Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil

Carolina Silva Nodari Marina Siebert Ursula da Silveira Matte Afonso Luís Barth About the authors

Abstract

Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

Keywords:
GES-5; Serratia marcescens; Whole-genome sequencing

Nowadays carbapenemase production is the main carbapenem resistance mechanism among Enterobacteriaceae.11 Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm!. Trends Mol Med. 2012;18:263-272. The Guiana Extended-Spectrum (GES) β-lactamase family comprises several Ambler class A enzymes with distinguished β-lactam hydrolysis profiles. The original GES were classified as extended-spectrum β-lactamases (ESBL), but amino acid substitutions in the GES-type ESBLs enhanced their activity against carbapenems.22 Walsh TR. Clinically significant carbapenemases: an update. Curr Opin Infect Dis. 2008;21:367-371. The GES-5 is the GES-carbapenemase which hydrolyses imipenem most efficiently.33 Bae IK, Lee YN, Jeong SH, et al. Genetic and biochemical characterization of GES-5, an extended-spectrum class A beta-lactamase from Klebsiella pneumoniae. Diagn Microbiol Infect Dis. 2007;58:465-468. Here we report the draft genome of the first GES-5-producing Serratia marcescens reported in Brazil.

As part of a surveillance study,44 Rozales FP, Ribeiro VB, Magagnin CM, et al. Emergence of NDM-1-producing Enterobacteriaceae in Porto Alegre, Brazil. Int J Infect Dis. 2014;25:79-81. isolates with reduced susceptibility to carbapenems were submitted to Real Time (RT) High Resolution Melting (HRM) Multiplex PCR with specific primers for blaNDM, blaKPC, blaVIM, blaGES, blaOXA-48-like and blaIMP.55 Monteiro J, Widen RH, Pignatari AC, et al. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother. 2012;67:906-909. One isolate, obtained from an ascitic fluid of a female patient in a tertiary hospital in Porto Alegre (Brazil) in October 2014, and identified as S. marcescens by the Vitek2 system, presented an amplicon with a Temperature of Melting (Tm) similar to the blaGES positive control in the RT-PCR – 85.32 °C and 85.52 °C, respectively. Antimicrobial susceptibility was determined by Etest and the isolate presented high resistance levels to carbapenems (MIC > 32 mg/L) and polymyxins (MIC > 256 mg/L), but remained susceptible to fluoroquinolones and tigecycline.

Whole genome sequencing (WGS) was performed using the Ion Torrent PGM™ system, with a 400-bp-read kit and a 316™ Chip. Library was previously obtained by enzymatic fragmentation. We obtained 997,155 reads with a mean read length of 232 bp. The reads were assembled in contigs using SPAdes.66 Bankevich A, Nurk S, Antipov D, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455-477. The assembling revealed a 5,378,959 bp length genome, distributed in 208 contigs (≥500 bp), with a total GC content of 59%.

The contigs were annotated using the NCBI pipeline77 Tatusova T, DiCuccio M, Badretdin A, et al. Prokaryotic genome annotation pipeline. In: The NCBI Handbook [Internet]. 2nd ed; 2013. and manually curated using Artemis,88 Carver T, Harris SR, Berriman M, et al. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics. 2012;28:464-469. when necessary. We also submitted the contigs to ResFinder Database.99 Zankari E, Hasman H, Cosentino S, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640-2644. Annotation revealed 3657 CDS, as well as 70 tRNA and 14 rRNA genes. As expected, we were able to identify the presence of blaGES-5 (locus tag AN414_25255) after annotation, as well as genes coding for other β-lactamases (blaCTX-M-2 and blaOXA-2, locus tags AN414_24540 and AN414_25310, respectively), and aminoglycoside modifying enzymes (aac(3)-IIa and aac(6′)-Ic, locus tags AN414_24600 and AN414_08220, respectively). We were also able to observe the presence of a gene coding for an efflux pump to tetracyclines (tet(41), locus tag AN414_07940). The location of the resistance determinants in the genome (chromosome or plasmid borne) was not determined. To the best of our knowledge, this is the first report of a GES-5-producing S. marcescens in Brazil. We also demonstrated that NGS platforms can be used as a valuable tool to evaluate resistance determinants among Enterobacteriaceae.

Accession number: This whole-genome shotgun project has been deposited at GenBank under the accession number LNZT00000000. The version described in this paper is in the first version, LNZT01000000. The BioProject ID is PRJNA294719.

References

  • 1
    Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm!. Trends Mol Med 2012;18:263-272.
  • 2
    Walsh TR. Clinically significant carbapenemases: an update. Curr Opin Infect Dis 2008;21:367-371.
  • 3
    Bae IK, Lee YN, Jeong SH, et al. Genetic and biochemical characterization of GES-5, an extended-spectrum class A beta-lactamase from Klebsiella pneumoniae. Diagn Microbiol Infect Dis 2007;58:465-468.
  • 4
    Rozales FP, Ribeiro VB, Magagnin CM, et al. Emergence of NDM-1-producing Enterobacteriaceae in Porto Alegre, Brazil. Int J Infect Dis. 2014;25:79-81.
  • 5
    Monteiro J, Widen RH, Pignatari AC, et al. Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother. 2012;67:906-909.
  • 6
    Bankevich A, Nurk S, Antipov D, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455-477.
  • 7
    Tatusova T, DiCuccio M, Badretdin A, et al. Prokaryotic genome annotation pipeline. In: The NCBI Handbook [Internet] 2nd ed; 2013.
  • 8
    Carver T, Harris SR, Berriman M, et al. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 2012;28:464-469.
  • 9
    Zankari E, Hasman H, Cosentino S, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67:2640-2644.

Publication Dates

  • Publication in this collection
    Apr-Jun 2017

History

  • Received
    21 Mar 2016
  • Accepted
    12 Aug 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br