AAC Accepted Manuscript Posted Online 4 March 2019

Antimicrob. Agents Chemother. doi:10.1128/AAC.00106-19

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

1 Proteus mirabilis producing the OXA-58 carbapenemase in Poland

3 E. Literacka1, R. Izdebski2, A. Baraniak2, D. Żabicka1, A. Schneider3, P. Urbanowicz2, M.

4 Herda1, W. Hryniewicz1, M. Gniadkowski2

1
6 Department of Epidemiology and Clinical Microbiology, National Medicines Institute, 00-

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7 725 Warsaw, Poland
2
8 Department of Molecular Microbiology, National Medicines Institute, 00-725 Warsaw,

9 Poland
3
10 Microbiological Laboratory, University Hospital of Lord’s Transfiguration, 61-848 Poznań,

11 Poland

12

13 *Corresponding author. Tel: +48 22 851 46 70; Fax: +48 22 841 29 49; E-mail:

14 e.literacka@nil.gov.pl

15 Keywords: Enterobacterales, Proteus mirabilis, carbapenemase, class D carbapenemase,

16 OXA-58

17

18 OXA-58 represents one of the class D carbapenemase types that are major carbapenem

19 resistance determinants in Acinetobacter spp. (1-3), being extremely rare in Enterobacterales.

20 Its first report in diverse enterobacteria was from Sierra Leone (4), followed by recent papers

21 on Proteus mirabilis from Belgium (5) and Germany (6), the latter one describing specific

22 small-size OXA-58-encoding plasmids in four clonally and epidemiologically non-related

23 strains. Here we present a Polish isolate with an almost identical plasmid, raising questions on

24 the cryptic epidemiology of OXA-58-producing P. mirabilis in Europe.

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25 Carbapenem-resistant, Carba NP test-positive (7) P. mirabilis NMI1213/17 was recovered in

26 February 2017 from blood of a 37-year-old haematological patient in a hospital in Poznań.

27 OXA-58 was indicated by the eazyplex SuperBug CRE assay (AmplexDiagnostics, Giessen,

28 Germany), and confirmed by PCR and sequencing. Antimicrobial MICs were evaluated by

29 the broth microdilution or agar dilution (for fosfomycin) according to EUCAST

30 (http://eucast.org). The β-lactam susceptibility pattern comprised resistance to penicillins

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31 (incl. temocillin), their β-lactam inhibitor combinations and carbapenems (ertapenem,

32 imipenem and meropenem MICs, 8, 32 and 16μg/ml, respectively) and susceptibility to

33 oxyimino-compounds (1, 2). NMI1213/17 was resistant to fluoroquinolones and

34 chloramphenicol, and susceptible to amikacin, gentamicin, tobramycin, co-trimoxazole and

35 fosfomycin.

36 The isolate was subjected to whole-genome sequencing (WGS), using the MiSeq platform

37 (Illumina, San Diego, USA). Contigs were assembled and the draft genome obtained using

38 SPAdes 3.12.0 (8), annotation was done with PROKKA 1.11 (9), and resistance genes were

39 identified by ResFinder 3.0 (10). blaOXA-58 was on a 6,351bp-long contig with identical

40 termini, suggesting a circular DNA molecule, confirmed by PCR and Sanger sequencing. The

41 entire plasmid pPOZ-OXA-58 was of 6,224bp (GenBank accession No. MK086028), and

42 when compared with p10797-OXA-58 (6,219bp) from Germany (KU871396) (6), it was

43 almost identical, with two putative rep genes, repA and rep, blaOXA-58 flanked by remnants of

44 ISAba3 elements, the spectinomycin/streptomycin resistance gene aadA14, and a mobA-like

45 gene (6). The only differences were insertion of five adenine nucleotides in rep in pPOZ-

46 OXA-58, and one single-nucleotide polymorphism in the ISAba3 tnpA gene. As revealed by

47 Lange et al. (6), the rep and aadA14 genes are present also in the 5,198-bp plasmid pCCK647

48 (AJ884726) from Pasteurella multocida from Belgium (11). However, the only pCCK647

49 replicase gene rep is presumably non-active in pPOZ-OXA-58 due to the (A)5 frameshift

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50 insertion; therefore, its replication might rely on repA, and proceed in a broader host range, as

51 demonstrated by successful electroporation of the plasmid into Escherichia coli TOP10

52 (Invitrogen, Carlsbad, CA). P. mirabilis NMI1213/17 contained only one more acquired

53 resistance gene, cat, encoding chloramphenicol O-acetyltransferase, identical to those in

54 several P. mirabilis WGS entries (e.g. CP015347). The gyrA and parC genes specified DNA

55 gyrase and topoisomerase IV subunits, respectively, with frequent quinolone resistance

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56 substitutions, S83I in GyrA and S80R in ParC. P. mirabilis NMI1213/17 was not related by

57 PFGE to any of the four German P. mirabilis OXA-58 strains (obtained by courtesy of N.

58 Pfennigwerth and F. Lange).

59 Among carbapenemase-producing Enterobacterales OXA-58-positive organisms represent a

60 marginal, though puzzling epidemiological story. Only six such isolates, exclusively P.

61 mirabilis, have been reported in Europe so far, of which German and Polish isolates shared

62 unique plasmids with the truncated blaOXA-58-carrying ISAba3-associated transposon (3). The

63 plasmids might spread horizontally by mobilised transfer (6), and occurrence of the isolates in

64 distant locales might suggest hidden dissemination of unknown scale and future potential.

65 Accession number. The nucleotide sequence obtained in this study has been submitted to the

66 GenBank database and assigned accession number No. MK086028.

67 Acknowledgments

68 This work was supported by the grant SPUB MIKROBANK from the Polish Ministry of

69 Science and Higher Education, Narodowy Program Ochrony Antybiotyków from the Polish

70 Ministry of Health, and grant DS-4.47/2018 from the National Medicines Institute. All of the

71 authors have no conflicts of interest to declare.

72 References

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74 2. Poirel L, Naas T, Nordmann P. 2010. Diversity, epidemiology, and genetics of class D

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77 58, a novel class D β-lactamase involved in resistance to carbapenems in

78 Acinetobacter baumannii. Antimicrob Agents Chemother 49:202-208.

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81 blaVIM carbapenemase genes in hospital Enterobacteriaceae isolates from Sierra

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103 Antimicrob Agents Chemother 49:3046-3049.

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105 Figure legends

106 Figure 1. Scheme of the pPOZ-OXA-58 plasmid structure (6,224bp; GenBank acc. No.

107 MK086028). Black arrows represent genes or putative genes, and black boxes represent the

108 IR-L and IR-R sequences of two truncated ISAba3 elements, respectively. Two differences

109 with the plasmid p10797-OXA-58 (KU871396) are indicated above the scheme, namely the

110 (A)5 insertion into the original (A)6 tract at the position 174-179 of the rep coding region, and

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111 the T to G silent mutation at the position 72 of the ISAba3 tnpA coding region. Sizes of the

112 individual arrows/boxes and their positions only roughly correspond to the actual plasmid

113 structure.

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