Four carbapenem-resistant KP and one carbapenem-resistant S. marcescens were obtained from blood cultures from patients hospitalized at Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo a tertiary teaching hospital in Sao Paulo, Brazil comprised of 2,200 beds.
The five isolates were from the Microbiology Laboratory samples bank and were involved in previous studies, when informed consent term was obtained. This is a retrospective study and all methods were performed in accordance with the guidelines and regulations, approved by the Ethics Committee of Hospital das Clinicas of University of São Paulo, Brazil. Data was consolidated and analyzed, and patients’ identity was confidential. The approval numbers are 1.310.231, 2.158.859 and 2.452.282.
Vitek II (BioMerieux-France) was used to identify the isolates species until February 2015, since then we have been using the Matrix-assisted laser desorption ionization-time of flight (MALD-TOF/Bruker) as well.
The minimum inhibitory concentration (MIC) was tested by Sensititre (ThermoFisher Diagnostics-) to Aztreonam, Meropenem, Imipenem, Colistin, Amikacin and Tigecycline. The susceptibility test results were interpreted according to the criteria recommended by CLSI. Escherichia coli isolate ATCC 25922 was used as the quality control of the test.
Additional phenotypic analysis was done to characterize the resistance using disk diffusion (DD) method, cutoff according to CLSI: Meropenem commercial disks containing 10µg were added with 0.05 M of Ethylenediaminetetraacetic acid (Sigma-Aldrich, St. 115 Louis, MO) and/or 20 μg ml−1of phenylboronic acid (Sigma-Aldrich, St. Louis, MO). According to Migliavacca et al (2002)(19), a difference of ≥4mm in the zone of inhibition diameter was used as criteria to determine whether the isolate produced serine carbapenemase, metallo-beta-lactamase or both in presence of EDTA, PBA or either, respectively, when compared to the MPM disk alone. E. coli ATCC 25922 was used as negative quality control.
To detect carbapenemases genes, we performed PCR using previously described primers for blaKPC-2 and blaNDM-1 genes(20). The amplicons were submitted to Sanger Sequencing using MegaBACE 1000 (ABI 3730 DNA Analyser; Applied Biosystems, Alameda, CA) to confirm the gene identity.
Whole Genome Sequencing (WGS) was done by Illumina MiSeq. For the WGS, total DNA was extracted with Illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare Life Sciences). DNA quality was verified using the NanoDrop spectrophotometer (Thermo Scientific, Delaware, USA). The whole genome was sequenced by MiSeq IlluminaTM. Libraries were prepared with the commercial kit Nextera XT IlluminaTM according to manufacturer's instructions. The quality of the files generated in the sequencing was evaluated by FastQC v.0.11.3 and Trimmomatic v.0:33. The genome assembly was performed using Velvet Optimiser v.2.2.5. The genome was annotated with Prokka v.1:11. The ST of the isolates was checked by MLSTfinder tool (Larsen et al, 2012) and confirmed in the database PubMLST (http://pubmlst.org). The gene blaKPC-2 was manually investigated using Artemis v.16.0.0.