Positive signal intensities were transformed in a binary code. The binary code corresponding to the
core genome was converted to a hexadecimal code as previously described [7]. Pulsed-field gel electrophoresis (PFGE) PFGE was performed on 162 isolates of our collection, as previously described [8, 31]. In detail, chromosomal DNA was prepared in 2% (wt/vol) low melting point agarose plugs this website and digested with SpeI restriction enzyme at 37°C overnight. Samples were run on 1% (wt/vol) agarose gel in 0.5X TBE buffer at 14°C on a CHEF DR-III PFGE system (Bio-Rad, Hertsfordshire, United Kingdom). PFGE run settings were: initial switching time 5 s; final switching time 45 s; gradient 6 V; run time 21 h. PFGE band patterns were compared as described previously [4] and the PFGE clusters were defined according to the criteria established by Tenover and coworkers [32]. In detail, isolates with band pattern with >85% similarity were refer to as genetically related clones. Multilocus sequence typing (MLST) A total of 80 P. aeruginosa independent isolates were typed. MLST was performed as described by Maatallah and co-workers [33]. Briefly, genomic DNA was isolated by using the “DNeasy Blood & Tissue kit” (Qiagen,
Valencia, CA, USA) following the manufacturer’s guidelines. DNA amplification of the seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) was performed with a MiniOpticon real-time PCR detection system (Bio-Rad Laboratories, Munich, Germany) using the QuantiTect Capmatinib nmr SYBR Green PCR mix (Qiagen, Valencia, CA, USA). Standard primers [34] were employed as previously described [33]. The specificity of the amplification products was
determined by a final melting curve analysis. DNA products were purified and sequenced on both strands by Eurofins MWG Operon these GmbH (Ebersberg, Germany) with published primers [33]. Sequences were compared to publicly available MLST databases, accessible on the P. aeruginosa MLST website (http://pubmlst.org/paeruginosa). Each isolate was assigned a sequence type (ST) number according to its allelic profile. Genetic distance between MLST profiles was calculated as defined at http://pubmlst.org/analysis/. Evaluation of typing methods The discriminatory index (DI), which indicates the probability for two strains, sampled randomly from a population, to belong to a different type was calculated as previously described [35]. In order to quantify the congruence between typing methods the adjusted Rand coefficient was calculated, using the algorithm available at http://I-BET-762 mw comparingpartitions.info. The first coefficient quantifies the global agreement between two methods, while the second indicates the probability that two strains are coherently classified as the same clone by both methods [35, 36]. Identification of AT cluster of clones The relatedness between the AT-genotypes was inferred with the eBURST clustering algorithm (http.//eBURST.mlst.net).