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Emergence of ST654 Pseudomonas aeruginosa co-harbouring blaNDM-1 and blaGES-5 in novel class I integron In1884 from Bulgaria

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a common cause of hospital-acquired infections. Carbapenem resistance, in particular, represents a substantial problem in terms of treatment of infections due to this pathogen and leads to increased mortality, longer duration of hospital stay and increased healthcare costs [1].

The class B New Delhi metallo-β-lactamase 1 (NDM-1) carbapenemase has spread to and has been described in many bacterial species, including P. aeruginosa [2] Another carbapenem-hydrolysing enzyme, the Guiana extended-spectrum β-lactamase-5 (GES-5), initially found in Escherichia coli in Greece and belonging to class A β-lactamase family, has also been detected in P. aeruginosa in Brazil, China, Spain and South Africa [3].

Here, we report the first detection of NDM-1-producing P. aeruginosa isolates in Bulgaria and the chromosomal co-harbouring of blaNDM-1 and blaGES-5 genes in these MDR P. aeruginosa in novel class I integron In1884.

Five carbapenem-resistant (CR) P. aeruginosa strains were isolated from clinical samples of patients in two Bulgarian hospitals. The first two isolates, named Pae1250 and Pae1251, were recovered in September 2017 from urine samples (>105 CFU/mL) of two different patients hospitalised in Alexandrovska University Hospital in Sofia. In August 2018, we detected three other CR P. aeruginosa isolates (Pae1252, Pae1255 and Pae1257) from tracheobronchial aspirates of three patients hospitalised in ‘St. Ivan Rilski’ University Hospital in Sofia. A CR Klebsiella pneumoniae (Kpn1256) was also isolated from the patient from whom CR P. aeruginosa Pae1255 was collected.

Antibiotic susceptibility testing was performed using Etest (bioMérieux, la Balme-les-Grottes, France). Colistin susceptibility was tested by broth microdilution method using MICRONAUT plate (MERLIN Diagnostika GmbH, Bornheim, Germany) and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines v9.0 ( All six strains were resistant to carbapenems and had minimum inhibitory concentration (MIC) values of imipenem and meropenem >32 mg/L. They were also resistant to ceftazidime, cefepime, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, ciprofloxacin, levofloxacin, amikacin, tobramycin and gentamicin, but were susceptible to colistin (MIC range 1–2 mg/L). Genomic DNA from all strains (five P. aeruginosa and one K. pneumoniae) was extracted using the MasterPure™ DNA Purification Kit (Epicentre Technologies Inc.) and sequenced (via 2 × 250 bp, MiSeq, Illumina, San Diego, USA), and long-read sequencing (MinION, Oxford Nanopore Technologies, Oxford, UK) of Pae1250 and Pae1255 was done according to the manufacturer’s instructions. Data analysis was performed using an in-house tool (BacPipe) [4], and single-nucleotide polymorphism (SNP) calling and genetic context analysis was performed using CLC Genomics Workbench v.9.5.1 (Qiagen, Hilden, Germany).

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