Chef 3 pfge system

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.

Analysis of genetic similarity between A. baumannii strains was performed using pulsed-field gel electrophoresis (PFGE) in accordance with a previously published protocol [34 (link)]. Electrophoresis was conducted using the CHEF III PFGE system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Gel Compar II 6.5 (Applied Maths, Sint-Martens-Latem, Belgium) was used for cluster analysis using the Dice coefficient and unweighted pair group method with arithmetic mean. Isolates with more than 95% similarity were clustered together as identical.

PFGE was used to determine the possible horizontal transfer of E. coli strains among patients. All isolates were analysed using the standardized PFGE protocol developed at the Centers for Disease Control and Prevention by the PulseNet program http://ttp://www.cdc.gov/pulsenet/pathogens/ecoli.html (accessed: 11.02.2013). Genomic DNA was digested with 10 U XbaI (ThermoScientific, ABO, Gdansk, Poland). The resulting DNA fingerprinting was analysed using the CHEF III PFGE system (BioRad, Warsaw, Poland) in 0.5 Tris–borate–EDTA buffer at 14°C at 6 V for 20 h with a ramped pulse time of 2.2–54.2 s. The GelCompar (Applied Maths) was used for cluster analysis using the Dice coefficient and the unweighted pair group method with arithmetic mean.