Laura Mataseje

Carbapenem-resistant Gram-negative bacilli in Canada 2009–10: results from the Canadian Nosocomial Infection Surveillance Program (CNISP)

OBJECTIVES To investigate the occurrence and molecular mechanisms associated with carbapenemases in carbapenem-resistant Gram-negative isolates from Canadian cases. METHODS Twenty hospital sites across Canada submitted isolates for a 1 year period starting 1 September 2009. All Enterobacteriaceae with MICs ≥ 2 mg/L and Acinetobacter baumannii and Pseudomonas aeruginosa with MICs ≥ 16 mg/L of carbapenems were submitted to the National Microbiology Laboratory (NML) where carbapenem MICs were confirmed by Etest and isolates were characterized by PCR for carbapenemase genes, antimicrobial susceptibilities, PFGE and plasmid isolation. RESULTS A total of 444 isolates (298 P. aeruginosa, 134 Enterobacteriaceae and 12 A. baumannii) were submitted to the NML of which 274 (61.7%; 206 P. aeruginosa, 59 Enterobacteriaceae and 9 A. baumannii) met the inclusion criteria as determined by Etest. Carbapenemase genes were identified in 30 isolates: bla(GES-5) (n = 3; P. aeruginosa), bla(KPC-3) (n = 7; Enterobacteriaceae), bla(NDM-1) (n = 2; Enterobacteriaceae), bla(VIM-2) and bla(VIM-4) (n = 8; P. aeruginosa) bla(SME-2) (n = 1; Enterobacteriaceae) and bla(OXA-23) (n = m9; A. baumannii). PFGE identified a cluster in each of Enterobacteriaceae, P. aeruginosa and A. baumannii corresponding to isolates harbouring carbapenemase genes. Three KPC plasmid patterns (IncN and FllA) were identified where indistinguishable plasmid patterns were identified in unrelated clinical isolates. CONCLUSIONS Carbapenemases were rare at the time of this study. Dissemination of carbapenemases was due to both dominant clones and common plasmid backbones.

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.

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.

Plasmid comparison and molecular analysis of Klebsiella pneumoniae harbouring blaKPC from New York City and Toronto

OBJECTIVES This study examined Klebsiella pneumoniae clinical isolates and their bla(KPC) plasmids to determine potential relatedness of the isolates and their plasmids harbouring carbapenem resistance mechanisms. METHODS K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae from New York City (NYC) (n = 19) and Toronto (n = 2) were typed by PFGE and multilocus sequence typing (MLST). bla(KPC)-harbouring plasmids were transformed into Escherichia coli DH10B(TM), restricted using EcoRI and analysed for bla content and replicon (rep) type. Susceptibility profiles for clinical and transformed strains were determined by automated microbroth dilution using CLSI breakpoints. Outer membrane protein (OMP) genes were analysed by sequencing of ompk35 and ompk36. RESULTS PFGE analysis identified 17 related strains (≥ 80% similarity; 11 KPC-2, 6 KPC-3) where ST258 was the dominant clonal type. All clinical isolates contained both bla(SHV) and bla(TEM-1) and, with the exception of one isolate, were multidrug resistant (MDR). Transformed KPC plasmids (n = 21) carried TEM-1 (n = 18) and were MDR (n = 5). Three plasmid clusters, repFIIA (n = 10), repR (n = 3) and an unknown type (n = 3), were observed. repFllA plasmids were observed from both NYC and Toronto strains. OMP gene analysis revealed premature stop codons in ompk35 and numerous deletions and insertions in ompk36. CONCLUSIONS The dissemination of bla(KPC) is due both to carriage of similar KPC-harbouring plasmids within genetically distinct K. pneumoniae and to clonal spread of K. pneumoniae with unrelated KPC plasmids.

Characterization of Cefoxitin-Resistant Escherichia coli Isolates from Recreational Beaches and Private Drinking Water in Canada between 2004 and 2006

ABSTRACT A total of 142 cefoxitin-resistant Escherichia coli isolates from water sources were collected across Canada. Multidrug resistance was observed in 65/142 (45.8%) isolates. The blaCMY-2 gene was identified in 110/142 (77.5%) isolates. Sequencing of the chromosomal ampC promoter region showed mutations from the wild type, previously shown to hyperproduce AmpC. CMY-2-producing plasmids predominantly belonged to replicon groups I1-Iγ, A/C, and K/B. The majority of the E. coli isolates belonged to the nonvirulent phylogenetic groups A and B1.

Comparison of Antimicrobial Resistance Profiles among Extended-Spectrum-β-Lactamase-Producing and Acquired AmpC β-Lactamase-Producing Escherichia coli Isolates from Canadian Intensive Care Units

ABSTRACT Resistance profiles were compared among 18 extended-spectrum-β-lactamase-producing (ESBL) and 27 acquired AmpC β-lactamase-producing Escherichia coli isolates collected from Canadian intensive care units from 2005 to 2006. ESBL-producing E. coli isolates were more likely to be gentamicin resistant (P < 0.03), fluoroquinolone resistant (P < 0.0001), and multidrug resistant (P < 0.0001) than AmpC-producing E. coli isolates.

Complete sequences of a novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type plasmid from Klebsiella pneumoniae identified in Canada

OBJECTIVES Emergence of plasmids harbouring bla(NDM-1) is a major public health concern due to their association with multidrug resistance and their potential mobility. METHODS PCR was used to detect bla(NDM-1) from clinical isolates of Providencia rettgeri (PR) and Klebsiella pneumoniae (KP). Antimicrobial susceptibilities were determined using Vitek 2. The complete DNA sequence of two bla(NDM-1) plasmids (pPrY2001 and pKp11-42) was obtained using a 454-Genome Sequencer FLX. Contig assembly and gap closures were confirmed by PCR-based sequencing. Comparative analysis was done using BLASTn and BLASTp algorithms. RESULTS Both clinical isolates were resistant to all β-lactams, carbapenems, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to tigecycline. Plasmid pPrY2001 (113 295 bp) was isolated from PR. It did not show significant homology to any known plasmid backbone and contained a truncated repA and novel repB. Two bla(NDM-1)-harbouring plasmids from Acinetobacter lwoffii (JQ001791 and JQ060896) shared 100% similarity to a 15 kb region that contained bla(NDM-1). pPrY2001 also contained a type II toxin/antitoxin system. pKp11-42 (146 695 bp) was isolated from KP. It contained multiple repA genes. The plasmid backbone had the highest homology to the IncFIIk plasmid type (51% coverage, 100% nucleotide identity). The bla(NDM-1) region was unique in that it was flanked upstream by IS3000 and downstream by a novel transposon designated Tn6229. pKp11-42 also contained a number of mutagenesis and plasmid stability proteins. CONCLUSIONS pPrY2001 differed from all known plasmids due to its novel backbone and repB. pKp11-42 was similar to IncFIIk plasmids and contained a number of genes that aid in plasmid persistence.

Polyclonal Outbreak of KPC-3-Producing Enterobacter cloacae at a Single Hospital in Montréal, Québec, Canada

ABSTRACT From September 2010 to December 2011, 26 KPC-3-producing Enterobacter cloacae isolates were identified from 16 patients at a single hospital. Analyses revealed the bla KPC gene to be localized on multiple plasmids in a diverse nonclonal E. cloacae genetic background. These findings highlight the potential complexity of a KPC outbreak at a single hospital.

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.

Carbapenem-hydrolyzing Oxacillinase-48 and Oxacillinase-181 in Canada, 2011

To the Editor: In 2001, a Klebsiella pneumoniae isolate from a patient in Turkey was found to harbor a novel class D carbapenem-hydrolyzing oxacillinase, OXA-48 (1). Although this enzyme hydrolyzes carbapenems at a low level and shows weak activity against expanded-spectrum cephalosporins, it is often associated with other β-lactamases and is multidrug resistant. Reports of Enterobacteriaceae harboring OXA-48 have been described across Europe, the Mediterranean area, and the Middle East (2). In addition, OXA-181, which differs from OXA-48 by 4 aa substitutions, has been described in India (2). We describe the emergence of OXA-48 and OXA-181 in Canada. Hospital and provincial public health laboratories in Canada voluntarily submitted Enterobacteriaceae isolates to the National Microbiology Laboratory. Isolates submitted by the laboratories were not susceptible to carbapenems and were to be tested by PCR for carbapenemase genes (KPC, NDM, IMP, VIM, OXA-48, and GES) (3). During April–November 2011, a total of 4 isolates (3 K. pneumoniae, 1 Escherichia coli) tested positive for the blaOXA-48–type gene. Sequencing, using the primers preOXA-48A and -48B (4), revealed that 3 of the isolates (K. pneumoniae 11-882 and 11-2720 and E. coli 11-1498) possessed the blaOXA-48 gene, and the other isolate (K. pneumoniae 11-2568) possessed the blaOXA-181 gene. We conducted additional β-lactamase PCR and sequencing as described (3) (Table). The Modified Hodge test (using a 10-μg disk of ertapenem and meropenem) showed that all isolates were strongly positive for carbapenemase production. Table Patient data and antimicrobial drug susceptibility profiles for isolates of Klebsiella pneumoniae and Escherichia coli harboring OXA-type carbapenemases, Canada, 2011* The Table shows clinical data and antimicrobial drug susceptibility profiles for the K. pneumoniae and E. coli harboring OXA-type carbapenemases. Half of the isolates were from men >65 years of age. Three of the case-patients had a history of travel to regions where OXA-48–type carbapenemases are endemic (Lebanon, India, and Dubai), and while still abroad (shortly before being hospitalized in Canada), 2 case-patients had sought medical attention. Pulsed-field gel electrophoresis of 3 K. pneumoniae isolates digested with XbaI showed unique fingerprint patterns (data not shown). Multilocus sequence typing (www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) of K. pneumoniae 11-2568, 11-882, and 11-2720 showed that they belonged to sequence type (ST) 147, ST395, and ST831, respectively. ST395 has been identified in K. pneumoniae from Morocco, Amsterdam, and France harboring blaOXA-48 (2). E. coli 11-1498 was shown to belong to sequence type 38, previously described in a nosocomial outbreak and in isolated cases of OXA-48 in France; travel to Morocco and Egypt was linked to those cases (5,6). Phylogenetic grouping of the E. coli isolate revealed that it belonged to group D, which is associated with virulent extraintestinal strains (7). Antimicrobial drug susceptibilities were determined by using Vitek 2 (GN-25) (bioMerieux, Canada Inc., St Laurent, Quebec, Canada) and interpreted by using the 2012 guidelines (M100-S22) of the Clinical Laboratory Standards Institute (www.clsi.org/source/orders/free/m100-s22.pdf) (Table). All but 1 isolate (K. pneumoniae 11-2720) was multidrug resistant (defined as resistant to >3 antimicrobial drug classes). All isolates were resistant to ampicillin, cefazolin, and pipericillin/tazobactam. Two isolates were resistant to broad-range cephalosporins, both of which contained the CTX-M–type extended-spectrum β-lactamase. All isolates were also resistant to ertapenem, 2 of 4 were resistant to imipenem, and 1 was resistant to meropenem. Results were confirmed by Etest (bioMerieux, Canada Inc.) and Clinical Laboratory Standards Institute disk diffusion, with the exception of results for K. pneumoniae 11-882, which showed susceptibility to meropenem and imipenem by Etest and intermediate susceptibility by disk diffusion (Table). All isolates were susceptible to tigecycline and colistin. PCR mapping, using previously described primers (8), identified the blaOXA-48 gene located on the transposon Tn1999. The blaOXA-181 gene was found downstream of ISEcp1, as described (4). We isolated plasmid DNA by using QIAGEN Plasmid Mini Kits (QIAGEN, Mississauga, ON, Canada) and attempted to transfer the plasmid harboring the blaOXA-48–type gene, using electroporation into E. coli DH10B, as described (3). Plasmid transfer of blaOXA-48–type genes was successful only for K. pneumoniae 11-882 and 11-2568. PCR revealed the transfer of blaTEM-1 and blaOXA-48 along with a plasmid of ≈114 Kb (p48-11-882). In addition, blaOXA-181 was transferred along with a plasmid of ≈4 Kb (p181-11-2568) with no additional β-lactamases present. Replicon typing (9,10), using primers for incompatibility group IncR (IncRrv 5′-GTGTGCTGTGGTTATGCCTCA-3′), showed that p48-11-882 belonged to IncR and to the OXA-48–associated IncL/M. Plasmid p181-11-2568 was negative for all replicons tested. Transformants were resistant to ampicillin, cefazolin, and pipericillin/tazobactam but susceptible to third- and fourth-generation cephalosporins. This finding is not surprising because OXA-48 and OXA-181 show weak activity against expanded-spectrum cephalosporins (1,4). Resistance to third- and fourth-generation cephalosporins in clinical isolates is likely caused by a combination of additional mechanisms, such as porin mutations, or additional β-lactamases, such as CTX-M–type, extended-spectrum β-lactamases (Table). We report the emergence of OXA-48 and OXA-181 in North America. The emergence of carbapenem-resistant Enterobacteriaceae in Canada is of concern because they are difficult to detect in the laboratory, and treatment options are lacking. The history of travel by the patients in our study to regions where carbapenem-resistant Enterobacteriaceae are endemic highlights the necessity for understanding the potential risk factors associated with acquiring these multidrug-resistant pathogens.