IMI-2 carbapenemase in a clinical Klebsiella variicola isolated in the UK

Sir, IMI carbapenemases, named based on their ability to hydrolyse imipenem, are Ambler class A enzymes. blaIMI-1 was first identified on the chromosome of two Enterobacter cloacae isolates from the USA in 1984,1 and subsequently small numbers of IMI-positive isolates have been identified in China, Finland, France, Ireland, Norway, Singapore, Tahiti (French Polynesia) and the USA.2,3 In contrast, IMI-2 and IMI-3 have been described as plasmid-mediated enzymes in Enterobacter asburiae and E. cloacae, respectively.4,5 Twelve IMI variants have now been identified (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047) and to date the only report of an IMI carbapenemase identified outside of the genus Enterobacter has been in a single isolate of Escherichia coli from Spain.6 Here we report an IMI-2 carbapenemase in a Klebsiella variicola strain isolated in the UK. The strain was initially isolated from an intensive therapy unit patient in 2011 from a soft tissue infection of the buttock and referred to the Specialist Antimicrobial Chemotherapy Unit, Public Health Wales, for investigation of carbapenem resistance. The patient had no known recent travel history. The strain was identified as a Klebsiella pneumoniae by BD Phoenix™ (BD Diagnostics, Oxford, UK). MICs of ertapenem, meropenem and imipenem were >32 mg/L, with susceptibility to cefotaxime and ceftazidime (MICs ≤0.25 mg/L) as determined by Liofilchem® MIC test strips (Launch Diagnostics, Longfield, UK). Disc inhibition tests (BioConnections, Knypersley, UK) gave positive results for class A and AmpC enzyme activity, but the isolate was negative for KPC, OXA-48 and OXA-23 carbapenemase and plasmid-mediated AmpC genes by in-house PCR. The isolate was subsequently referred to PHE’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit for further investigation, where MICs were determined against AMRHAI’s standard panel of Gram-negative antibiotics, which includes ertapenem, meropenem and imipenem, using agar dilution, and interpreted using EUCAST criteria. Identification as a K. pneumoniae was confirmed by MALDI-TOF MS (Bruker Microflex LT, Bruker Daltonik GmbH, Bremen, Germany). To investigate the underlying carbapenem resistance mechanism(s) the strain was used as a plasmid donor in electro-transformation to E. coli Alpha-Select cells (Bioline, London, UK), with transformants selected on LB agar containing 100 mg/L ampicillin onto which a 10 μg ertapenem disc was placed. WGS of the clinical isolate and transformant was performed using a HiSeq sequencer (Illumina) and the resulting data were analysed using an in-house bioinformatics pipeline, as previously described.7 Transformant reads were assembled into contigs using SPAdes 3.5.0 (http://cab.spbu.ru/software/spades/). Plasmid contigs were extracted from the E. coli Alpha-Select genome by in silico subtraction and assembled; replicon typing was performed using PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/). The strain was resistant to the carbapenems (MICs: ertapenem >16 mg/L, meropenem >32 mg/L and imipenem >128 mg/L), but fully susceptible to the cephalosporins (MICs: cefotaxime ≤0.125 mg/L and ceftazidime 0.5 mg/L), suggestive of a class A carbapenemase (Table 1). MICs for IMI-2-producing K. variicola, the IMI-2 transformant and E. coli Alpha-Select MICs for IMI-2-producing K. variicola, the IMI-2 transformant and E. coli Alpha-Select WGS identified the strain as a K. variicola, which cannot be reliably differentiated from its close relative K. pneumoniae by biochemical tests or MALDI-TOF MS.8,9,K. variicola was originally isolated from plants,8 and has been reported as a frequent cause of bloodstream infections, with increased mortality compared with infections with K. pneumoniae.10 WGS also identified a blaIMI-2 carbapenemase gene. The only other resistance genes detected were blaLEN-2, oqxAB and fosA, which are all intrinsic in close relative K. pneumoniae, and were located on contigs alongside chromosomal genes. The total genomic DNA of the IMI plasmid transformant was sequenced to >50× coverage with reads assembling into a single contig, which determined that blaIMI-2 was located on a 77 843 bp IncFIIY plasmid (hereafter referred to as pJF-787, GenBank accession number KX868552). There are currently no other complete IMI-2 plasmid sequences deposited in GenBank for comparison; however, the genetic environment of blaIMI-2 in pJF-787 was identical to an E. coli 48.5 kb IncF plasmid with ISEc36 and LysR-type regulator gene blaIMI-2R found upstream of blaIMI-2 (JN412066).6 pJF-787 did not encode any other known resistance genes. A 56 kb region of plasmid pJF-787 shared high similarities (97% identity) with other published IncFIIY plasmids, of which many also harboured a carbapenemase gene: blaNDM (CP018366, KC887917, KC887916, KJ812998, KF732966, JQ314407, KP900016, KP868647, KR351290, CP017285, CP017850 and CP017283), blaIMI (KT780723 and KT225520), blaIMP (CP016763) or blaKPC (KU318419). Shared sequences include the genes encoding the replicon, conjugation and transfer functions as well as those encoding the plasmid SOS inhibition system and restriction endonuclease, suggesting that plasmid pJF-787 is likely to be transferable. Outside of this region the sequences surrounding the direct environment of blaIMI-2 varied considerably from other published IncFIIY plasmid sequences. To the best of our knowledge this is the first report of an IMI carbapenemase outside of the E. cloacae complex in the UK, and only the second such report worldwide. We also report the first complete IMI-2 plasmid sequence. IMI carbapenemases are rarely identified from clinical isolates in the UK, accounting for only 22 out of >8990 carbapenemase-producing Enterobacteriaceae confirmed by the national reference laboratory since screening for carbapenemases began in the early 2000s (2010, n = 2; 2011, n = 1; 2012, n = 1; 2013, n = 2; 2014, n = 4; 2015, n = 5; 2016, n = 7; K. L. Hopkins and N. Woodford, AMRHAI, unpublished data). WGS of 10 of these indicates that IMI-2 was the most common variant identified (7 isolates), but IncFIIY plasmids were only detected in 3 isolates and shared only 55%–84% coverage and 52%–82% identity when compared with pJF-787 (M. Doumith, AMRHAI, unpublished data). This study further highlights the possible role of ISEc36 in the mobilization of blaIMI-2 between different plasmid backbones, and its association with an IncFIIY plasmid backbone may facilitate spread to other Enterobacteriaceae. IMI carbapenemases should no longer be considered as limited to the E. cloacae complex and should be sought in any bacterial isolates expressing a class A carbapenemase phenotype, but that are negative for a KPC gene in molecular tests. IMI carbapenemases are not currently covered by any of the commercial molecular assays for carbapenemase detection; therefore, such isolates should be referred to a reference laboratory for further investigation. We wish to thank AMRHAI, PHE and the Specialist Antimicrobial Chemotherapy Unit, Public Health Wales staff for their role in the characterization of this isolate. This study was carried out as part of our routine work. PHE authors: none to declare. However, PHE’s AMRHAI Reference Unit has received financial support for conference attendance, lectures, research projects or contracted evaluations from numerous sources, including: Accelerate Diagnostics, Achaogen Inc., Allecra Therapeutics, Amplex, AstraZeneca UK Ltd, Basilea Pharmaceutica, Becton Dickinson Diagnostics, bioMérieux, Bio-Rad Laboratories, BSAC, Cepheid, Check-Points B.V., Cubist Pharmaceuticals, Department of Health, Enigma Diagnostics, Food Standards Agency, GlaxoSmithKline Services Ltd, Henry Stewart Talks, IHMA Ltd, Kalidex Pharmaceuticals, Melinta Therapeutics, Merck Sharpe & Dohme Corp., Meiji Seika Pharmo Co. Ltd, Mobidiag, Momentum Biosciences Ltd, Nordic Pharma Ltd, Norgine Pharmaceuticals, Rempex Pharmaceuticals Ltd, Roche, Rokitan Ltd, Smith & Nephew UK Ltd, Trius Therapeutics, VenatoRx Pharmaceuticals and Wockhardt Ltd. Specialist Antimicrobial Chemotherapy Unit authors: none to declare. However, the Specialist Antimicrobial Chemotherapy Unit has received funding for projects from Accelerate Diagnostics. S. D.: none to declare.