (African, n = 4), and Clade IV (South American, n = 5). Isolates within each clade were clonal; however, whole genome sequencing may be helpful in identifying clusters within healthcare facilities. LAY SUMMARY: The fungal pathogen Candida auris has caused many hospital outbreaks and is often multidrug resistant. All four major strains of C. auris were identified in Canada from 2012 to 2019. Genomic epidemiology may be useful for identifying and reducing transmission of C. auris within hospitals.

Integrated surveillance of extended-spectrum beta-lactamase (ESBL)-producing Salmonella and Escherichia coli from humans and animal species raised for human consumption in Canada from 2012 to 2017.

Resistance to beta-lactam antimicrobials caused by extended-spectrum beta-lactamase (ESBL)-producing organisms is a global health concern. The objectives of this study were to (1) summarise the prevalence of potential ESBL-producing Escherichia coli (ESBL-EC) and Salmonella spp. (ESBL-SA) isolates from agrifood and human sources in Canada from 2012 to 2017, and (2) describe the distribution of ESBL genotypes among these isolates. All data were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). CIPARS analysed samples for the presence of ESBLs through phenotypic classification and identified beta-lactamase genes (blaTEM, blaSHV, blaCTX, blaOXA, blaCMY-2) using polymerase chain reaction (PCR) and whole genome sequencing (WGS). The prevalence of PCR-confirmed ESBL-EC in agrifood samples ranged from 0.5% to 3% across the surveillance years, and was detected most frequently in samples from broiler chicken farms. The overall prevalence of PCR-confirmed ESBL-SA varied between 1% and 4% between 2012 and 2017, and was most frequently detected in clinical isolates from domestic cattle. The TEM-CMY2 gene combination was the most frequently detected genotype for both ESBL-EC and ESBL-SA. The data suggest that the prevalence of ESBL-EC and ESBL-SA in Canada was low (i.e. <5%), but ongoing surveillance is needed to detect emerging or changing trends.

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.

Importation of Extensively Drug-Resistant Salmonella enterica Serovar Typhi Cases in Ontario, Canada.

A strain of extensively drug-resistant (XDR) Salmonella enterica serovar Typhi has caused a large ongoing outbreak in Pakistan since 2016. In Ontario, Canada, 10 cases of mainly bloodstream infections (n = 9) were identified in patients who traveled to Pakistan. Whole-genome sequencing showed that Canadian cases were genetically related to the Pakistan outbreak strain. The appearance of XDR typhoid cases in Ontario prompted a provincial wide alert to physicians to recommend treatment with carbapenems or azithromycin in suspected typhoid cases with travel history to Pakistan.

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.

The use of genome sequencing to investigate an outbreak of hospital-acquired mucormycosis in transplant patients.

We report three cases of hospital-acquired mucormycosis in heart and lung transplant patients over a 6-month period. Traditional epidemiological investigation tools were used to look for a common link between patients to explain the outbreak. Genome sequencing of each fungal strain was used to supplement the investigation. By disproving a close genetic link between infecting strains of mucormycosis, we were able to conclude the outbreak investigation. Genome sequencing is a novel tool that can be used in addition to traditional epidemiologic investigations to help determine linkage of patients during outbreak investigations.

Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014.

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM(r)) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM(r) strains. The genomes of 213 AZM(r) and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM(r) (MICs ≥ 256 µg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM(r) collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 µg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM(r) (MICs = 2 to 4 and 8 to 32 µg/ml, respectively). Low-level AZM(r) was also associated with mtrR promoter mutations, including the -35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM(r) strains arise independently and can then rapidly expand clonally in a region through local sexual networks.

In vitro potency and combination testing of antimicrobial agents against Neisseria gonorrhoeae.

Antimicrobial resistant Neisseria gonorrhoeae is a major concern to public health due to decreased susceptibility to frontline antimicrobials. To find agents that are active against N. gonorrhoeae, we tested antimicrobials alone or in combination by Etest gradient strips. The potencies (as assessed by minimum inhibitory concentrations) of twenty-five antimicrobials were evaluated against nine reference strains of N. gonorrhoeae (WHO F, G, K, L, M, N, O, P and ATCC 49226). Potency was greatest for netilmicin, quinupristin-dalfopristin, ceftriaxone, ertapenem and piperacillin-tazobactam. Combinations of azithromycin, moxifloxacin, or gentamicin with ceftriaxone, doripenem, or aztreonam were tested against reference isolates and the fractional inhibitory concentration index (FICI) was calculated. All nine combinations resulted in indifference (>0.5 FICI ≤ 4). Combinations with FICI < 1 were further evaluated in nine clinical isolates which supported the finding of indifference. No antagonism was observed in any of the combinations tested. This is the first report in which the six combinations of azithromycin, moxifloxacin or gentamcin in combination with doripenem or aztreonam were tested in N. gonorrhoeae. These data on antimicrobials with higher potency and combinations that did not show antagonism can help to guide larger scale susceptibility studies for antimicrobial resistant N. gonorrhoeae.

Effect of Variants of Penicillin-Binding Protein 2 on Cephalosporin and Carbapenem Susceptibilities in Neisseria gonorrhoeae.

To characterize the relationship between penicillin-binding protein 2 (PBP2/penA) and susceptibility to extended-spectrum cephalosporins (ESCs) and carbapenem antibiotics, we compared 17 PBP2 variants in Neisseria gonorrhoeae. Nonmosaic and mosaic variants of PBP2 caused decreased susceptibility to ESCs and, to a lesser extent, to carbapenems. An A501P substitution in mosaic XXXIV_A501P conferred decreased susceptibility to ESCs but restored carbapenem susceptibility to wild-type levels. These results could aid the molecular surveillance of antimicrobial resistance to these agents.

Whole-genome phylogenomic heterogeneity of Neisseria gonorrhoeae isolates with decreased cephalosporin susceptibility collected in Canada between 1989 and 2013.

A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections.

Using whole-genome sequence data to examine the epidemiology of antimicrobial resistance in Escherichia coli from wild meso-mammals and environmental sources on swine farms, conservation areas, and the Grand River watershed in southern Ontario, Canada.

Antimicrobial resistance (AMR) threatens the health of humans and animals and has repeatedly been detected in wild animal species across the world. This cross-sectional study integrates whole-genome sequence data from Escherichia coli isolates with demonstrated phenotypic resistance that originated from a previous longitudinal wildlife study in southern Ontario, as well as phenotypically resistant E. coli water isolates previously collected as part of a public health surveillance program. The objective of this work was to assess for evidence of possible transmission of antimicrobial resistance determinants between wild meso-mammals, swine manure pits, and environmental sources on a broad scale in the Grand River watershed, and at a local scale-for the subset of samples collected on both swine farms and conservation areas in the previous wildlife study. Logistic regression models were used to assess potential associations between sampling source, location type (swine farm vs. conservation area), and the occurrence of select resistance genes and predicted plasmids. In total, 200 isolates from the following sources were included: water (n = 20), wildlife (n = 73), swine manure pit (n = 31), soil (n = 73), and dumpsters (n = 3). Several genes and plasmid incompatibility types were significantly more likely to be identified on swine farms compared to conservation areas. Conversely, internationally distributed sequence types (e.g., ST131), extended-spectrum beta-lactamase- and AmpC-producing E. coli were isolated in lower prevalences (<10%) and were almost exclusively identified in water sources, or in raccoon and soil isolates obtained from conservation areas. Differences in the odds of detecting resistance genes and predicted plasmids among various sources and location types suggest different primary sources for individual AMR determinants, but, broadly, our findings suggest that raccoons, skunks and opossums in this region may be exposed to AMR pollution via water and agricultural sources, as well as anthropogenic sources in conservation areas.

Combining analytical epidemiology and genomic surveillance to identify risk factors associated with the spread of antimicrobial resistance in Salmonella enterica subsp. enterica serovar Heidelberg.

Antimicrobial resistance (AMR) has become a critical threat to public health worldwide. The use of antimicrobials in food and livestock agriculture, including the production of poultry, is thought to contribute to the dissemination of antibiotic resistant bacteria (ARB) and the genes and plasmids that confer the resistant phenotype (ARG). However, the relative contribution of each of these processes to the emergence of resistant pathogens in poultry production and their potential role in the transmission of resistant pathogens in human infections, requires a deeper understanding of the dynamics of ARB and ARG in food production and the factors involved in the increased risk of transmission.

Correlation between Phenotypic and In Silico Detection of Antimicrobial Resistance in Salmonella enterica in Canada Using Staramr.

Whole genome sequencing (WGS) of Salmonella supports both molecular typing and detection of antimicrobial resistance (AMR). Here, we evaluated the correlation between phenotypic antimicrobial susceptibility testing (AST) and in silico prediction of AMR from WGS in Salmonella enterica (n = 1321) isolated from human infections in Canada. Phenotypic AMR results from broth microdilution testing were used as the gold standard. To facilitate high-throughput prediction of AMR from genome assemblies, we created a tool called Staramr, which incorporates the ResFinder and PointFinder databases and a custom gene-drug key for antibiogram prediction. Overall, there was 99% concordance between phenotypic and genotypic detection of categorical resistance for 14 antimicrobials in 1321 isolates (18,305 of 18,494 results in agreement). We observed an average sensitivity of 91.2% (range 80.5-100%), a specificity of 99.7% (98.6-100%), a positive predictive value of 95.4% (68.2-100%), and a negative predictive value of 99.1% (95.6-100%). The positive predictive value of gentamicin was 68%, due to seven isolates that carried aac(3)-IVa, which conferred MICs just below the breakpoint of resistance. Genetic mechanisms of resistance in these 1321 isolates included 64 unique acquired alleles and mutations in three chromosomal genes. In general, in silico prediction of AMR in Salmonella was reliable compared to the gold standard of broth microdilution. WGS can provide higher-resolution data on the epidemiology of resistance mechanisms and the emergence of new resistance alleles.

Rural Raccoons (Procyon lotor) Not Likely to Be a Major Driver of Antimicrobial Resistant Human Salmonella Cases in Southern Ontario, Canada: A One Health Epidemiological Assessment Using Whole-Genome Sequence Data.

Non-typhoidal Salmonella infections represent a substantial burden of illness in humans, and the increasing prevalence of antimicrobial resistance among these infections is a growing concern. Using a combination of Salmonella isolate short-read whole-genome sequence data from select human cases, raccoons, livestock and environmental sources, and an epidemiological framework, our objective was to determine if there was evidence for potential transmission of Salmonella and associated antimicrobial resistance determinants between these different sources in the Grand River watershed in Ontario, Canada. Logistic regression models were used to assess the potential associations between source type and the presence of select resistance genes and plasmid incompatibility types. A total of 608 isolates were obtained from the following sources: humans (n = 58), raccoons (n = 92), livestock (n = 329), and environmental samples (n = 129). Resistance genes of public health importance, including bla CMY-2, were identified in humans, livestock, and environmental sources, but not in raccoons. Most resistance genes analyzed were significantly more likely to be identified in livestock and/or human isolates than in raccoon isolates. Based on a 3,002-loci core genome multi-locus sequence typing (cgMLST) scheme, human Salmonella isolates were often more similar to isolates from livestock and environmental sources, than with those from raccoons. Rare instances of serovars S. Heidelberg and S. Enteritidis in raccoons likely represent incidental infections and highlight possible acquisition and dissemination of predominantly poultry-associated Salmonella by raccoons within these ecosystems. Raccoon-predominant serovars were either not identified among human isolates (S. Agona, S. Thompson) or differed by more than 350 cgMLST loci (S. Newport). Collectively, our findings suggest that the rural population of raccoons on swine farms in the Grand River watershed are unlikely to be major contributors to antimicrobial resistant human Salmonella cases in this region.

First reported outbreak of the emerging pathogen Candida auris in Canada.

BACKGROUND: Candida auris was first described in Japan in 2009 and has since been detected in over 40 countries. The yeast is concerning for multiple reasons, primarily: (1) challenges with accurate identification; (2) reported multidrug resistance; (3) published mortality rates of 30%-60%; and (4) persistence in the environment associated with human transmission. We report the emergence of a healthcare-associated cluster in the Greater Vancouver area in 2018 and describe the measures implemented to contain its transmission. METHODS: Cases were identified through passive and ring surveillance of affected wards. Positive isolates were sent to provincial and national reference laboratories for confirmation and genomic characterization. Extensive infection control measures were implemented immediately after the initial case was identified. RESULTS: Four cases were identified during the outbreak. In a 4-month period, over 700 swabs were collected in order to screen 180 contacts. Whole genome sequencing concluded that all isolates clustered together and belonged to the South Asian clade. No isolates harbored FKS gene mutations associated with resistance to echinocandins. Infection control measures, including surveillance, education, cleaning and/or disinfection, patient cohorting, isolation, and hand hygiene, effectively contained the outbreak; it was declared over within 2 months. CONCLUSIONS: The spread of C auris in healthcare facilities has not spared Canadian institutions. Our experience demonstrates that strict infection control measures combined with microbiological screening can effectively halt transmission in healthcare centers. The necessity of active prospective screening remains unclear.