Four carbapenem-resistant K. pneumoniae and one carbapenem-resistant S. marcescens were obtained from blood cultures during the years 2012 and 2016. The patients were hospitalized at Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, a tertiary teaching hospital in São Paulo, Brazil. These isolates belong to the Microbiology Laboratory Biobank.
Vitek II (bioMerieux, Marcy-l'Étoile, France) was used to identify the isolates KP1411, KP4301, KP158 and SM1581 species. The isolate KP4990 was identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Billerica, Massachusetts, USA).
To confirm the susceptibility pattern, the minimum inhibitory concentrations (MICs) were determined using Sensititre Gram Negative GNX3F AST Plate (TREK Diagnostic Systems, Cleveland, OH, USA). The antimicrobials tested were aztreonam, meropenem, imipenem, ceftazidime, colistin, amikacin, tigecycline and ciprofloxacin [20]. The susceptibility test results were interpreted according to the criteria recommended by Clinical Laboratory Standards Institute (CLSI) M100 [21]. Isolates were considered susceptible if the MIC of carbapenems were ≤1mg/mL, intermediate if MIC=2mg/mL and resistant with MICs ≥4mg/mL [22]. Escherichia coli ATCC25922 was used as quality control strain and Klebsiela pneumoniae ATC700603 as carbapenem-resistant Enterobacteriaceae control strain.
To precisely detect the genes coding for the carbapenemases KPC and NDM, we performed PCR using previously described primers for blaKPC-2 and blaNDM-1 [22,23,24]. The amplicons were submitted to Sanger Sequencing using MegaBACE 1000 (ABI 3730 DNA Analyser; Applied Biosystems, Alameda, CA) to confirm the gene identity.
To determine the genotypic profile including resistance and virulence genes, whole genome sequencing (WGS) was performed by Illumina MiSeq. For WGS, total DNA was extracted with Illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare Life Sciences, Marlborough, USA). DNA quality was verified using the NanoDrop spectrophotometer (Thermo Scientific, Delaware, USA). The quality of the files generated in the sequencing was evaluated by FastQC v.0.11.3 and Trimmomatic v.0:33. Genome assembly was performed using Velvet Optimiser v.2.2.5 and annotated with Prokka v.1:11 [25,26,27]. The sequence type (ST) of the isolates was determined with MLSTfinder tool [28] and confirmed in the database PubMLST (https://pubmlst.org/kpneumoniae/info/primers.shtml). The gene blaKPC-2 was manually investigated using Artemis v.16.0.0.
Additional phenotypic analysis was done to determine the resistance to carbapenems using the disk diffusion (DD) method described by Migliavacca et al. [29], and the CLSI breakpoints for carbapenems. Meropenem (MPM) commercial disks containing 10 µg (Sensidisc DME, Araçatuba, BRA) were added with 0.05 M of ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, USA) and/or 20 μg/mL of phenylboronic acid (PBA) (Sigma-Aldrich, St. Louis, USA). According to Migliavacca et al. [29], a difference of ≥4mm in the zone of inhibition diameter was used as criteria to determine whether the isolate produce a serine carbapenemase, a metallo-β-lactamase or both, in presence of EDTA and/or PBA, respectively, when compared to the MPM disk alone. E. coli ATCC25922 was used as negative quality control.