DNA concentration was measured by NANODROP 1000 Spectrophotometer (Thermo Scientific). The amplified product was stored at -20°C until detection. Analysis of rep-PCR products was performed using a DiversiLab system, in which the amplified fragments of various sizes and intensities are separated and detected using a microfluidic LabChip by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The relatedness GS-1101 molecular weight of the isolates was analysed
by means of the DiversiLab software, version 3.4, using the Pearson correlation coefficient, to determine distance matrices, and the unweighted-pair group method with arithmetic mean (UPGMA). Further analysis was performed by the Kullback–Leibler distance correlation coefficient. In general, “different” was defined as <95% pattern similarity and 2 or more band differences for organisms defined as homogeneous, and three or more band differences for organisms defined as heterogeneous. “Similar” was defined as >95% and <97.% pattern similarity and one band difference for homogeneous organisms, or up to two band differences for heterogeneous organisms. “Indistinguishable” was defined as >97% pattern similarity and
no band differences, including no variation in the intensities of individual bands, although overall intensities may differ [13]. Genotyping by PFGE The 23 O. anthropi isolates were grown overnight and suspended in SE buffer (75 mM NaCl, 25 mM EDTA, pH 7.5). The cell suspensions (4 McFarland units) were mixed with Selleck LY333531 an equal volume of 2% low-melting point agarose, moulded into plugs at 4°C, and lysed with lysis buffer (1% N-lauryl sarcosine, 0.5 M EDTA, pH 8) supplemented with Proteinase K (500 μg/mL).
The DNA contained in each plug was digested by 20 U of SpeI restriction enzyme in accordance with the manufacturer’s instructions. Macrorestriction fragments were separated using the CHEF-DR III PFGE system (Bio-Rad) at 10°C for 20 h, at a field strength of 6 V/cm, with an initial Sodium butyrate switch time of 5 s and a final switch time of 35 s. A lambda ladder of phage DNA concatemers was used as a size marker. O. anthropi ATCC 49188 T and O. intermedium LMG 3301 T were also genotyped by PFGE, and fragment patterns were compared according to the criteria AZD5363 cost described by Tenover [14]. MALDI-TOF MS and data analysis A single colony grown overnight on TSI (Triple Sugar Iron) agar was spotted in duplicate onto the MALDI target (MSP 96 target polished steel (MicroScoutTarget) plate; (Bruker Daltonik, Bremen, Germany) and air-dried at room temperature. After air-drying, each spot was overlaid with 1 μL of HCCA (a-cyano-4-hydroxycinnamic acid) matrix solution saturated with organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid) and air-dried completely before MALDI-TOF MS. MALDI-TOF MS was carried out using a MALDI Microflex LT.