When the Trojan horse is unable to reach inside the city: investigation of the mechanism of resistance behind the first reported cefiderocol-resistant E. coli in Canada.

Gram-negative metallo-ß-lactamase-producing bacteria can be extremely problematic, especially when found to be extensively drug-resistant (XDR). Cefiderocol is a novel antimicrobial that has been shown to overcome most carbapenemases, with very rare resistance reported to date. Within our institution, two multidrug-resistant and one XDR strains were isolated from a patient who recently emigrated from India. Each isolate underwent whole-genome sequencing to resolve plasmids and determine phylogenetics, strain typing, and mechanisms of resistance. The XDR E. coli was ST167, harbored NDM-5, cirA and PBP3 mutations, consistent with cefiderocol resistance. Our study suggests that the NDM region is required in conjunction with cirA and PBP3 mutations. It is not clear why; however, our study did determine a potential novel iron-transport region unique to the cefiderocol-resistant isolate. This is the first characterized cefiderocol-resistant E.coli reported from Canada. Health centers should be on alert for this clone.IMPORTANCEThe development of cefiderocol, a novel siderophore cephalosporin, has provided additional options to the treatment of extensively drug-resistant (XDR) Gram-negative bacteria. Resistance to cefiderocol is poorly understood and only recently described. Here, we describe a case of a patient with recent travel to India harboring three Escherichia coli isolates, one resistant and two susceptible to cefiderocol. Two isolates are highly similar genetically, allowing the mechanism of resistance to be described more closely. The importance of this manuscript contributes both globally to the understanding of cefiderocol resistance in E. coli as well as nationally as this is the first resistant case reported in Canada. This is especially concerning as cefiderocol is not currently approved in Canada. The implications of reporting emerging resistance to new antimicrobials for XDR Gram negatives are impactful to infectious disease specialists, clinical microbiologists, physicians, and public health.

Genetic and Phenotypic Characterization of the First Canadian Case of Ambler Class A Carbapenemase FRI-8.

This study investigated the mechanism of carbapenem resistance in an Enterobacter cloacae complex positive by the modified carbapenem inactivation method (mCIM) but negative by the Rosco Neo-Rapid Carb Kit, ß CARBA, and conventional PCR for common carbapenemase genes (KPC, NDM, OXA-48, IMP, VIM, GES, and IMI/NMC). Using whole genome sequencing (WGS) data we confirmed the identification of Enterobacter asburiae (ST1639) and the presence of blaFRI-8 located on a 148kb IncFII(Yp) plasmid. This is the first occurrence of a clinical isolate harboring the FRI-8 carbapenemase and the second occurrence of FRI in Canada. This study highlights the need to use both WGS and phenotypic screening methods for detection of carbapenemase-producing strains if we consider the growing diversity of carbapenemases.

Absence of transmission of NDM and OXA-48 carbapenemase genes in a chronic care unit of a long-term care facility.

Infection prevention and control measures are used to contain outbreaks of carbapenemase-producing Enterobacteriaceae. We report the absence of transmission of Klebsiella pneumoniae carrying New Delhi metallo-ß-lactamase and oxacillinase-48 genes among 19 screened contacts of an index case after 14 months of routine practices in a long-term care facility.

Analysis of an IncR Plasmid Carrying blaNDM-1 Linked to an Azithromycin Resistance Region in Enterobacter hormaechei Involved in an Outbreak in Quebec.

In the context of a recent rise in prevalence of NDM-encoding carbapenemase-producing Enterobacterales (CPE) in the province of QC, Canada, the genetic environment of blaNDM-1 was investigated. Three NDM-producing clinical isolates of Enterobacter hormaechei recovered from hospitalized patients involved in a putative outbreak were further characterized by whole-genome sequencing (WGS). Two isolates were confirmed by pulsed-field gel electrophoresis and WGS to be closely related. In addition to a ∼128 kb IncFII conjugative multidrug-resistance (MDR) plasmid, these isolates possessed a ∼45 kb mobilizable IncR MDR plasmid containing 2 MDR regions: a complex class 1 integron harboring blaNDM-1 and 7 other AMR genes, and the IS26-mph(A)-mrx-mphR(A)-IS6100 azithromycin resistance unit. The predicted antimicrobial resistance (AMR) genes correlated with the antimicrobial susceptibility testing results. The multidrug-resistant phenotype in addition to the presence of two important mobile genetic elements, suggest a potent role as a reservoir of antibiotic resistance for such a small IncR plasmid. IMPORTANCE Analyzing the genetic environment of clinically relevant MDR genes can provide information on the way in which such genes are maintained and disseminated. Understanding this phenomenon is of interest for clinicians as it can also provide insight on where these genes might have been sourced, possibly supporting outbreak investigations.

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales at a Hospital System in Toronto, Ontario, Canada, 2007 to 2018.

At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received health care at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters, namely, A to G, involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0 to 6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18 to 21 SNVs away from cluster B, suggesting possible undetected interhospital transmission. SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

Emergence of Morganellaceae Harboring blaIMP-27 Metalloenzyme in Canada.

In 2018 to 2019, PCR for carbapenemases in routine Gram-negative isolates submitted to the National Microbiology Laboratory revealed an increase in IMP-type metalloenzyme-positive isolates, mostly among Morganellaceae Whole-genome sequencing revealed that 23 Morganellaceae harbored blaIMP-27 within a chromosomal Tn7 element. Phylogenomics indicated diversity of isolates but also the presence of a few clonal isolates dispersed geographically. These isolates may be difficult to detect due to carbapenem susceptibility and false-negative results in phenotypic testing.IMPORTANCE Over the last decade or so, the frequency of isolation of clinical carbapenemase-producing organisms (CPOs) has increased among health care-associated infections. This may seriously compromise antimicrobial therapy, as carbapenems are considered the last line of defense against these organisms. The ability of carbapenemases to hydrolyze most ß-lactams in addition to the co-occurrence of mechanisms of resistance to other classes of antimicrobials in CPOs can leave few options for treating infections. The class B metalloenzymes are globally distributed carbapenemases, and the most commonly found include the NDM, VIM, and IMP types. Our study describes a sudden emergence of IMP-27-harboring Morganellaceae during 2018 to 2019 in Canada. There is a paucity of literature on IMP-27 isolates, and our data bolster the information on the genetic context, antimicrobial profiles, and phylogenomics of this group of CPOs.

Arrival of the rare carbapenemase OXA-204 in Canada causing a multispecies outbreak over 3 years.

OBJECTIVES: To investigate a persistent multispecies OXA-204 outbreak occurring simultaneously in multiple distant hospitals in the province of Quebec, Canada. METHODS: OXA-204 carbapenemase-producing Enterobacterales (CPE) isolated from multiple hospitals between January 2016 and October 2018 were included in the study. An epidemiological inquiry was conducted in order to elucidate possible transmission routes and a putative source. Isolates were characterized by standardized antibiotic susceptibility testing and by WGS, using Illumina short-read data and MinION long-read data. RESULTS: The outbreak comprised 65 patients and 82 isolates from four hospital sites. Most patients were ≥65 years old, had multiple comorbidities and had received antibiotics recently. The infection to colonization ratio was 1:20. No persistent environmental reservoir was identified. The most frequent organism was Citrobacter freundii (n = 78), followed by Klebsiella spp. (n = 3) and Escherichia coli (n = 1). WGS analysis showed 77/78 C. freundii isolates differing by 0-26 single nucleotide variants (SNVs). Results of WGS analysis showed blaOXA-204 was present on three plasmids types (IncX1, IncA/C2 and IncFII/FIB/A/C2) and on a prophage. All C. freundii isolates harboured multiple copies of blaOXA-204, both on the chromosome and a plasmid. Plasmid IncFII/FIB/A/C2 was observed in all three species. CONCLUSIONS: Transfer of OXA-204 plasmids likely occurred between species within the same patient, highlighting the plasticity of these plasmids and potential for widespread dissemination. OXA-204 carbapenemase has been introduced into Quebec and has rapidly disseminated. Although the infection to colonization ratio was low in this outbreak, this carbapenemase has been associated with severe infection elsewhere.

Two Hypervirulent Klebsiella pneumoniae Isolates Producing a blaKPC-2 Carbapenemase from a Canadian Patient.

This report describes two hypervirulent Klebsiella pneumoniae isolates that produced K. pneumoniae carbapenemase (KPC), which were identified from a rectal swab and a urine culture upon hospital admission. The patient had recently traveled to Greece, where he was hospitalized. The isolates were sequence type 86 and contained an IncHI1B IncFIBK hypervirulent plasmid and an IncFIIK plasmid harboring KPC.

Results from the Canadian Nosocomial Infection Surveillance Program for detection of carbapenemase-producing Acinetobacter spp. in Canadian hospitals, 2010-16.

Objectives: Globally there is an increased prevalence of carbapenem-resistant Acinetobacter spp. (CRAs) and carbapenemase-producing Acinetobacter spp. (CPAs) in the hospital setting. This increase prompted the Canadian Nosocomial Infection Surveillance Program (CNISP) to conduct surveillance of CRA colonizations and infections identified from patients in CNISP-participating hospitals between 2010 and 2016. Methods: Participating acute care facilities across Canada submitted CRAs from 1 January 2010 to 31 December 2016. Patient data were collected from medical records using a standardized questionnaire. WGS was conducted on all CRAs and data underwent single nucleotide variant analysis, resistance gene detection and MLST. Results: The 7 year incidence rate of CRA was 0.02 per 10 000 patient days and 0.015 per 1000 admissions, with no significant increase observed over the surveillance period (P > 0.73). Ninety-four CRA isolates were collected from 58 hospitals, of which 93 (98.9%) were CPA. Carbapenemase OXA-235 group (48.4%) was the most common due to two separate clusters, followed by the OXA-23 group (41.9%). Patients with a travel history were associated with 38.8% of CRA cases. The all-cause 30 day mortality rate for infected cases was 24.4 per 100 CRA cases. Colistin was the most active antimicrobial agent (95.8% susceptibility). Conclusions: CRA remains uncommon in Canadian hospitals and the incidence did not increase from 2010 to 2016. Almost half of the cases were from two clusters harbouring OXA-235-group enzymes. Previous medical treatment during travel outside of Canada was common.

Characterization of OXA-48-like carbapenemase producers in Canada, 2011-14.

Objectives: Since the first identification of the OXA-48 carbapenemase in 2001, Enterobacteriaceae harbouring OXA-48-like enzymes have been reported globally. Here, we applied WGS to characterize the molecular epidemiology of these bacterial isolates. Methods: Enterobacteriaceae non-susceptible to carbapenems isolated from patients between 2011 and 2014 were voluntarily submitted to the Canadian National Microbiology Laboratory where they were screened for carbapenemase genes. WGS was conducted on OXA-48-like producers using the Illumina MiSeq platform. WGS data were used for single nucleotide variant (SNV) analysis, MLST analysis, detection of resistance genes and partial plasmid characterization. Susceptibilities were determined using Vitek2 and Etest. Patient data provided from sites were reviewed. Results: Sixty-seven non-duplicated cases were identified among Escherichia coli (n = 21) and Klebsiella pneumoniae (n = 46). Recent international travel was observed in 40.4% of cases. OXA-181 (52.2%) and OXA-48 (31.3%) were the most common variants, one E. coli OXA-48 producer was found to harbour the acquired colistin resistance gene mcr-1. The dominant STs were ST38 and ST410 in E. coli and ST14 in K. pneumoniae. Three common plasmid types were observed among isolates: IncL/M associated with OXA-48 producers, and ColKP3 and IncX3 associated with OXA-181/232 producers. Conclusions: Enterobacteriaceae with OXA-48-like carbapenemases are emerging in Canada. This study highlights the complexity of OXA-48-types identified in Canada owing to travel and the successful clones and plasmids harbouring the OXA-48-like enzyme.

Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids.

Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A blaNMC-A or blaIMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, blaNMC-A was highly associated with Enterobacter ludwigii Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A (n = 10), IMI-1 (n = 5), and IMI-9 (n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.

Results from the Canadian Nosocomial Infection Surveillance Program on Carbapenemase-Producing Enterobacteriaceae, 2010 to 2014.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing globally; here we report on the investigation of CPE in Canada over a 5-year period. Participating acute care facilities across Canada submitted carbapenem-nonsusceptible Enterobacteriaceae from 1 January 2010 to 31 December 2014 to the National Microbiology Laboratory. All CPE were characterized by antimicrobial susceptibilities, pulsed-field gel electrophoresis, multilocus sequence typing, and plasmid restriction fragment length polymorphism analysis and had patient data collected using a standard questionnaire. The 5-year incidence rate of CPE was 0.09 per 10,000 patient days and 0.07 per 1,000 admissions. There were a total of 261 CPE isolated from 238 patients in 58 hospitals during the study period. blaKPC-3 (64.8%) and blaNDM-1 (17.6%) represented the highest proportion of carbapenemase genes detected in Canadian isolates. Patients who had a history of medical attention during international travel accounted for 21% of CPE cases. The hospital 30-day all-cause mortality rate for the 5-year surveillance period was 17.1 per 100 CPE cases. No significant increase in the occurrence of CPE was observed from 2010 to 2014. Nosocomial transmission of CPE, as well as international health care, is driving its persistence within Canada.

New Delhi metallo-ß-lactamase in Jamaica.

INTRODUCTION: The global dissemination of the New Delhi metallo-beta-lactamase (NDM) gene among certain strains of bacteria has serious implications since the infections caused by such organisms pose a therapeutic challenge. Although the NDM gene has been detected in various parts of the world, this is the first report of its detection in the English-speaking Caribbean. The NDM producing Klebsiella pneumoniae was isolated from an Indian patient who had recently relocated to Jamaica. METHODOLOGY: Identification and susceptibility testing of the K. pneumoniae isolate was performed using the Vitek 2 automated system) in keeping with Clinical and Laboratory Standards Institute (CLSI) standards. It was identified as a metallobetalactamase producer using the Rosco KPC+MBL kit. Genotypic screening for common betalactamase (including carbapenemase) genes, was carried out  using two multiplex PCRs: one for SHV-, TEM-, CTX-M-, OXA-1-, and CMY-2-types, and one for VIM-, KPC-, IMP-, OXA-48, GES-, and NDM-types. Strain typing was conducted by pulsed-field gel electrophoresis (PFGE) using XbaI and multi-locus sequencing (MLS). Plasmid isolation and analysis was also performed. RESULTS: K. pneumoniae (N11-02395), not previously associated with the dissemination of the NDM in India, Sweden or the UK, was found to harbor the NDM-1 gene on plasmid pNDM112395. CONCLUSION: The identification of the NDM-1 gene underscores the need for effective surveillance and infection control measures to identify and prevent spread of multidrug resistant Gram negative bacilli. Strict infection control measures implemented for this patient helped to prevent the spread of this organism to other patients.

Characterization of Escherichia coli urinary tract infection isolates in remote northern Saskatchewan communities: the Northern Antibiotic Resistance Partnership.

Antimicrobial resistance is a growing concern especially in many remote northern communities of Canada where antimicrobials are liberally used. In this study, 1418 Escherichia coli urinary tract infection (UTI) isolates, obtained over a 2.5-year period (October 2005-March 2008), from 3 remote northern sites in Saskatchewan, Canada, were identified. Antimicrobial susceptibility testing of the first 544 clinically significant isolates revealed high prevalence of resistance to trimethoprim-sulfamethoxazole (TMP-SXT) (30.7%). Pulsed-field gel electrophoresis (PFGE) of 165 TMP-SXT-resistant isolates revealed a heterogeneous population. Multilocus sequence typing identified 7 STs from 9 identified PFGE clusters, which included separate PFGE clusters of fluoroquinolone-resistant and -susceptible ST131 isolates. The majority of TMP-SXT-resistant isolates (85.5%) were found to carry class 1 integrons, and plasmids from 62 (81%) of 77 representative isolates were successfully transformed into E. coli DH10B. Overall, ampicillin was the most common plasmid-encoded resistance phenotype transferred with TMP-SXT at 60% (37/62). Further characterization of 52 plasmids by restriction fragment length polymorphism and replicon typing revealed the presence of many plasmid lineages, suggesting that the elevated rates of TMP-SXT resistance in these communities are most likely attributed to the horizontal transfer of class 1 integrons. Results from this study emphasize the importance of continued surveillance of remote northern communities in order to optimize the efficacy of empiric UTI treatment.

Comparison of community-associated and health care-associated methicillin-resistant Staphylococcus aureus in Canada: results of the CANWARD 2007-2009 study.

This study assessed the demographics, antimicrobial susceptibility, and molecular epidemiology of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and health care-associated MRSA (HA-MRSA) in Canadian hospitals between 2007 and 2009. Among 3589 S. aureus, 889 (24.8%) were MRSA; 224 (25.2%) were CA-MRSA genotypes and 644 (72.4%) were HA-MRSA genotypes. The prevalence of CA-MRSA genotypes increased from 19.5% in 2007 to 31.9% in 2009 (P < .001). CMRSA10/USA300 (73.7%) was the predominant CA-MRSA epidemic type; the most common HA-MRSA epidemic type was CMRSA2/USA100/800 (83.5%). CA-MRSA genotypes carried SCCmec type IVa (98.2%) and were largely agr type I (73.2%). Most HA-MRSA genotypes were SCCmec type II (81.2%) and agr type II (83.4%). Panton-Valentine leukocidin was detected in 201/224 (89.7%) CA-MRSA genotypes and 3/644 (0.5%) HA-MRSA genotypes. An increase in vancomycin minimum inhibitory concentration (MIC) was observed in HA-MRSA genotypes overall, with 1.3% (4/305) of strains in 2007 and 4.6% (7/152) in 2009 exhibiting vancomycin MICs of 2 µg/mL. No MRSA resistance occurred with linezolid, daptomycin, or tigecycline. In conclusion, CA-MRSA genotypes represented 25.2% of all MRSA and continue to increase in prevalence in Canadian hospitals.

Characterization of Acinetobacter baumannii and meropenem-resistant Pseudomonas aeruginosa in Canada: results of the CANWARD 2007-2009 study.

A total of 66 (0.35% of overall isolates) Acinetobacter baumannii and 102 (0.55%) meropenem-resistant Pseudomonas aeruginosa were identified among 18 538 isolates collected from medical centers across Canada during the 2007-2009 period. A. baumannii was most frequently recovered from patients in intensive care units (ICUs; 42.4%) and was isolated mostly from blood cultures (53.0%) and respiratory tract specimens (33.3%). Colistin, meropenem, and amikacin were the most active agents against A. baumannii strains (≥ 92.4% coverage). Gentamicin, levofloxacin, and tigecycline were also active against this bacterial species (MIC(50) 1, 0.12, and 0.5 µg/mL, respectively). Multidrug resistance (MDR; resistance to ≥ 3 antimicrobial classes) was noted in only 4 strains (6.1%), and molecular typing revealed 6 clusters of 2 isolates per cluster that displayed >85% similarity on the dendrogram. Meropenem-resistant P. aeruginosa isolates were primarily obtained from patients in ICUs (40.2%) and the most prevalent specimen types were those collected from the respiratory tract (63.7%), followed by blood cultures (18.6%). Most of the meropenem-resistant P. aeruginosa were resistant to all antimicrobial agents tested, and low susceptibility rates were observed for levofloxacin (8.8%) and gentamicin (28.4%). Amikacin and colistin were active against 67.7% and 88.2% of the isolates, respectively. A total of 68.6% (n = 70) of meropenem-resistant P. aeruginosa were MDR. Pulsed-field gel electrophoresis analysis revealed 94 unique isolates and 2 small clusters (6 and 4 isolates, 1 hospital each). In summary, MDR A. baumannii are rare in Canada and, conversely, meropenem-resistant P. aeruginosa were mostly MDR; however, there was minimal clonal spread among these nonfermentative bacilli.

Comparison of CMY-2 plasmids isolated from human, animal, and environmental Escherichia coli and Salmonella spp. from Canada.

A total of 244 CMY-2 plasmids from 5 separate studies involving Escherichia coli and Salmonella human clinical cases as well as E. coli from feedlots and water sources were examined. Genetically similar CMY-2 plasmids isolated from either E. coli or Salmonella from human, animal, and environmental sources are widely distributed across Canada and cluster into replicon types I1, A/C, and K/B and an unidentified group.