Gelcompar

Analysis of the genetic similarity between SA isolates was performed by PFGE method in accordance with a previously published protocol [11 (link)]. Restriction enzyme digestion was performed with 25 U of SmaI enzyme in Tango buffer (ThermoScientific, USA). Electrophoresis was conducted in a CHEFIII PFGE unit applying the parameters: block 1- starting pulse 5 s, ending pulse 12 s, voltage 6 V/cm, run time 11 h, block 2- starting pulse 20s, ending pulse 60 s, voltage 6 V/cm, run time 13 h. The GelCompar (Applied Maths, Sint-Martens-Latem, Belgium) was used for cluster analysis using the Dice coefficient and unweighted pair group method with arithmetic mean. Isolates that clustered ≥ 95% were considered as an epidemic clone. S. aureus strain ATCC 11632 was used as reference.

We performed routine identification using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and biochemical species identification. For MALDI-TOF mass spectrometry, we used Biotyper MBT Smart (Bruker Corporation, https://www.bruker.com) with flexControl and MBT Compass version 4.1 software. We considered scores >2.0 high confidence identification and scores of 1.7–2.0 low confidence identification. We used VITEK 2 gram negative identification card (bioMérieux, https://www.biomerieux.com) for biochemical species identification. Phenotypic antimicrobial resistance profiles were determined using disk diffusion. We interpreted breakpoints according to Clinical and Laboratory Standards Institute (9 ) standards for Bcc (ceftazidime, trimethoprim/sulfamethoxazole, and meropenem) or Enterobacteriaceae (aminoglycosides, ciprofloxacin, piperacillin/tazobactam, and other β-lactams). We used XbaI to digest DNA before using previously described pulsed-field gel electrophoresis (PFGE) molecular typing principles (10 (link)). We used GelCompar (Applied Maths, http://www.applied-maths.com) to analyze PFGE results.

All isolates of Klebsiella pneumoniae were analyzed using the standardized PFGE protocol developed at the Centers for Disease Control and Prevention by the PulseNet program (version for Escherichia coli). Data from previous studies showed that K. pneumoniae strains isolated in Polish hospitals (mainly from ICUs for adults and neonates and pediatric units) often belong to one clone. For this reason, a PFGE study was performed for this species to check for clonal spreading of strains coming from oral bacteriota samples and HAI cases. Genomic DNA was prepared in situ in agarose blocks and was subsequently digested with restriction enzymes: XbaI (25 U per block, Thermo Scientific. The digested products were separated on a CHEF III PFGE system (Bio-Rad, Warsaw, Poland) in 0.5 × Tris-borate-EDTA buffer at 14 °C at 6 V for 22 h with a starting pulse of 2 s and a final pulse of 35 s. GelCompar (Applied Maths, Kortrijk, Belgium) was used for cluster analysis with the unweighted pair group method with an arithmetic mean and the Dice coefficient. Similarity must be > 90% for the pattern to be considered to belong to the same pulsotype. The results of the PFGE analysis are presented for each patient with a number that they were assigned on recruitment (1–56). The term “case” refers to HAI diagnoses.