Characterization of a novel blaIMP gene, blaIMP-58, using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark

    Characterisation of a novel bla IMP gene, blaIMP −58 , using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark Dennis Back Holmgaard, Frank Hansen, Henrik Hasman, Ulrik S. Justesen, Anette M. Hammerum PII: DOI: Reference:

S0732-8893(16)30326-1 doi: 10.1016/j.diagmicrobio.2016.10.002 DMB 14209

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

16 July 2016 21 September 2016 4 October 2016

Please cite this article as: Holmgaard Dennis Back, Hansen Frank, Hasman Henrik, Justesen Ulrik S., Hammerum Anette M., Characterisation of a novel blaIMP gene, blaIMP −58 , using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.10.002

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Characterisation of a novel blaIMP gene, blaIMP-58, using whole genome

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sequencing in a Pseudomonas putida isolate detected in Denmark

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Dennis Back HOLMGAARD1, Frank HANSEN2, Henrik HASMAN2, Ulrik S. JUSTESEN1, Anette M. HAMMERUM2

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Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark

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Contact information corresponding author: Dennis Back Holmgaard

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Rigshospitalet

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Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark

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Department of Clinical Microbiology Blegdamsvej 9

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2100 Copenhagen Ø

E-mail: [email protected]

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Telephone +45 35456409

Keywords: Carbapenemase, metallo-beta-lactamases, multiresistant

ACCEPTED MANUSCRIPT Carbapenems are potent antimicrobial agents reserved for the treatment of serious infections caused by multi-resistant enterobacteriacae and Pseudomonas aeruginosa. The overall frequency of carbapenem resistant strains, attributed to metallo-β –lactamases (MBL) in Denmark, remains low. However, incidence

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has seen an increase in recent years. MBLs catalyze the hydrolysis and inactivation of a broad range of β –

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lactam antibiotics rendering them inactive against penicillins, cephalosporins and carbapenems. In Denmark VIM- producing strains and to lesser extents NDM-producing isolates have been responsible for producing P. aeruginosa was reported in Denmark 1.

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the majority of carbapenemase producing P. aeruginosa cases. Recently, however, a case of an IMP-7

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In October 2015, a 65-year-old woman was admitted to the Department of Infectious Diseases, Rigshospitalet, Copenhagen, Denmark. The patient was primarily hospitalised at another hospital in the Copenhagen area in early October 2015 with fever and cauda equine syndrome; i.e. lower back pain, saddle anaesthesia and urinary retention. A diagnosis of spondylodiscitis was made and the patient was transferred to Rigshospitalet, where the patient underwent surgery two days later. Streptococcus

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anginosus was detected in five of five tissue samples. The patient was initially treated with cefuroxime 3 g x 3 and linezolid 600 mg x 2 intravenously daily for three days, which was subsequently, substituted with benzylpenicillin 3 million international units x 3 daily, once a final confirmation of S. anginosus in

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monoculture was confirmed. After ten days of treatment the patient developed lower abdominal pain and underwent acute surgery. A diagnosis of perforated appendicitis was made. Severe complications arose

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during and after the operation and as a result the patient died the following day. A meropenem resistant Pseudomonas putida isolate was detected from a catheter urine sample retrieved on day eight of

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treatment. The isolate was not considered clinically significant. A search in the local laboratory information system was performed for P. putida with a similar resistance profile within the last year. Thirty isolates were identified, but none were meropenem resistant. The patient had no prior history of hospitalisation outside of Denmark for a period of 12 months leading up to this admission. The origin of the meropenem resistant P. putida remains unknown. Using the multiplex PCR described by Ellington et al., the meropenem-resistant P. putida isolate was found to be positive for blaIMP2. The minimum inhibitory concentrations (MICs) for the meropenem-resistant P. putida isolate were determined for 31 antimicrobial agents (Table 1) by the microbroth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, except for the MIC for tigecycline, streptomycin and spectinomycin, which were determined using MIC Test Strips (Liofilchem, Roseto degli Abruzzi, Italy). P. aeruginosa ATCC 27853 was used as quality control. All susceptibility results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/clinical breakpoints/;accessed 20 January 2016), except for

ACCEPTED MANUSCRIPT chloramphenicol, fosfomycinand tetracycline , for which no clinical breakpoints have been defined. The MICs are shown in Table 1. Genomic DNA was extracted using a DNeasy Blood & Tissue Kit (QIAGEN, Copenhagen, Denmark) and fragment libraries were constructed using a Nextera Kit (Illumina, Little

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Chesterford, UK) followed by 251-bp paired-end sequencing (MiSeqTM; Illumina) according to

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manufacturer’s instructions. The paired-end Illumina data were assembled using CLC Genomic Workbench v.8.5 (QIAGEN, Aarhus, Denmark). The ResFinder web server (http://www.genomicepidemiology.org) was

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used to identify acquired antimicrobial resistance genes in the whole genome sequence data, using a threshold of 100% identity for the genes encoding β-lactamases and 98.00% identity for all other genes. ResFinder detects the presence of resistance genes, but not functional integrity and expression or

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resistance due to acquired variation in housekeeping genes.

This study identified a novel IMP-22 enzyme derivate in a P. putida designated IMP-58 (GenBank accession number KU647281) differing from IMP-22 by one amino acid change (V49F). The resulting change is displayed in Figure 1. IMP-22 or IMP-2 variants have not previously been reported in Denmark. We were unable to determine the origin of the isolate, but IMP-22 has previously been reported in urban

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wastewater in Italy and in hospital settings in Austria, Italy and Spain 3-5. It is conceivable that the isolate was acquired prior to the patient's admission to hospital. We were unable to determine definitively

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whether the gene was located on a plasmid or encoded on a chromosome. However, analysis of the draft genome data showed that the contig carrying the blaIMP-58 as well as an additional contig carrying an incP(6)

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replicon both had an average read coverage approximately 10 times higher than the average read coverage of the remaining (chromosomal) contigs indicating these two contigs being part of the same entity. Unfortunately, it was not possible to link the two contigs directly, but subsequent agarose gel analysis did

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reveal that the strain contained one plasmid of approximately 50 kilo bases, which further support plasmid localization of blaIMP-58. IMP-58 retains a very high degree of activity against all β-lactams as previously reported 3,5. In addition to resistance to β-lactams, six other well-known resistance genes were detected i.e. aadA2, aacA4, aac(6')Ib-cr, cmlA1, tet(C)and sul1 (Table 1). The isolate was only susceptible to amikacin, gentamicin, tobramycin and colistin. The emergence of a novel IMP-22 variant (IMP-58) in Denmark, in a hospital setting, is important and disturbing, as the overall occurrence of MBLs in Denmark remains low. This is especially true for the IMP group, which has only previously been reported once in Denmark 1. This patient was not deemed a high risk patient as she had no prior hospitalisation outside of Denmark within the last 12 months and did not receive broad spectrum antibiotics prior to this finding. This emphasises the need for continued surveillance to limit the spread of resistant bacteria.

ACCEPTED MANUSCRIPT Acknowledgements:

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Karin Sixhøj Pedersen is thanked for her excellent technical assistance.

ACCEPTED MANUSCRIPT Funding Part of this work was supported by the Danish Ministry of Health as part of The Integrated Surveillance of

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ESBL/AmpC-producing E. coli and Carbapenemase-Producing Bacteria.

Transparency declarations

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None to declare.

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Ethical approval

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Ethical approval for access to patient data was approved by the Danish Patient Safety Authority ref nr. 33013- 1442/1/

ACCEPTED MANUSCRIPT Table 1: Genotyping and phenotypic resistance profile of the IMP-58 producing Pseudomonas putida isolate Susceptibility

Resistance gene(s) aadA2, aacA4

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>1024

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>16/8 >64 >128/2 >64 64/4 >256 >32 >32 >16 >16 >32 >16 >8 >8 >2

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≤4 ≤1 ≤1 32

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MIC, minimum inhibitory concentration

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MIC (mg/L)

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Antimicobial group Aminoglycosides Amikacin Gentamicin Tobramycin Streptomycin Aminocyclitols Spectinomycin β-lactams Ampicillin/sulbactam Ticarcillin Ticarcillin/clavulanic acid Piperacillin Piperacillin/tazobactam Carbenicillin Ceftriaxone Cefotaxime Ceftazidime Cefepime Cefoperazone Aztreonam Imipenem Meropenem Doripenem Fluoroquinolones Ciprofloxacin Levofloxacin Lomefloxacin Fosfomycin Fosfomycin Phenicols Chloramphenicol Tetracyclines Doxycyclin Minocycline Tetracycline Tigecycline Polymyxins Colistin Polymyxin B Folate pathway inhibitors Trimethoprim/Sulfamethoxazole

aadA2, aacA4

blaIMP-58, blaOXA-10

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aac(6')Ib-cr >2 >8 >4

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128 cmlA1 8 tet(C) >16 >16 >8 8

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0.5 0.5

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sul1 >4/76

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Figure 1 – Amino acid comparison of the IMP-22 (NG 049186) with the IMP-58 variant (GenBank accession number KU647281). Site of change in amino acid composition is displayed in yellow.

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Hammerum AM, Jakobsen L, Hansen F, et al. Characterisation of an IMP-7-producing ST357 Pseudomonas aeruginosa isolate detected in Denmark using whole genome sequencing. Int J Antimicrob Agents. 2015;45(2):200-201. Ellington MJ, Kistler J, Livermore DM, Woodford N. Multiplex PCR for rapid detection of genes encoding acquired metallo-beta-lactamases. J Antimicrob Chemother. 2007;59(2):321-322. Pellegrini C, Mercuri PS, Celenza G, et al. Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location. J Antimicrob Chemother. 2009;63(5):901-908. Miró E, Agüero J, Larrosa MN, et al. Prevalence and molecular epidemiology of acquired AmpC βlactamases and carbapenemases in Enterobacteriaceae isolates from 35 hospitals in Spain. Eur J Clin Microbiol Infect Dis. 2013;32(2):253-259. Duljasz W, Gniadkowski M, Sitter S, Wojna A, Jebelean C. First organisms with acquired metallobeta-lactamases (IMP-13, IMP-22, and VIM-2) reported in Austria. Antimicrob Agents Chemother. 2009;53(5):2221-2222.

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Highlights

- The variant is characterized by a single amino acid substitution

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- The isolate retains a very high degree of activity against all β-lactams

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- A novel IMP-22 variant is presented in a pseudomonas putida isolate