Detection of GES-5 carbapenemase in Klebsiella pneumoniae, a newcomer in France. Hygiene unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France

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1 AAC Accepted Manuscript Posted Online 4 January 2017 Antimicrob. Agents Chemother. doi: /aac Copyright 2017 American Society for Microbiology. All Rights Reserved Detection of GES-5 carbapenemase in Klebsiella pneumoniae, a newcomer in France Rémy A. Bonnin, 1,2,3* Agnès B. Jousset, 1,2,3 Noémie Urvoy, 1,2 Lauraine Gauthier, 1,2,3 Linda Tlili, 1,2 Elodie Creton, 1,2,3 Garance Cotellon, 1,2,3 Fabienne Arthur, 4 Laurent Dortet, 1,2,3 Thierry Naas 1,2,3* EA7361, Université Paris-Sud, Université Paris-Saclay, LabEx Lermit, Bacteriology- Hygiene unit, APHP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France 2 EERA Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur- APHP-Université Paris Sud, Paris, France 3 Associated French National Reference Center for Antibiotic Resistance Carbapenemase-producing Enterobacteriaceae, 4 Microbiology department, Biocéane, Le Havre, France Keywords : β-lactamase, carbapenemase, ESBL Word count : 637 * Corresponding authors. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, Le Kremlin-Bicêtre Cedex, France. Phone: Fax: thierry.naas@aphp.fr and remy.bonnin@u-psud.fr

2 To the editor, Carbapenemase-producing Klebsiella pneumoniae (CPKp) are increasingly reported worldwide and represent a major health threat (1). Carbapenemases frequently encountered in CPKp belong to Ambler s class A (KPC enzymes), class B (NDM, VIM and IMP enzymes) and class D (OXA-48-like enzymes) (1). GES-1, is an Extended Spectrum Beta Lactamase (ESBL) initially described in 2000 in a K. pneumoniae isolate from French Guiana (2). Hitherto, 31 variants have been identified ( including several with carbapenem-hydrolyzing activity (GES-2, -4, -5, -6, -14, -14, -15, , -20 and - 24), which is due to a substitution of Glycine at position 170 by an Asparagine or a Serine (3 5). GES carbapenemases have rarely been reported in Enterobacteriaceae (GES-5 and GES-6) (3, 6). Here, we report, the first description of a GES-5 producing K. pneumoniae in France. This isolate was recovered from a man previously hospitalized in Bangkok, Thailand, for an acute ischemic cerebrovascular stroke. The patient was transferred to the hospital of Le Havre (France), where a rectal swab upon admission revealed a K. pneumoniae isolate MON with reduced susceptibility to carbapenems (MICs of 0.25, 0.38 and 0.5 µg/ml for imipenem, meropenem and ertapenem, respectively). This isolate was also resistant to penicillins, all cephalosporins, cephamycins, aztreonam, to fluoroquinolones, aminoglycosides (gentamicin, netilmicin and tobramycin), sulfamethoxazol/trimethoprim, chloramphenicol, and tetracycline. This isolate remained susceptible to amikacin (MIC of 1 µg/µl) and colistin (MIC of 0.5 µg/µl). The biochemical test CARBA-NP, revealed a carbapenemase activity in K. pneumoniae MON (7). This carbapenem-hydrolyzing activity was confirmed by several other confirmatory assays, such as by MALDI-TOF (8), by the commercially

3 available beta-carba test (BioRad, Marnes-la-coquette, France) (9) and the recently described Carbapenem Inactivation Method (CIM) (10). However, PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae (bla NDM, bla IMP, bla VIM, bla KPC, bla OXA-48 ) as previously described (11). Whole genome sequencing using Illumina s technology was performed (pairend; 2 x 150 bp) as previously described (13). A total of 2,352,841 reads were mapped allowing to build 206 contigs with an average length of 26,670 bp in size (from 500 to 509,094 bp) representing 5,493,972 bp (mean coverage of 64X). Resfinder webserver (14), revealed three β-lactamases (SHV-11, SHV-12 and GES- 5), eight aminoglycoside modifying enzymes (AMEs) as well as genes involved in resistance to phenicols, trimethoprim, sulfonamides and erythromycin (Table 1). High-level resistance to fluoroquinolones resulted from mutations in chromosomallyencoded gyrase (S83F and D87Y in GyrA subunit) and topoisomerase IV (S79I in ParC subunit). In addition, a frame shift in ompk35 gene leading to a stop codon at position 32 (K32Stop) and two insertions in OmpK36 (TS at position 183 and FSGNGE at position 264) likely result in inactive porins, thus resulting in decreased susceptibility to carbapenems as previously shown (15). Multilocus sequence typing (MLST, analysis using the Pasteur Institute s MLST scheme (16) revealed that this isolate belonged to ST42 ( ), an ST type described in South Asia (17). This ST does not belong to an epidemic clone but seems to have played a role in the genesis of ST258 (18, 19). Mating out assays performed as previously described (6) yielded transconjugants, possessing both bla SHV-12 and bla GES-5 genes or possessing only bla SHV-12 genes but none with only bla GES-5 gene, suggesting plasmid pmon-ges-5 may be mobilized only but not self-transferable. The conjugation frequency for the

4 77 pmon-shv-12 plasmid was transconjugants per donor cell and the presence of the bla GES-5 gene was found in 42% (21/50) of the transconjugants. Plasmid extraction, performed as previously described (20), revealed the presence of two plasmids of ca. 120-kb (carrying the bla SHV-12 gene) and 70-kb (carrying the bla GES-5 gene) in size determined as described (21). Close genetic context analysis revealed that the bla GES-5 was carried by a class 1 integron in first position. Promoter analysis revealed two promoters PcH1 (TGGACA - N 17 - TAAACT) and P2 (TTGTTA - N 14 - TACAGT) upstream of the bla GES-5 gene. The latter is likely inactive due to a shorter spacer (N=14). This report constitutes the first identification of a GES-5-producing Enterobacteriaceae isolate in France (22). This report highlights the usefulness of biochemical tests for the identification of minor carbapenemases (Carba NP test, MALDI-TOF techniques, CIM or BYG test) as the commercially available tests fail to detect them (23). ACKNOWLEDGMENTS This work was funded by a grant from the Ministère de l Education Nationale et de la Recherche (EA7361), Université Paris Sud. RAB, LD and TN are members of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR- 10-LABX-33)

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9 Table 1. Resistome of K. pneumoniae MON Antibiotic family Resistance %identity Accession number Enzyme In silico deduced Observed genes Name Function resistance c resistance β-lactams bla GES AF GES-5 Carbapenemase All β-lactams All beta-lactams except AZT bla SHV DQ SHV-12 ESBL All β-lactams except carbapenems and cephamycins bla SHV X98101 SHV-1 penicillinase AMX, TIC Aminoglycosides aada JN ANT(3 )- Ia adenyltransferase STR, SPE All tested aminoglycosides stra M96392 phosphotransferase STR Except amikacin strb M96392 phosphotransferase STR aada JQ ANT(3 )- adenyltransferase STR, SPE Ia aph(3 )-Ia V00359 APH(3 )Ia phosphotransferase KAN, NEO aaca AY AAC(6 )-I Acetyltransferase KAN, TOB, NET, AMI aac(3)-iid 99.9 EU AAC(3 )- Acetyltransferase GEN, TOB, NET IIb aac(6 )-II 99.6 Z54241 Acetyltransferase GEN, TOB, NET Quinolones gyra 98 a NZ_CDMU GyrA Gyrase CIP, LVX, NA Fluoroquinolones parc 99 a NZ_CDMU ParC Topoisomerase CIP, LVX, NA Macrolides, mph(a) D16251 MphA phosphotransferase ERY Macrolides b Lincosamides & streptogramines Phenicols cata1-like 99.9 V00622 CatA1-like Acetyltransferase CHL Chloramphenicol Sulphonamides sul CP Sul1 Efflux SUL Sulfonamide Cyclines tet(a) 99.9 AJ TetA Efflux TET Tetracycline Trimethorpim dfra AB DfrA12 Dihydrofolate Reductase TRI Trimethoprim

10 a Percentage of identity in comparison with a quinolone-susceptible strain, ie. Klebsiella pneumoniae ATCC b intrinsic resistance c AMX, amoxicillin ; AZT, azteronam ; CHL, chloramphenicol ; CIP, ciprofloxacine ; ERY, erythromycine ; GEN, gentamicin, KAN, Kanamycin ; LVX, levofloxacine ; NA, nalidixic acid ; NEO, neomycin, NET, netilmicin ; STR, streptomycin ; SPE, spectinomycine ; SUL, sulfonamide ; TET, tetracycline ; TIC, ticarcillin ; TOB, Tobramycine ; TRI, trimethoprim Downloaded from on July 5, 2018 by guest