Characterization of blaKPC-3-positive plasmids from an Enterobacter aerogenes isolated from a corvid in Canada

Sir, Currently, KPC enzymes are the most prevalent carbapenemases in Enterobacteriaceae, reported from hospitals in North America.1 Considering the current epidemiological situation, in this study, we examined the wild corvids as indicators of environmental contamination by carbapenemase-producing Enterobacteriaceae (CPE) in Canada. A total of 242 (53.9%) Gram-negative bacteria with reduced susceptibility to carbapenems were identified among 449 faecal samples originating from wild corvid birds (Corvus brachyrhynchos and Corvus corax), collected in five geographically distinct regions across Canada.2 However, only an Enterobacter aerogenes isolate (Eaer-4382) showed carbapenemase activity by MALDI-TOF MS meropenem hydrolysis assay.3 Eaer-4382 was resistant to penicillins, penicillin/sulbactam combinations, piperacillin/tazobactam, cephalosporins and aztreonam but susceptible to carbapenems, as determined by the broth dilution method4 and interpreted according to EUCAST criteria (www.eucast.org). The isolate was also resistant to various non-β-lactam antibiotics (Table S1, available as Supplementary data at JAC Online). PCR screening for various carbapenemase-encoding genes followed by sequencing revealed the presence of the blaKPC-3 gene in Eaer-4382. Eaer-4382 was capable of transferring the blaKPC-3 gene to the Escherichia coli MT102 laboratory strain by conjugation, using the filter-mating method.5 S1 nuclease analysis revealed the presence of multiple plasmids in Eaer-4382, including molecules of ∼90, ∼120 and ∼300 kb.6 Moreover, plasmid analysis showed two types of transconjugants carrying either the ∼90 kb plasmid pEaer-4382s or the ∼120 kb plasmid pEaer-4382b. Both plasmids hybridized with the blaKPC probe. The genomic DNA of E. aerogenes Eaer-4382 and plasmids from E. coli transconjugants were sequenced on the Illumina NextSeq platform (Illumina Inc., San Diego, CA, USA). Assembling of the reads, filling of sequence gaps and analysis of the sequences were performed as described previously.7 Analysis of Illumina data using the MLST website (https://pubmlst.org/kaerogenes/) assigned isolate Eaer-4382 to ST93, which belongs to clonal complex 3 (CC3). E. aerogenes ST93 was originally identified from a clinical sample in China in 2014 (https://pubmlst.org/kaerogenes/). Additionally, analysis of sequencing data by ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) showed that Eaer-4382 exhibited several genes for resistance to β-lactams, aminoglycosides, fluoroquinolones, chloramphenicol, sulphonamides, tetracycline and trimethoprim (Table S1). Analysis of plasmid sequences carried by transconjugants demonstrated that pEaer-4382b (GenBank accession no. KY093013) is a 134 521 bp molecule with a sequence closely related to the IncFII-type plasmids. The plasmid showed highest similarity to a plasmid (71% coverage, 99% identity) from the Raoultella planticola FDAARGOS_64 strain (GenBank accession no. CP026048) recovered in the USA. pEaer-4382b included a contiguous plasmid backbone of 94 320 bp (nt 1–9593 and 49 795–134 521) and an acquired sequence of 40 201 bp (nt 9594–49 794) (Figure 1a). The IncFII-type plasmid backbone harboured regions responsible for replication [FIIk (K7) allele (repA gene) and FIB-like replicon (repA2 gene)], conjugative transfer and plasmid maintenance. (a) Overview of the plasmid pEaer-4382b of an E. aerogenes isolate from a corvid. Innermost circle shows the main regions of the plasmid. Similarities with other plasmids (pEaer-4382s and R. planticola FDAARGOS_64 strain) are shown in the next circles; each colour represents a unique plasmid. In the outer circle, indicative genes and the direction of transcription are shown by arrows. Replicons of the plasmid are indicated as pink arrows. Genes responsible for plasmid transfer and maintenance are shown in green and orange, respectively. (b) Linear map of the acquired region of the plasmid pEaer-4382b. Arrows show the direction of transcription of ORFs, while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes, IS elements and transposases are shown in red, yellow and green, respectively. The intI1 gene is shaded blue and remaining genes are shown in white. Thin lines below the map correspond to highly similar sequences from other plasmids. (c) Comparison of iteron regions of pEaer-4382s and pEaer-4382b. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. (a) Overview of the plasmid pEaer-4382b of an E. aerogenes isolate from a corvid. Innermost circle shows the main regions of the plasmid. Similarities with other plasmids (pEaer-4382s and R. planticola FDAARGOS_64 strain) are shown in the next circles; each colour represents a unique plasmid. In the outer circle, indicative genes and the direction of transcription are shown by arrows. Replicons of the plasmid are indicated as pink arrows. Genes responsible for plasmid transfer and maintenance are shown in green and orange, respectively. (b) Linear map of the acquired region of the plasmid pEaer-4382b. Arrows show the direction of transcription of ORFs, while truncated ORFs appear as rectangles (arrows within rectangles indicate the direction of transcription). Resistance genes, IS elements and transposases are shown in red, yellow and green, respectively. The intI1 gene is shaded blue and remaining genes are shown in white. Thin lines below the map correspond to highly similar sequences from other plasmids. (c) Comparison of iteron regions of pEaer-4382s and pEaer-4382b. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. In the acquired sequence, the class 1 integron In1177, whose variable region comprised the aadB, aphA16, aadA32 and blaOXA-2 cassettes, was found at the boundary of the IncFII-type backbone. In1177 was previously found in plasmid pCAV1335-92 from the Klebsiella oxytoca CAV1335 strain (GenBank accession no. CP011614). The 3′-CS of In1177 was disrupted by IS26 (Figure 1b). Upstream of IS26, a 19 798 bp sequence (nt 15 521–35 318) harbouring coding sequences for an alcohol dehydrogenase, ABC transporters and proteins with unknown function was identified. A similar sequence has also been observed in A/C2 plasmids pEc9, pEc19, pEc78 and pKp55, which were characterized from IMP-producing Enterobacteriaceae isolates of wildlife origin.7 The KPC-3-encoding transposon Tn4401b was found downstream of the latter sequence. The IRR of Tn4401b was deleted due to insertion of a Tn5403 transposon, which was found at the boundary of the plasmid backbone. In the plasmid from the FDAARGOS_64 strain, Tn5403 was identified at a similar position. Plasmid pEaer-4382s (GenBank accession no. KY093014), being 108 772 bp in size, is a derivative of pEaer-4382b (100% coverage, 99% identity). Only two differences between the two plasmids were observed. A 25 638 bp segment (nt 9712–35 349 in pEaer-4382b), including the In1177 and the 19 798 bp sequence of wildlife origin, was not present in pEaer-4382s. In addition, an 8 bp duplication (ACAAGATA) was identified in the region between the resD and parA genes. This duplication that resulted in a different number of iterons (Figure 1c), which presumably play a role in plasmid replication and maintenance, may explain the coexistence of both IncFII-type plasmids with identical replicons in the same cell. To our knowledge, this is the first description of a KPC-producing Enterobacteriaceae, isolated in Canada, from a sample of wildlife origin. Although the isolation of CPE in wildlife is rare,8 the identification of even one specimen containing CPE from a wild corvid is of great concern showing the potential dissemination of resistance genes throughout the environment. Furthermore, sequencing data demonstrated that blaKPC-3-carrying plasmids, characterized during this study, did not exhibit extensive similarity with KPC-encoding plasmids from other studies. This finding further punctuates the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants. We would like to thank M. Elderkin, S. McBurney and M. Phinney for their contribution to sample collection. We would also like to thank Jaroslav Hrabak, Martina Masarikova, Katarina Murgasova and Jana Hofirkova for their assistance in the laboratory and help with data interpretation. Furthermore, we thank the Core Facility Genomics of CEITEC for their support with obtaining scientific data presented in this paper. This work was supported by the Czech Research Health Council (grant 15-28663A), the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno (225/2017/FVHE) and the National Sustainability Programs I (NPU I; grant LO1503) and II (NPU II; grant LQ1601) provided by the Ministry of Education, Youth and Sports of the Czech Republic. The Core Facility Genomics of CEITEC was supported by the National Center for Medical Genomics research infrastructure (LM2015091) funded by the Ministry of Education, Youth and Sports of the Czech Republic. None to declare.

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