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

Characterization of OXA-48-like carbapenemase producers in Canada, 2011–14 Abstract 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. Introduction The continued emergence and dissemination of carbapenem-hydrolysing β-lactamases in Enterobacteriaceae has been observed globally over the last 15–20 years. Of the Ambler class A carbapenemases, Klebsiella pneumoniae carbapenemase (KPC) is the most commonly identified in many countries and is endemic to the USA, Columbia, Argentina, Greece, Italy and Israel.1 The Ambler class B enzyme, New Delhi metallo-β-lactamase (NDM-1), emerged in 2009 and has since been reported globally; its dissemination has been commonly associated with intercontinental travel.1 Carbapenem-hydrolysing class D β-lactamases (CHDL), such as OXA-23, OXA-24, OXA-58 and OXA-143, are commonly associated with Acinetobacter spp.2,3 However, in 2001 a K. pneumoniae isolated from a patient in Turkey was found to harbour a novel CHDL, named OXA-48.4 Since then reports of Enterobacteriaceae harbouring OXA-48 have been described across Europe, the Mediterranean and the Middle East.5–10 Similarly, OXA-181, which differs from OXA-48 by four amino acid substitutions, was initially described in 2011 from India.11 Other OXA-48-like variants, such as OXA-204,12 OXA-232,13 OXA-244, OXA-24514 and OXA-370,15 have also been described in Enterobacteriaceae species. OXA-48-like carbapenemases hydrolyse penicillins and carbapenems but not extended-spectrum cephalosporins;4 however, they are often associated with ESBLs, and provide the strain with a similar resistance phenotype to cephalosporins as seen in ESBLs.16 In contrast, a number of OXA-48-types that lack carbapenemase activity have been described, namely OXA-163, OXA-247 and OXA-405. They differ by only 1–2 amino acid substitutions but contain a deletion in the active site which is thought to be responsible for their lack of carbapenemase activity.17–19 The first description of OXA-48-type producers in Canada was from four cases in 2011.20 Here, we describe the molecular and epidemiological characteristics of OXA-48-like producers submitted to the National Microbiology Laboratory (NML) in Canada from 2011 to 2014. Materials and methods The study Enterobacteriaceae isolates non-susceptible to a carbapenem (as determined by the submitting site) isolated from patients from 2011 to 2014 were voluntarily submitted to the NML by Canadian hospitals and provincial public health laboratories for carbapenemase gene detection. Duplicated isolates were defined as isolates of the same species collected from a single patient in the same year and were excluded. Isolates of different species in same year or same species in different years from same patients were included. Multiplex PCR for carbapenemase genes (blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 and blaGES) was conducted as previously described.21 Regional distribution was defined as follows: Western (British Columbia, Alberta, Saskatchewan and Manitoba), Central (Ontario and Quebec), Eastern (Nova Scotia, Prince Edward Island, New Brunswick and Newfoundland). Antimicrobial susceptibility testing Antimicrobial susceptibilities were determined using Vitek 2 (AST-GN25; bioMerieux, St Laurent, Canada) using 2016 CLSI breakpoints.22 Tigecycline breakpoints were based on FDA criteria for Enterobacteriaceae [susceptible (S), ≤2 mg/L; intermediate (I), 4 mg/L; resistant (R), ≥8 mg/L]. Custom-made broth microdilution plates (0.25–16 mg/L range) were used to determine colistin susceptibilities with CLSI breakpoints for Acinetobacter spp. (S ≤ 2 mg/L, R > 2 mg/L). Student’s t-test was used for comparison of the antimicrobial resistance profiles of the K. pneumoniae versus the Escherichia coli with the significance level set at P ≤ 0.05. Short-read WGS analysis Total cellular DNA was prepared using Epicentre MasterPureTM Complete kits (Mandel Scientific, Guelph, ON, Canada). Libraries were created with TruSeq Nano DNA HT sample preparation kits (Illumina, San Diego, CA, USA). Paired-end, 301 bp indexed reads were generated on an Illumina MiSeqTM platform (Illumina). All raw read data were uploaded to the Sequence Read Archive (SRA) hosted by NCBI under the BioProject PRJNA390933. An average of 130× coverage was achieved. De novo assemblies of Illumina reads were done using Spades version 3.5. Assembled sequence data were analysed using the batch uploader mode at the Centre for Genomic Epidemiology website (https://cge.cbs.dtu.dk/services/) and data were produced from the ResFinder, PlasmidFinder and MLST tools. Plasmid analysis was performed by mapping the sequencing reads against a database of all Gammaproteobacteria plasmids available from NCBI (https://www.ncbi.nlm.nih.gov/genome/browse/#tabs-plasmids) to identify plasmids associated with OXA-48-like carbapenemases. Phylogenetic analysis based on core single nucleotide variants (SNVs) SNV analysis was conducted using a previously published pipeline.23 The following parameters were set: map/base quality 30, alternate fraction 0.75, minimum read coverage of identification of variants 15 and SNV density filtering of 2 SNVs within a 20 bp window. Maximum likelihood phylogenetic trees were built using PhyML version 3.1.24 Images were rendered in FigTree (version 1.4.1) (http://tree.bio.ed.ac.uk/software/figtree). References used were assembled multifasta contigs of genomes from the current study, which represented specific ST groups (E. coli ST38: N11-01498; E. coli ST410: N14-00405; K. pneumoniae ST14: N14-02136). Results Molecular summary and patient demographics During 2011 (n = 4), 2012 (n = 22), 2013 (n = 15) and 2014 (n = 26), a total of 67 non-duplicate isolates producing OXA-48-like carbapenemases from 62 patients were submitted to the NML. These cases were only identified in E. coli (n = 21) and K. pneumoniae (n = 46). Variant gene types identified were blaOXA-48 (31.3%, n = 21), blaOXA-181 (52.2%, n = 35), blaOXA-232 (a 1 bp variant of blaOXA-181; 14.9%, n = 10) and blaOXA-244 (a 1 bp variant of blaOXA-48; 1.5%, n = 1). Of these, two blaOXA-181 containing isolates were found to co-produce either blaNDM-1 or blaNDM-4, and three blaOXA-232 containing isolates were found to co-produce blaNDM-1 or blaNDM-5. One isolate co-produced the mobile colistin resistance gene mcr-1, which has been previously reported.25 Patient demographics available for gender (n = 57, 92%) showed 50.9% and 49.1% of cases to be from male or female patients, respectively. Available age ranges (n = 57) were 22–64 years (38.6%) or ≥65 years (61.4%). Organisms were isolated predominantly from urine (37.9%), rectal (28.8%) or respiratory (10.6%) samples. Where infection or colonization was determined (n = 37), 45.9% of cases were considered infections. Travel history was collected for 57 patients, of whom 23 (40.4%) indicated travel outside of Canada in the previous 12 months from date of positive culture. With the exception of a patient harbouring blaOXA-48 with travel history to Pakistan, all isolates from patients with travel history to the Indian subcontinent (n = 11), Nigeria (n = 1), Ukraine (n = 1) or the USA (n = 1) harboured blaOXA-181/-232. These patients harboured either E. coli (n = 3) belonging to ST940 (n = 2) or ST410 (n = 1), or K. pneumoniae (n = 11) belonging to ST14 (n = 6), ST147 (n = 3), ST16 (n = 1) or ST1847 (n = 1). Individuals with travel history to North Africa (n = 2; Egypt, Libya) or the Middle East [n = 6; Lebanon (n = 2), Syria (n = 1), Saudi Arabia (n = 2), United Arab Emirates (n = 1)] harboured blaOXA-48. These patients harboured either E. coli (n = 5) belonging to ST38 (n = 3) or ST624 (n = 1), or K. pneumoniae (n = 3) belonging to ST101, ST15 or ST831. Figure 1 shows the distribution of OXA-like-types across Canada by region and ST. The dominant clones in E. coli were ST38 (28.6%) and ST410 (28.6%). All ST410 isolates were blaOXA-181 producers and were equally observed in both Western and Central Canada. ST38 were all blaOXA-48 producers from Central Canada with the exception of one blaOXA-244 from the Western region. For K. pneumoniae the dominant clone was ST14 observed in 54.3% of isolates. Of these 72% were blaOXA-181 producers from the West and the remaining were blaOXA-232 from both West and Central regions. Figure 1. View largeDownload slide Graphic representation of the distribution of OXA-like-types and STs across three Canadian regions. The inner circle represents the distribution between regions, the second circle represents the OXA-type and the outer circle represents the ST. Figure 1. View largeDownload slide Graphic representation of the distribution of OXA-like-types and STs across three Canadian regions. The inner circle represents the distribution between regions, the second circle represents the OXA-type and the outer circle represents the ST. Characterization of antimicrobial resistance The antimicrobial susceptibilities for E. coli and K. pneumoniae are shown in Table 1. All isolates were resistant to ertapenem; however, only 61.2% were resistant to meropenem. Imipenem susceptibilities were not determined. Resistance to piperacillin/tazobactam, ceftazidime and cefotaxime was observed in 100%, 82.1% and 91.1% of isolates, respectively. Overall, 85% of the isolates harboured genes associated with ESBLs. Of the 15 E. coli harbouring ESBLs, blaCTX-M-15 (n = 8),blaCTX-M-24 (n = 3), blaCTX-M-14 (n = 2), and blaCTX-M-15 plus blaTEM-33 (n = 2) were identified. For K. pneumoniae harbouring ESBLs (n = 44) the majority contained blaCTX-M-15 (n = 42), followed by blaSHV-28 (n = 2). Additionally, resistance to trimethoprim/sulfamethoxazole (91%), ciprofloxacin (87%), gentamicin (75%) and tobramycin (68%) was often observed. Tigecycline and colistin resistance was observed in 30% and 6% of isolates, respectively. Overall, significantly more K. pneumoniae isolates were resistant to meropenem, tobramycin, nitrofurantoin, ciprofloxacin and tigecycline (P = <0.0001–0.0028) than E. coli. This is due to the higher proportion of blaOXA-181/-232 producers in K. pneumoniae isolates as blaOXA-181/-232 producers were also significantly more resistant to all of the above (P = <0.0001–0.0223) compared with blaOXA-48 producers, with the exception of meropenem, which was not significant (P = 0.1761). Table 1. Antimicrobial susceptibilities of OXA-48-type producing E. coli and K. pneumoniae in Canada   OXA-181, OXA-232   OXA-48, OXA-244a   Organism (no. tested)/antimicrobial  %Sb  %I  %R  range (mg/L)  %S  %I  %R  range (mg/L)  Escherichia coli (n = 21)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  0  100  ≥32  0  0  100  ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  0  0  100  ≥32  30  20  50  ≤4 to ≥32   cefpodoxime  0  0  100  ≥8  30  0  70  1 to ≥8   cefotaxime  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ceftazidime  0  0  100  ≥64  80  0  20  ≤1 to 16   ceftriaxone  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  4 to ≥8   meropenem  27  55  18  0.5 to ≥16  10  60  30  1 to ≥16   amikacin  82  0  18  ≤2 to ≥64  100  0  0  ≤2   gentamicin  18  0  82  ≤1 to ≥16  40  0  60  ≤1 to ≥16   tobramycin  27  9  64  ≤1 to ≥16  60  30  10  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  60  0  40  ≤0.25 to ≥4   tigecycline  100  0  0  ≤0.5  100  0  0  ≤0.5   nitrofurantoin  36  64  0  ≤16 to 64  70  30  0  ≤16 to 64   SXT  9  0  91  ≤20 to ≥320  10  0  90  ≤20 to ≥320   colistin  100  N/A  0  <0.25  90  n/a  10  <0.25 to 4  Klebsiella pneumoniae (n = 46)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  3  97  16 to ≥32  8  0  92  ≤2 to ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  3  12  85  8 to ≥64  59  8  33  ≤4 to ≥64   cefpodoxime  3  3  94  2 to ≥8  25  0  75  ≤0.25 to ≥8   cefotaxime  0  6  94  4 to ≥64  25  0  75  ≤1 to ≥64   ceftazidime  3  0  97  ≤1 to ≥64  25  0  75  ≤1 to ≥64   ceftriaxone  0  3  97  2 to ≥64  17  0  83  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  2 to ≥8   meropenem  12  3  85  1 to ≥16  34  8  58  0.5 to ≥16   amikacin  62  6  32  ≤2 to ≥64  92  0  8  ≤2 to ≥64   gentamicin  9  0  91  ≤1 to ≥16  50  0  50  ≤1 to ≥16   tobramycin  0  9  91  8 to ≥16  25  25  50  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  17  8  75  0.5 to ≥4   tigecycline  27  21  52  ≤0.5 to ≥8  75  8  17  ≤0.5 to ≥8   nitrofurantoin  0  6  94  64 to ≥512  8  25  67  32 to ≥512   SXT  3  0  97  ≤20 to >320  25  0  75  40 to ≥320   colistin  94  N/A  6  <0.25 to 16  92  N/A  8  <0.25 to 8    OXA-181, OXA-232   OXA-48, OXA-244a   Organism (no. tested)/antimicrobial  %Sb  %I  %R  range (mg/L)  %S  %I  %R  range (mg/L)  Escherichia coli (n = 21)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  0  100  ≥32  0  0  100  ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  0  0  100  ≥32  30  20  50  ≤4 to ≥32   cefpodoxime  0  0  100  ≥8  30  0  70  1 to ≥8   cefotaxime  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ceftazidime  0  0  100  ≥64  80  0  20  ≤1 to 16   ceftriaxone  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  4 to ≥8   meropenem  27  55  18  0.5 to ≥16  10  60  30  1 to ≥16   amikacin  82  0  18  ≤2 to ≥64  100  0  0  ≤2   gentamicin  18  0  82  ≤1 to ≥16  40  0  60  ≤1 to ≥16   tobramycin  27  9  64  ≤1 to ≥16  60  30  10  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  60  0  40  ≤0.25 to ≥4   tigecycline  100  0  0  ≤0.5  100  0  0  ≤0.5   nitrofurantoin  36  64  0  ≤16 to 64  70  30  0  ≤16 to 64   SXT  9  0  91  ≤20 to ≥320  10  0  90  ≤20 to ≥320   colistin  100  N/A  0  <0.25  90  n/a  10  <0.25 to 4  Klebsiella pneumoniae (n = 46)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  3  97  16 to ≥32  8  0  92  ≤2 to ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  3  12  85  8 to ≥64  59  8  33  ≤4 to ≥64   cefpodoxime  3  3  94  2 to ≥8  25  0  75  ≤0.25 to ≥8   cefotaxime  0  6  94  4 to ≥64  25  0  75  ≤1 to ≥64   ceftazidime  3  0  97  ≤1 to ≥64  25  0  75  ≤1 to ≥64   ceftriaxone  0  3  97  2 to ≥64  17  0  83  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  2 to ≥8   meropenem  12  3  85  1 to ≥16  34  8  58  0.5 to ≥16   amikacin  62  6  32  ≤2 to ≥64  92  0  8  ≤2 to ≥64   gentamicin  9  0  91  ≤1 to ≥16  50  0  50  ≤1 to ≥16   tobramycin  0  9  91  8 to ≥16  25  25  50  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  17  8  75  0.5 to ≥4   tigecycline  27  21  52  ≤0.5 to ≥8  75  8  17  ≤0.5 to ≥8   nitrofurantoin  0  6  94  64 to ≥512  8  25  67  32 to ≥512   SXT  3  0  97  ≤20 to >320  25  0  75  40 to ≥320   colistin  94  N/A  6  <0.25 to 16  92  N/A  8  <0.25 to 8  AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; SXT, trimethoprim/sulfamethoxazole; N/A, not applicable. a E. coli (OXA-181, n = 10; OXA-232, n = 1; OXA-48, n = 9; OXA-244, n = 1) and K. pneumoniae (OXA-181, n = 25; OXA-232, n = 9; OXA-48, n = 12). b S, susceptible; I, intermediate; R, resistant. Phylogenetic analysis of the isolates A phylogenetic SNV analysis of the core genome was conducted for all E. coli and K. pneumoniae. All isolate clustering corresponded to STs (data not shown). Separate phylogenetic analyses were then conducted for the dominant ST clusters: E. coli ST38 (n = 6) and ST410 (n = 6), and K. pneumoniae ST14 (n = 24) (Figure 2). The E. coli ST38 cluster was quite diverse in that there were between 23 and ∼3000 SNV differences between isolates. The isolates with the closest genetic relationship (23 SNV differences) were identified from patients in different provinces >14 months apart. The E. coli ST410 cluster differed by as many as 1600 SNVs. A sub-cluster of four isolates differed by 5–21 SNVs, all of which were from different patients and provinces isolated in the same year. The K. pneumoniae ST14 cluster was much more clonal than other STs, exhibiting a maximum of 141 SNV difference between isolates which came from three different provinces. One sub-cluster contained 75% (18/24) of ST14 and showed between 0 and 62 SNVs. These isolates were all from the same province and harboured blaOXA-181. Unfortunately, we were unable to obtain additional epidemiological data to link patients to specific hospital sites. Interestingly, two different patients from two separate provinces contained K. pneumoniae ST14 that only differed by 4 SNVs. Both patients had recent travel history to India where they had sought medical care but it was not known if the two patients received care from the same location in India and no epidemiological link between Canadian hospitals could be made. Figure 2. View largeDownload slide Phylogenetic single nucleotide variant (SNV) tree representing core genome of all E.coli ST38, E. coli ST410 and K. pneumoniae ST14 isolates. Approximate numbers of SNVs between isolates are indicated and represent the shortest SNV difference between isolates or clusters. Province abbreviations: ON, Ontario; BC, British Columbia; QC, Quebec; AB, Alberta; MB, Manitoba. Shaded circles represent multiple isolates from the same patient taken in different years of the study. Figure 2. View largeDownload slide Phylogenetic single nucleotide variant (SNV) tree representing core genome of all E.coli ST38, E. coli ST410 and K. pneumoniae ST14 isolates. Approximate numbers of SNVs between isolates are indicated and represent the shortest SNV difference between isolates or clusters. Province abbreviations: ON, Ontario; BC, British Columbia; QC, Quebec; AB, Alberta; MB, Manitoba. Shaded circles represent multiple isolates from the same patient taken in different years of the study. There were three patients who had two isolates of the same species collected >1 year apart. All of these cases were K. pneumoniae ST14 and the shortest SNV difference between isolates from the same patient was 9 SNVs. Comparison of plasmid content The four most common plasmids in E. coli were IncF variants, ColKP3, IncX3 and IncI1-type plasmids. K. pneumoniae isolates commonly harboured IncF variants, Col-type variants, IncR and IncL/M plasmids (Table S1, available as Supplementary data at JAC Online). Plasmid mapping showed that 14/21 (66.7%) blaOXA-48-containing isolates exhibited 96%–99.9% identity to a known IncL/M OXA-48 plasmid (accession no. LN864820). Of these, 10 were found in K. pneumoniae belonging to eight different STs (ST15, ST37, ST45, ST101, ST307, ST392, ST395, ST514) and four were E. coli belonging to four different STs (ST38, ST156, ST538, ST624). Of the blaOXA-181/232-producing isolates, 13/45 (28.9%) isolates mapped with 100% identity to a known ColKP3 OXA-232 plasmid (accession no. NZ_CP012562). Of these, 12 were K. pneumoniae [ST14 (n = 5), ST16 (n = 1), ST147 (n = 2), ST231 (n = 1), ST395 (n = 1), ST1847 (n = 2)] that contained either blaOXA-181 (n = 8) or blaOXA-232 (n = 4) and one was an E. coli ST167 that harboured blaOXA-232. Nine blaOXA-181-containing isolates (9/45, 20%) mapped with >99.9% identity to a known IncX3 OXA-181 plasmid (accession no. KX523903). All isolates were E. coli and belonged to ST410 (n = 6), ST940 (n = 2) or ST1284 (n = 1). There were two patients who had two isolates from different species collected within the same year. In both cases a K. pneumoniae and E. coli were collected. In the first patient both isolates harboured blaOXA-48 and mapped with 100% identity to an IncL/M plasmid (accession no. KU159085.1). In the second patient both isolates harboured blaOXA-232 and mapped with 100% identity to a ColKP3 plasmid (accession no. NZ_CP012562). Both cases suggested possible plasmid transfer within patients. Characterization of chromosomal OXA-48-like genetic regions Contigs harbouring the blaOXA-48-like carbapenemases were extracted from WGS assembly data and aligned to determine the homology within OXA-48-like regions. Twenty-seven percent (18/67) of OXA-48-like contigs aligned with >99% identity to K. pneumoniae chromosomal DNA, suggesting chromosomal integration of the blaOXA-48-like gene. Figure S1 shows the chromosomal locations of the blaOXA181-232 integration sites across the collection. The most common structure was the insertion of blaOXA-181 downstream of a tRNA structure and upstream of chromosomal genes lysR and a sodium symporter observed in 16 isolates, all of which were K. pneumoniae ST14 from a single province. Although we are unable to determine if these were from the same hospital, there were isolates that were 2 years apart and had 139 SNV differences, suggesting a common ancestor maintaining stability in this province over the course of the study. Overall, the chromosomal integration of blaOXA-181/232 represented 39.1% (18/46) of K. pneumoniae isolates from the reported cases. Discussion Over the course of the study four different OXA-48-like variants were identified, with OXA-181 being the most common followed by OXA-48. Isolates from Central Canada were predominantly OXA-48, whereas OXA-181 was predominantly from the West. Similar to a recent study describing OXA-48-like-containing isolates from the UK,16 we also observed ST38 and ST410 as the most common E. coli ST and ST14 as the most common K. pneumoniae ST. With one exception, all isolates from patients who had travelled to the Indian subcontinent harboured blaOXA-181/-232 and those who had travelled to North Africa or the Middle East harboured blaOXA-48. Both OXA-18111 and OXA-4826 have been well described as endemic to India and the North African/Middle Eastern regions, respectively. There were no dominant STs associated with patients with travel history. These data support the contributing role of OXA-48-type carbapenemase dissemination in part to international travel to endemic regions. Core SNV phylogeny analysis revealed that among the most prevalent STs submitted, K. pneumoniae ST14 appeared to be more phylogenetically related than either of the E. coli ST38 and K. pneumoniae ST410 clusters due to the number of SNVs across the collections in conjunction with the overall time and location where samples were collected. There are limited studies on the definitions of phylogenetic relatedness of non-outbreak and outbreak cases based on SNV differences in E. coli and K. pneumoniae.27–29 In the current study, we had two isolates from two different provinces with four SNV differences, suggesting a common ancestor, yet the epidemiological information provided could not provide a link with the patients. In contrast, we observed nine SNV differences within the same patient where isolates were collected a year apart. More data need to be collected with strong epidemiological information in order to determine definitions for what is considered related in terms of SNV differences. It was our goal to be able to generate phylogenetic comparisons in order to report clustering of isolates back to provinces so that they could perform in-depth epidemiological investigations into acquisition of OXA-48-types within and between hospital sites; this type of epidemiological data are not always available at the national level in Canada. All isolates in this study were resistant to ertapenem and 83.6% of them were non-susceptible to meropenem. Concurrent with previous reports, high levels of resistance to third-generation cephalosporins was due to co-carriage of extended-spectrum β-lactamases16 such as CTX-M-type, which here we found in 85% of the isolates (Table S1). The highest rates of susceptibility were to amikacin (76.1%), tigecycline (59.7%) and colistin (94%), though treatment with these drugs may not be commonly recommended due either to site of infection or toxicity.30,31 Similar to a recent surveillance report on OXA-48-like carbapenemases from the UK,16 we found that blaOXA-48 genes were commonly observed in both K. pneumoniae and E. coli from numerous STs and were often associated with an IncL/M plasmid carriage. In blaOXA-181/232-containing isolates we found that the carbapenemase gene was often associated with carriage of either previously described ColKP316,32 or IncX3 plasmids. In fact all E. coli ST410, which were identified in four different provinces from patients with no known epidemiological links and only one with travel history (Nigeria), mapped with >99% identity to an known IncX3 plasmid harbouring blaOXA-181, suggesting an association of a clonal group with this plasmid type. IncX plasmids are narrow host range plasmids of Enterobacteriaceae, particularly associated with OXA-48-like variants first identified from an E. coli ST41033 and subsequently reported in ST410.16 In contrast to plasmid carriage, we showed three different examples of chromosomal integration, representing 39.1% of blaOXA-181/232-harbouring isolates. This is suggested to be a factor in the dissemination of the OXA-48-type carbapenemases in Canada specifically among K. pneumoniae ST14 in Western Canada. Limitations to the study included the voluntary submission of isolates to the study, the lack of comprehensive patient information to identify risk factors and the inability to extract long contigs of DNA sequence from OXA-48-like plasmids due to short-read sequencing. Since completion of the study we have continued our surveillance of OXA-48-like isolates in Canada from voluntary submitting laboratories and have identified 40 cases from 2015 and 109 cases from 2016 representing 19.7% of CPE cases submitted during this time frame, which is an increase of 5.9% compared with the study period. Analysis of these isolates was beyond the scope of this paper but is ongoing. In conclusion, we have seen an increase in the number of OXA-48-like carbapenemases submitted for reference purposes to the NML from 2011 to 2014 and this trend appears to be continuing. In Canada the presence of patients with OXA-48-like enzymes is linked to patients who have travelled to known endemic regions. It should be noted, however, that a large proportion of Canadian OXA-48-like isolates come from patients with no travel history (59.6%), suggesting local transmission. Continued surveillance to better understand the epidemiology and risk factors for acquisition of these carbapenemase-producing isolates is required. Acknowledgements We gratefully acknowledge Romeo Hizon and Ken Fakrhuddin for their technical expertise, Adrian Zetner for his work on plasmid mapping, and the Genomics Core Facility of the NML for the whole genome sequencing. Funding This study was funded by the Public Health Agency of Canada. Transparency declarations None to declare. Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online. References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect  2014; 20: 821– 30. Google Scholar CrossRef Search ADS PubMed  2 Antunes NT, Lamoureaux TL, Toth M et al.   Class D β-lactamases: are they all carbapenemases? Antimicrob Agents Chemother  2014; 58: 2119– 25. Google Scholar CrossRef Search ADS PubMed  3 Evans BA, Amyes SG. OXA β-lactamases. Clin Microbiol Rev  2014; 27: 241– 63. Google Scholar CrossRef Search ADS PubMed  4 Poirel L, Heritier C, Tolun V et al.   Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob Agents Chemother  2004; 48: 15– 22. Google Scholar CrossRef Search ADS PubMed  5 Potron A, Kalpoe J, Poirel L et al.   European dissemination of a single OXA-48-producing Klebsiella pneumoniae clone. Clin Microbiol Infect  2011; 17: E24– 6. Google Scholar CrossRef Search ADS PubMed  6 Matar GM, Dandache I, Carrer A et al.   Spread of OXA-48-mediated resistance to carbapenems in Lebanese Klebsiella pneumoniae and Escherichia coli that produce extended spectrum β-lactamase. Ann Trop Med Parasitol  2010; 104: 271– 4. Google Scholar CrossRef Search ADS PubMed  7 Carrer A, Poirel L, Yilmaz M et al.   Spread of OXA-48-encoding plasmid in Turkey and beyond. Antimicrob Agents Chemother  2010; 54: 1369– 73. Google Scholar CrossRef Search ADS PubMed  8 Cuzon G, Ouanich J, Gondret R et al.   Outbreak of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in France. Antimicrob Agents Chemother  2011; 55: 2420– 3. Google Scholar CrossRef Search ADS PubMed  9 Walsh TR. Emerging carbapenemases: a global perspective. Int J Antimicrob Agents  2010; 36 Suppl 3: S8– 14. Google Scholar CrossRef Search ADS PubMed  10 Cremet L, Bourigault C, Lepelletier D et al.   Nosocomial outbreak of carbapenem-resistant Enterobacter cloacae highlighting the interspecies transferability of the blaOXA-48 gene in the gut flora. J Antimicrob Chemother  2012; 67: 1041– 3. Google Scholar CrossRef Search ADS PubMed  11 Castanheira M, Deshpande LM, Mathai D et al.   Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY antimicrobial surveillance program, 2006-2007. Antimicrob Agents Chemother  2011; 55: 1274– 8. Google Scholar CrossRef Search ADS PubMed  12 Potron A, Nordmann P, Poirel L. Characterization of OXA-204, a carbapenem-hydrolyzing class D β-lactamase from Klebsiella pneumoniae. Antimicrob Agents Chemother  2013; 57: 633– 6. Google Scholar CrossRef Search ADS PubMed  13 Potron A, Rondinaud E, Poirel L et al.   Genetic and biochemical characterisation of OXA-232, a carbapenem-hydrolysing class D β-lactamase from Enterobacteriaceae. Int J Antimicrob Agents  2013; 41: 325– 9. Google Scholar CrossRef Search ADS PubMed  14 Oteo J, Hernandez JM, Espasa M et al.   Emergence of OXA-48-producing Klebsiella pneumoniae and the novel carbapenemases OXA-244 and OXA-245 in Spain. J Antimicrob Chemother  2013; 68: 317– 21. Google Scholar CrossRef Search ADS PubMed  15 Sampaio JL, Ribeiro VB, Campos JC et al.   Detection of OXA-370, an OXA-48-related class D β-lactamase, in Enterobacter hormaechei from Brazil. Antimicrob Agents Chemother  2014; 58: 3566– 7. Google Scholar CrossRef Search ADS PubMed  16 Findlay J, Hopkins K, Loy R et al.   OXA-48-like carbapenemases in the UK: an analysis of isolates and cases from 2007 to 2014. J Antimicrob Chemother  2017; 72: 1340– 9. Google Scholar CrossRef Search ADS PubMed  17 Poirel L, Castanheira M, Carrer A et al.   OXA-163, an OXA-48-related class D β-lactamase with extended activity toward expanded-spectrum cephalosporins. Antimicrob Agents Chemother  2011; 55: 2546– 51. Google Scholar CrossRef Search ADS PubMed  18 Dortet L, Oueslati S, Jeannot K et al.   Genetic and biochemical characterization of OXA-405, an OXA-48-type extended-spectrum β-lactamase without significant carbapenemase activity. Antimicrob Agents Chemother  2015; 59: 3823– 8. Google Scholar CrossRef Search ADS PubMed  19 Gomez S, Pasteran F, Faccone D et al.   Intrapatient emergence of OXA-247: a novel carbapenemase found in a patient previously infected with OXA-163-producing Klebsiella pneumoniae. Clin Microbiol Infect  2013; 19: E233. Google Scholar CrossRef Search ADS PubMed  20 Mataseje LF, Boyd DA, Hoang L et al.   Carbapenem-hydrolyzing oxacillinase-48 and oxacillinase-181 in Canada, 2011. Emerg Infect Dis  2013; 19: 157– 60. Google Scholar CrossRef Search ADS PubMed  21 Mataseje LF, Abdesselam K, Vachon J et al.   Results from the Canadian nosocomial infection surveillance program on carbapenemase-producing Enterobacteriaceae, 2010 to 2014. Antimicrob Agents Chemother  2016; 60: 6787– 94. Google Scholar CrossRef Search ADS PubMed  22 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement M100-S26 . CLSI, Wayne, PA, USA, 2016. 23 Petkau A, Mabon P, Sieffert C et al.   SNVPhyl: a single nucleotide variant phylogenomics pipeline for microbial genomic epidemiology. BioRxiv  2016; doi:10.1101/092940. 24 Guindon S, Dufayard JF, Lefort V et al.   New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol  2010; 59: 307– 21. Google Scholar CrossRef Search ADS PubMed  25 Mulvey MR, Mataseje LF, Robertson J et al.   Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis  2016; 16: 289– 90. Google Scholar CrossRef Search ADS PubMed  26 Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother  2012; 67: 1597– 606. Google Scholar CrossRef Search ADS PubMed  27 Lee KI, Morita-Ishihara T, Iyoda S et al.   A geographically widespread outbreak investigation and development of a rapid screening method using whole genome sequences of enterohemorrhagic Escherichia coli O121. Front Microbiol  2017; 8: 701. Google Scholar CrossRef Search ADS PubMed  28 Joensen KG, Scheutz F, Lund O et al.   Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli. J Clin Microbiol  2014; 52: 1501– 10. Google Scholar CrossRef Search ADS PubMed  29 Pecora ND, Li N, Allard M et al.   Genomically informed surveillance for carbapenem-resistant Enterobacteriaceae in a health care system. MBio  2015; 6: e01030– 15. Google Scholar CrossRef Search ADS PubMed  30 Noskin GA. Tigecycline: a new glycylcycline for treatment of serious infections. Clin Infect Dis  2005; 41 Suppl 5: S303– 14. Google Scholar CrossRef Search ADS PubMed  31 Spapen H, Jacobs R, Van Gorp V et al.   Renal and neurological side effects of colistin in critically ill patients. Ann Intensive Care  2011; 1: 14– 20. Google Scholar CrossRef Search ADS PubMed  32 Potron A, Nordmann P, Lafeuille E et al.   Characterization of OXA-181, a carbapenem-hydrolyzing class D β-lactamase from Klebsiella pneumoniae. Antimicrob Agents Chemother  2011; 55: 4896– 9. Google Scholar CrossRef Search ADS PubMed  33 Liu Y, Feng Y, Wu W et al.   First report of OXA-181-producing Escherichia coli in China and characterization of the isolate using whole-genome sequencing. Antimicrob Agents Chemother  2015; 59: 5022– 5. Google Scholar CrossRef Search ADS PubMed  © Her Majesty the Queen in Right of Canada 2017. Reproduced with the permission of the Minister of Health. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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Oxford University Press
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© Her Majesty the Queen in Right of Canada 2017. Reproduced with the permission of the Minister of Health.
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Abstract

Abstract 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. Introduction The continued emergence and dissemination of carbapenem-hydrolysing β-lactamases in Enterobacteriaceae has been observed globally over the last 15–20 years. Of the Ambler class A carbapenemases, Klebsiella pneumoniae carbapenemase (KPC) is the most commonly identified in many countries and is endemic to the USA, Columbia, Argentina, Greece, Italy and Israel.1 The Ambler class B enzyme, New Delhi metallo-β-lactamase (NDM-1), emerged in 2009 and has since been reported globally; its dissemination has been commonly associated with intercontinental travel.1 Carbapenem-hydrolysing class D β-lactamases (CHDL), such as OXA-23, OXA-24, OXA-58 and OXA-143, are commonly associated with Acinetobacter spp.2,3 However, in 2001 a K. pneumoniae isolated from a patient in Turkey was found to harbour a novel CHDL, named OXA-48.4 Since then reports of Enterobacteriaceae harbouring OXA-48 have been described across Europe, the Mediterranean and the Middle East.5–10 Similarly, OXA-181, which differs from OXA-48 by four amino acid substitutions, was initially described in 2011 from India.11 Other OXA-48-like variants, such as OXA-204,12 OXA-232,13 OXA-244, OXA-24514 and OXA-370,15 have also been described in Enterobacteriaceae species. OXA-48-like carbapenemases hydrolyse penicillins and carbapenems but not extended-spectrum cephalosporins;4 however, they are often associated with ESBLs, and provide the strain with a similar resistance phenotype to cephalosporins as seen in ESBLs.16 In contrast, a number of OXA-48-types that lack carbapenemase activity have been described, namely OXA-163, OXA-247 and OXA-405. They differ by only 1–2 amino acid substitutions but contain a deletion in the active site which is thought to be responsible for their lack of carbapenemase activity.17–19 The first description of OXA-48-type producers in Canada was from four cases in 2011.20 Here, we describe the molecular and epidemiological characteristics of OXA-48-like producers submitted to the National Microbiology Laboratory (NML) in Canada from 2011 to 2014. Materials and methods The study Enterobacteriaceae isolates non-susceptible to a carbapenem (as determined by the submitting site) isolated from patients from 2011 to 2014 were voluntarily submitted to the NML by Canadian hospitals and provincial public health laboratories for carbapenemase gene detection. Duplicated isolates were defined as isolates of the same species collected from a single patient in the same year and were excluded. Isolates of different species in same year or same species in different years from same patients were included. Multiplex PCR for carbapenemase genes (blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 and blaGES) was conducted as previously described.21 Regional distribution was defined as follows: Western (British Columbia, Alberta, Saskatchewan and Manitoba), Central (Ontario and Quebec), Eastern (Nova Scotia, Prince Edward Island, New Brunswick and Newfoundland). Antimicrobial susceptibility testing Antimicrobial susceptibilities were determined using Vitek 2 (AST-GN25; bioMerieux, St Laurent, Canada) using 2016 CLSI breakpoints.22 Tigecycline breakpoints were based on FDA criteria for Enterobacteriaceae [susceptible (S), ≤2 mg/L; intermediate (I), 4 mg/L; resistant (R), ≥8 mg/L]. Custom-made broth microdilution plates (0.25–16 mg/L range) were used to determine colistin susceptibilities with CLSI breakpoints for Acinetobacter spp. (S ≤ 2 mg/L, R > 2 mg/L). Student’s t-test was used for comparison of the antimicrobial resistance profiles of the K. pneumoniae versus the Escherichia coli with the significance level set at P ≤ 0.05. Short-read WGS analysis Total cellular DNA was prepared using Epicentre MasterPureTM Complete kits (Mandel Scientific, Guelph, ON, Canada). Libraries were created with TruSeq Nano DNA HT sample preparation kits (Illumina, San Diego, CA, USA). Paired-end, 301 bp indexed reads were generated on an Illumina MiSeqTM platform (Illumina). All raw read data were uploaded to the Sequence Read Archive (SRA) hosted by NCBI under the BioProject PRJNA390933. An average of 130× coverage was achieved. De novo assemblies of Illumina reads were done using Spades version 3.5. Assembled sequence data were analysed using the batch uploader mode at the Centre for Genomic Epidemiology website (https://cge.cbs.dtu.dk/services/) and data were produced from the ResFinder, PlasmidFinder and MLST tools. Plasmid analysis was performed by mapping the sequencing reads against a database of all Gammaproteobacteria plasmids available from NCBI (https://www.ncbi.nlm.nih.gov/genome/browse/#tabs-plasmids) to identify plasmids associated with OXA-48-like carbapenemases. Phylogenetic analysis based on core single nucleotide variants (SNVs) SNV analysis was conducted using a previously published pipeline.23 The following parameters were set: map/base quality 30, alternate fraction 0.75, minimum read coverage of identification of variants 15 and SNV density filtering of 2 SNVs within a 20 bp window. Maximum likelihood phylogenetic trees were built using PhyML version 3.1.24 Images were rendered in FigTree (version 1.4.1) (http://tree.bio.ed.ac.uk/software/figtree). References used were assembled multifasta contigs of genomes from the current study, which represented specific ST groups (E. coli ST38: N11-01498; E. coli ST410: N14-00405; K. pneumoniae ST14: N14-02136). Results Molecular summary and patient demographics During 2011 (n = 4), 2012 (n = 22), 2013 (n = 15) and 2014 (n = 26), a total of 67 non-duplicate isolates producing OXA-48-like carbapenemases from 62 patients were submitted to the NML. These cases were only identified in E. coli (n = 21) and K. pneumoniae (n = 46). Variant gene types identified were blaOXA-48 (31.3%, n = 21), blaOXA-181 (52.2%, n = 35), blaOXA-232 (a 1 bp variant of blaOXA-181; 14.9%, n = 10) and blaOXA-244 (a 1 bp variant of blaOXA-48; 1.5%, n = 1). Of these, two blaOXA-181 containing isolates were found to co-produce either blaNDM-1 or blaNDM-4, and three blaOXA-232 containing isolates were found to co-produce blaNDM-1 or blaNDM-5. One isolate co-produced the mobile colistin resistance gene mcr-1, which has been previously reported.25 Patient demographics available for gender (n = 57, 92%) showed 50.9% and 49.1% of cases to be from male or female patients, respectively. Available age ranges (n = 57) were 22–64 years (38.6%) or ≥65 years (61.4%). Organisms were isolated predominantly from urine (37.9%), rectal (28.8%) or respiratory (10.6%) samples. Where infection or colonization was determined (n = 37), 45.9% of cases were considered infections. Travel history was collected for 57 patients, of whom 23 (40.4%) indicated travel outside of Canada in the previous 12 months from date of positive culture. With the exception of a patient harbouring blaOXA-48 with travel history to Pakistan, all isolates from patients with travel history to the Indian subcontinent (n = 11), Nigeria (n = 1), Ukraine (n = 1) or the USA (n = 1) harboured blaOXA-181/-232. These patients harboured either E. coli (n = 3) belonging to ST940 (n = 2) or ST410 (n = 1), or K. pneumoniae (n = 11) belonging to ST14 (n = 6), ST147 (n = 3), ST16 (n = 1) or ST1847 (n = 1). Individuals with travel history to North Africa (n = 2; Egypt, Libya) or the Middle East [n = 6; Lebanon (n = 2), Syria (n = 1), Saudi Arabia (n = 2), United Arab Emirates (n = 1)] harboured blaOXA-48. These patients harboured either E. coli (n = 5) belonging to ST38 (n = 3) or ST624 (n = 1), or K. pneumoniae (n = 3) belonging to ST101, ST15 or ST831. Figure 1 shows the distribution of OXA-like-types across Canada by region and ST. The dominant clones in E. coli were ST38 (28.6%) and ST410 (28.6%). All ST410 isolates were blaOXA-181 producers and were equally observed in both Western and Central Canada. ST38 were all blaOXA-48 producers from Central Canada with the exception of one blaOXA-244 from the Western region. For K. pneumoniae the dominant clone was ST14 observed in 54.3% of isolates. Of these 72% were blaOXA-181 producers from the West and the remaining were blaOXA-232 from both West and Central regions. Figure 1. View largeDownload slide Graphic representation of the distribution of OXA-like-types and STs across three Canadian regions. The inner circle represents the distribution between regions, the second circle represents the OXA-type and the outer circle represents the ST. Figure 1. View largeDownload slide Graphic representation of the distribution of OXA-like-types and STs across three Canadian regions. The inner circle represents the distribution between regions, the second circle represents the OXA-type and the outer circle represents the ST. Characterization of antimicrobial resistance The antimicrobial susceptibilities for E. coli and K. pneumoniae are shown in Table 1. All isolates were resistant to ertapenem; however, only 61.2% were resistant to meropenem. Imipenem susceptibilities were not determined. Resistance to piperacillin/tazobactam, ceftazidime and cefotaxime was observed in 100%, 82.1% and 91.1% of isolates, respectively. Overall, 85% of the isolates harboured genes associated with ESBLs. Of the 15 E. coli harbouring ESBLs, blaCTX-M-15 (n = 8),blaCTX-M-24 (n = 3), blaCTX-M-14 (n = 2), and blaCTX-M-15 plus blaTEM-33 (n = 2) were identified. For K. pneumoniae harbouring ESBLs (n = 44) the majority contained blaCTX-M-15 (n = 42), followed by blaSHV-28 (n = 2). Additionally, resistance to trimethoprim/sulfamethoxazole (91%), ciprofloxacin (87%), gentamicin (75%) and tobramycin (68%) was often observed. Tigecycline and colistin resistance was observed in 30% and 6% of isolates, respectively. Overall, significantly more K. pneumoniae isolates were resistant to meropenem, tobramycin, nitrofurantoin, ciprofloxacin and tigecycline (P = <0.0001–0.0028) than E. coli. This is due to the higher proportion of blaOXA-181/-232 producers in K. pneumoniae isolates as blaOXA-181/-232 producers were also significantly more resistant to all of the above (P = <0.0001–0.0223) compared with blaOXA-48 producers, with the exception of meropenem, which was not significant (P = 0.1761). Table 1. Antimicrobial susceptibilities of OXA-48-type producing E. coli and K. pneumoniae in Canada   OXA-181, OXA-232   OXA-48, OXA-244a   Organism (no. tested)/antimicrobial  %Sb  %I  %R  range (mg/L)  %S  %I  %R  range (mg/L)  Escherichia coli (n = 21)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  0  100  ≥32  0  0  100  ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  0  0  100  ≥32  30  20  50  ≤4 to ≥32   cefpodoxime  0  0  100  ≥8  30  0  70  1 to ≥8   cefotaxime  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ceftazidime  0  0  100  ≥64  80  0  20  ≤1 to 16   ceftriaxone  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  4 to ≥8   meropenem  27  55  18  0.5 to ≥16  10  60  30  1 to ≥16   amikacin  82  0  18  ≤2 to ≥64  100  0  0  ≤2   gentamicin  18  0  82  ≤1 to ≥16  40  0  60  ≤1 to ≥16   tobramycin  27  9  64  ≤1 to ≥16  60  30  10  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  60  0  40  ≤0.25 to ≥4   tigecycline  100  0  0  ≤0.5  100  0  0  ≤0.5   nitrofurantoin  36  64  0  ≤16 to 64  70  30  0  ≤16 to 64   SXT  9  0  91  ≤20 to ≥320  10  0  90  ≤20 to ≥320   colistin  100  N/A  0  <0.25  90  n/a  10  <0.25 to 4  Klebsiella pneumoniae (n = 46)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  3  97  16 to ≥32  8  0  92  ≤2 to ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  3  12  85  8 to ≥64  59  8  33  ≤4 to ≥64   cefpodoxime  3  3  94  2 to ≥8  25  0  75  ≤0.25 to ≥8   cefotaxime  0  6  94  4 to ≥64  25  0  75  ≤1 to ≥64   ceftazidime  3  0  97  ≤1 to ≥64  25  0  75  ≤1 to ≥64   ceftriaxone  0  3  97  2 to ≥64  17  0  83  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  2 to ≥8   meropenem  12  3  85  1 to ≥16  34  8  58  0.5 to ≥16   amikacin  62  6  32  ≤2 to ≥64  92  0  8  ≤2 to ≥64   gentamicin  9  0  91  ≤1 to ≥16  50  0  50  ≤1 to ≥16   tobramycin  0  9  91  8 to ≥16  25  25  50  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  17  8  75  0.5 to ≥4   tigecycline  27  21  52  ≤0.5 to ≥8  75  8  17  ≤0.5 to ≥8   nitrofurantoin  0  6  94  64 to ≥512  8  25  67  32 to ≥512   SXT  3  0  97  ≤20 to >320  25  0  75  40 to ≥320   colistin  94  N/A  6  <0.25 to 16  92  N/A  8  <0.25 to 8    OXA-181, OXA-232   OXA-48, OXA-244a   Organism (no. tested)/antimicrobial  %Sb  %I  %R  range (mg/L)  %S  %I  %R  range (mg/L)  Escherichia coli (n = 21)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  0  100  ≥32  0  0  100  ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  0  0  100  ≥32  30  20  50  ≤4 to ≥32   cefpodoxime  0  0  100  ≥8  30  0  70  1 to ≥8   cefotaxime  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ceftazidime  0  0  100  ≥64  80  0  20  ≤1 to 16   ceftriaxone  0  0  100  ≥64  30  0  70  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  4 to ≥8   meropenem  27  55  18  0.5 to ≥16  10  60  30  1 to ≥16   amikacin  82  0  18  ≤2 to ≥64  100  0  0  ≤2   gentamicin  18  0  82  ≤1 to ≥16  40  0  60  ≤1 to ≥16   tobramycin  27  9  64  ≤1 to ≥16  60  30  10  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  60  0  40  ≤0.25 to ≥4   tigecycline  100  0  0  ≤0.5  100  0  0  ≤0.5   nitrofurantoin  36  64  0  ≤16 to 64  70  30  0  ≤16 to 64   SXT  9  0  91  ≤20 to ≥320  10  0  90  ≤20 to ≥320   colistin  100  N/A  0  <0.25  90  n/a  10  <0.25 to 4  Klebsiella pneumoniae (n = 46)   ampicillin  0  0  100  ≥32  0  0  100  ≥32   AMC  0  3  97  16 to ≥32  8  0  92  ≤2 to ≥32   TZP  0  0  100  ≥32  0  0  100  ≥32   cefazolin  0  0  100  ≥32  0  0  100  ≥32   cefoxitin  3  12  85  8 to ≥64  59  8  33  ≤4 to ≥64   cefpodoxime  3  3  94  2 to ≥8  25  0  75  ≤0.25 to ≥8   cefotaxime  0  6  94  4 to ≥64  25  0  75  ≤1 to ≥64   ceftazidime  3  0  97  ≤1 to ≥64  25  0  75  ≤1 to ≥64   ceftriaxone  0  3  97  2 to ≥64  17  0  83  ≤1 to ≥64   ertapenem  0  0  100  4 to ≥8  0  0  100  2 to ≥8   meropenem  12  3  85  1 to ≥16  34  8  58  0.5 to ≥16   amikacin  62  6  32  ≤2 to ≥64  92  0  8  ≤2 to ≥64   gentamicin  9  0  91  ≤1 to ≥16  50  0  50  ≤1 to ≥16   tobramycin  0  9  91  8 to ≥16  25  25  50  ≤1 to ≥16   ciprofloxacin  0  0  100  ≥4  17  8  75  0.5 to ≥4   tigecycline  27  21  52  ≤0.5 to ≥8  75  8  17  ≤0.5 to ≥8   nitrofurantoin  0  6  94  64 to ≥512  8  25  67  32 to ≥512   SXT  3  0  97  ≤20 to >320  25  0  75  40 to ≥320   colistin  94  N/A  6  <0.25 to 16  92  N/A  8  <0.25 to 8  AMC, amoxicillin/clavulanic acid; TZP, piperacillin/tazobactam; SXT, trimethoprim/sulfamethoxazole; N/A, not applicable. a E. coli (OXA-181, n = 10; OXA-232, n = 1; OXA-48, n = 9; OXA-244, n = 1) and K. pneumoniae (OXA-181, n = 25; OXA-232, n = 9; OXA-48, n = 12). b S, susceptible; I, intermediate; R, resistant. Phylogenetic analysis of the isolates A phylogenetic SNV analysis of the core genome was conducted for all E. coli and K. pneumoniae. All isolate clustering corresponded to STs (data not shown). Separate phylogenetic analyses were then conducted for the dominant ST clusters: E. coli ST38 (n = 6) and ST410 (n = 6), and K. pneumoniae ST14 (n = 24) (Figure 2). The E. coli ST38 cluster was quite diverse in that there were between 23 and ∼3000 SNV differences between isolates. The isolates with the closest genetic relationship (23 SNV differences) were identified from patients in different provinces >14 months apart. The E. coli ST410 cluster differed by as many as 1600 SNVs. A sub-cluster of four isolates differed by 5–21 SNVs, all of which were from different patients and provinces isolated in the same year. The K. pneumoniae ST14 cluster was much more clonal than other STs, exhibiting a maximum of 141 SNV difference between isolates which came from three different provinces. One sub-cluster contained 75% (18/24) of ST14 and showed between 0 and 62 SNVs. These isolates were all from the same province and harboured blaOXA-181. Unfortunately, we were unable to obtain additional epidemiological data to link patients to specific hospital sites. Interestingly, two different patients from two separate provinces contained K. pneumoniae ST14 that only differed by 4 SNVs. Both patients had recent travel history to India where they had sought medical care but it was not known if the two patients received care from the same location in India and no epidemiological link between Canadian hospitals could be made. Figure 2. View largeDownload slide Phylogenetic single nucleotide variant (SNV) tree representing core genome of all E.coli ST38, E. coli ST410 and K. pneumoniae ST14 isolates. Approximate numbers of SNVs between isolates are indicated and represent the shortest SNV difference between isolates or clusters. Province abbreviations: ON, Ontario; BC, British Columbia; QC, Quebec; AB, Alberta; MB, Manitoba. Shaded circles represent multiple isolates from the same patient taken in different years of the study. Figure 2. View largeDownload slide Phylogenetic single nucleotide variant (SNV) tree representing core genome of all E.coli ST38, E. coli ST410 and K. pneumoniae ST14 isolates. Approximate numbers of SNVs between isolates are indicated and represent the shortest SNV difference between isolates or clusters. Province abbreviations: ON, Ontario; BC, British Columbia; QC, Quebec; AB, Alberta; MB, Manitoba. Shaded circles represent multiple isolates from the same patient taken in different years of the study. There were three patients who had two isolates of the same species collected >1 year apart. All of these cases were K. pneumoniae ST14 and the shortest SNV difference between isolates from the same patient was 9 SNVs. Comparison of plasmid content The four most common plasmids in E. coli were IncF variants, ColKP3, IncX3 and IncI1-type plasmids. K. pneumoniae isolates commonly harboured IncF variants, Col-type variants, IncR and IncL/M plasmids (Table S1, available as Supplementary data at JAC Online). Plasmid mapping showed that 14/21 (66.7%) blaOXA-48-containing isolates exhibited 96%–99.9% identity to a known IncL/M OXA-48 plasmid (accession no. LN864820). Of these, 10 were found in K. pneumoniae belonging to eight different STs (ST15, ST37, ST45, ST101, ST307, ST392, ST395, ST514) and four were E. coli belonging to four different STs (ST38, ST156, ST538, ST624). Of the blaOXA-181/232-producing isolates, 13/45 (28.9%) isolates mapped with 100% identity to a known ColKP3 OXA-232 plasmid (accession no. NZ_CP012562). Of these, 12 were K. pneumoniae [ST14 (n = 5), ST16 (n = 1), ST147 (n = 2), ST231 (n = 1), ST395 (n = 1), ST1847 (n = 2)] that contained either blaOXA-181 (n = 8) or blaOXA-232 (n = 4) and one was an E. coli ST167 that harboured blaOXA-232. Nine blaOXA-181-containing isolates (9/45, 20%) mapped with >99.9% identity to a known IncX3 OXA-181 plasmid (accession no. KX523903). All isolates were E. coli and belonged to ST410 (n = 6), ST940 (n = 2) or ST1284 (n = 1). There were two patients who had two isolates from different species collected within the same year. In both cases a K. pneumoniae and E. coli were collected. In the first patient both isolates harboured blaOXA-48 and mapped with 100% identity to an IncL/M plasmid (accession no. KU159085.1). In the second patient both isolates harboured blaOXA-232 and mapped with 100% identity to a ColKP3 plasmid (accession no. NZ_CP012562). Both cases suggested possible plasmid transfer within patients. Characterization of chromosomal OXA-48-like genetic regions Contigs harbouring the blaOXA-48-like carbapenemases were extracted from WGS assembly data and aligned to determine the homology within OXA-48-like regions. Twenty-seven percent (18/67) of OXA-48-like contigs aligned with >99% identity to K. pneumoniae chromosomal DNA, suggesting chromosomal integration of the blaOXA-48-like gene. Figure S1 shows the chromosomal locations of the blaOXA181-232 integration sites across the collection. The most common structure was the insertion of blaOXA-181 downstream of a tRNA structure and upstream of chromosomal genes lysR and a sodium symporter observed in 16 isolates, all of which were K. pneumoniae ST14 from a single province. Although we are unable to determine if these were from the same hospital, there were isolates that were 2 years apart and had 139 SNV differences, suggesting a common ancestor maintaining stability in this province over the course of the study. Overall, the chromosomal integration of blaOXA-181/232 represented 39.1% (18/46) of K. pneumoniae isolates from the reported cases. Discussion Over the course of the study four different OXA-48-like variants were identified, with OXA-181 being the most common followed by OXA-48. Isolates from Central Canada were predominantly OXA-48, whereas OXA-181 was predominantly from the West. Similar to a recent study describing OXA-48-like-containing isolates from the UK,16 we also observed ST38 and ST410 as the most common E. coli ST and ST14 as the most common K. pneumoniae ST. With one exception, all isolates from patients who had travelled to the Indian subcontinent harboured blaOXA-181/-232 and those who had travelled to North Africa or the Middle East harboured blaOXA-48. Both OXA-18111 and OXA-4826 have been well described as endemic to India and the North African/Middle Eastern regions, respectively. There were no dominant STs associated with patients with travel history. These data support the contributing role of OXA-48-type carbapenemase dissemination in part to international travel to endemic regions. Core SNV phylogeny analysis revealed that among the most prevalent STs submitted, K. pneumoniae ST14 appeared to be more phylogenetically related than either of the E. coli ST38 and K. pneumoniae ST410 clusters due to the number of SNVs across the collections in conjunction with the overall time and location where samples were collected. There are limited studies on the definitions of phylogenetic relatedness of non-outbreak and outbreak cases based on SNV differences in E. coli and K. pneumoniae.27–29 In the current study, we had two isolates from two different provinces with four SNV differences, suggesting a common ancestor, yet the epidemiological information provided could not provide a link with the patients. In contrast, we observed nine SNV differences within the same patient where isolates were collected a year apart. More data need to be collected with strong epidemiological information in order to determine definitions for what is considered related in terms of SNV differences. It was our goal to be able to generate phylogenetic comparisons in order to report clustering of isolates back to provinces so that they could perform in-depth epidemiological investigations into acquisition of OXA-48-types within and between hospital sites; this type of epidemiological data are not always available at the national level in Canada. All isolates in this study were resistant to ertapenem and 83.6% of them were non-susceptible to meropenem. Concurrent with previous reports, high levels of resistance to third-generation cephalosporins was due to co-carriage of extended-spectrum β-lactamases16 such as CTX-M-type, which here we found in 85% of the isolates (Table S1). The highest rates of susceptibility were to amikacin (76.1%), tigecycline (59.7%) and colistin (94%), though treatment with these drugs may not be commonly recommended due either to site of infection or toxicity.30,31 Similar to a recent surveillance report on OXA-48-like carbapenemases from the UK,16 we found that blaOXA-48 genes were commonly observed in both K. pneumoniae and E. coli from numerous STs and were often associated with an IncL/M plasmid carriage. In blaOXA-181/232-containing isolates we found that the carbapenemase gene was often associated with carriage of either previously described ColKP316,32 or IncX3 plasmids. In fact all E. coli ST410, which were identified in four different provinces from patients with no known epidemiological links and only one with travel history (Nigeria), mapped with >99% identity to an known IncX3 plasmid harbouring blaOXA-181, suggesting an association of a clonal group with this plasmid type. IncX plasmids are narrow host range plasmids of Enterobacteriaceae, particularly associated with OXA-48-like variants first identified from an E. coli ST41033 and subsequently reported in ST410.16 In contrast to plasmid carriage, we showed three different examples of chromosomal integration, representing 39.1% of blaOXA-181/232-harbouring isolates. This is suggested to be a factor in the dissemination of the OXA-48-type carbapenemases in Canada specifically among K. pneumoniae ST14 in Western Canada. Limitations to the study included the voluntary submission of isolates to the study, the lack of comprehensive patient information to identify risk factors and the inability to extract long contigs of DNA sequence from OXA-48-like plasmids due to short-read sequencing. Since completion of the study we have continued our surveillance of OXA-48-like isolates in Canada from voluntary submitting laboratories and have identified 40 cases from 2015 and 109 cases from 2016 representing 19.7% of CPE cases submitted during this time frame, which is an increase of 5.9% compared with the study period. Analysis of these isolates was beyond the scope of this paper but is ongoing. In conclusion, we have seen an increase in the number of OXA-48-like carbapenemases submitted for reference purposes to the NML from 2011 to 2014 and this trend appears to be continuing. In Canada the presence of patients with OXA-48-like enzymes is linked to patients who have travelled to known endemic regions. It should be noted, however, that a large proportion of Canadian OXA-48-like isolates come from patients with no travel history (59.6%), suggesting local transmission. Continued surveillance to better understand the epidemiology and risk factors for acquisition of these carbapenemase-producing isolates is required. Acknowledgements We gratefully acknowledge Romeo Hizon and Ken Fakrhuddin for their technical expertise, Adrian Zetner for his work on plasmid mapping, and the Genomics Core Facility of the NML for the whole genome sequencing. Funding This study was funded by the Public Health Agency of Canada. Transparency declarations None to declare. Supplementary data Table S1 and Figure S1 are available as Supplementary data at JAC Online. References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect  2014; 20: 821– 30. Google Scholar CrossRef Search ADS PubMed  2 Antunes NT, Lamoureaux TL, Toth M et al.   Class D β-lactamases: are they all carbapenemases? Antimicrob Agents Chemother  2014; 58: 2119– 25. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 1, 2018

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