Proteus mirabilis Producing the OXA-58 Carbapenemase in Poland

LETTER

OXA-58 represents one of the class D carbapenemase types that are major carbapenem resistance determinants in Acinetobacter spp. (1–3), being extremely rare in Enterobacterales. The first report of its presence in diverse enterobacteria was from Sierra Leone (4), followed by recent papers on Proteus mirabilis from Belgium (5) and Germany (6); the latter described specific small OXA-58-encoding plasmids in four clonally and epidemiologically nonrelated strains. Here we present a Polish isolate with an almost identical plasmid, raising questions regarding the cryptic epidemiology of OXA-58-producing P. mirabilis in Europe.

Carbapenem-resistant, Carba NP test-positive (7) P. mirabilis strain NMI1213/17 was recovered in February 2017 from the blood of a 37-year-old hematological patient in a hospital in Poznań, Poland. The presence of OXA-58 was indicated by the eazyplex SuperBug CRE assay (AmplexDiagnostics, Giessen, Germany) and confirmed by PCR and sequencing. Antimicrobial MICs were evaluated by the broth microdilution method or agar dilution method (for fosfomycin), according to EUCAST guidelines (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf). The β-lactam susceptibility pattern indicated resistance to penicillins (including temocillin), their β-lactamase inhibitor combinations, and carbapenems (with ertapenem, imipenem, and meropenem MICs of 8, 32, and 16 μg/ml, respectively) and susceptibility to oxyimino compounds (1, 2). Strain NMI1213/17 was resistant to fluoroquinolones and chloramphenicol and susceptible to amikacin, gentamicin, tobramycin, co-trimoxazole, and fosfomycin.

The isolate was subjected to whole-genome sequencing (WGS), using the MiSeq platform (Illumina, San Diego, CA, USA). Contigs were assembled and the draft genome was obtained using SPAdes 3.12.0 (8), annotation was done with PROKKA 1.11 (9), and resistance genes were identified by ResFinder 3.0 (10). The bla_OXA-58 gene was on a 6,351-bp contig with identical termini, suggesting a circular DNA molecule, as confirmed by PCR and Sanger sequencing. The entire plasmid pPOZ-OXA-58 was 6,224 bp (GenBank accession no. MK086028) (Fig. 1) and, when compared with p10797-OXA-58 (6,219 bp) from Germany (GenBank accession no. KU871396) (6), it was almost identical, with two putative rep genes (repA and rep), bla_OXA-58 flanked by remnants of ISAba3 elements, the spectinomycin/streptomycin resistance gene aadA14, and a mobA-like gene (6). The only differences were insertion of 5 adenine nucleotides in rep in pPOZ-OXA-58 and 1 single-nucleotide polymorphism in the ISAba3 tnpA gene. As revealed by Lange et al. (6), the rep and aadA14 genes are present also in the 5,198-bp plasmid pCCK647 (GenBank accession no. AJ884726) from Pasteurella multocida from Belgium (11). However, the only pCCK647 replicase gene, rep, is presumably nonactive in pPOZ-OXA-58 due to the 5-adenine frameshift insertion; therefore, its replication might rely on repA and proceed in a broader host range, as demonstrated by successful electroporation of the plasmid into Escherichia coli TOP10 (Invitrogen, Carlsbad, CA). P. mirabilis NMI1213/17 contained only one more acquired resistance gene, cat (encoding chloramphenicol O-acetyltransferase), identical to those in several P. mirabilis WGS entries (e.g., GenBank accession no. CP015347). The gyrA and parC genes specified DNA gyrase and topoisomerase IV subunits, respectively, with frequent quinolone resistance substitutions (S83I in GyrA and S80R in ParC). P. mirabilis NMI1213/17 was not related by pulsed-field gel electrophoresis to any of the four German P. mirabilis OXA-58 strains (obtained courtesy of N. Pfennigwerth and F. Lange).

FIG 1.

Scheme of the pPOZ-OXA-58 plasmid structure (6,224 bp; GenBank accession no. MK086028). Black arrows represent genes or putative genes, and black boxes represent the IR-L and IR-R sequences of two truncated ISAba3 elements. Two differences from the plasmid p10797-OXA-58 (GenBank accession no. KU871396) are indicated above the scheme, namely, the (A)₅ insertion into the original (A)₆ tract at positions 174 to 179 of the rep coding region and the T-to-G silent mutation at position 72 of the ISAba3 tnpA coding region. Sizes of the individual arrows and boxes and their positions only roughly correspond to the actual plasmid structure.

Among carbapenemase-producing Enterobacterales strains, OXA-58-positive organisms represent a marginal but puzzling epidemiological story. Only 6 such isolates, exclusively P. mirabilis, have been reported in Europe to date, of which German and Polish isolates shared unique plasmids with the truncated bla_OXA-58-carrying ISAba3-associated transposon (3). The plasmids might spread horizontally by mobilized transfer (6), and the occurrence of the isolates in distant locales might suggest hidden dissemination of unknown scale and future potential.

Accession number(s).

The nucleotide sequence obtained in this study has been submitted to the GenBank database and assigned accession no. MK086028.