Fpquest software

A PCR MP method was used according to the procedure described by Stojowska et al. [35 (link)], with the slight modification described by Gołębiewska et al. [36 (link)]. Comparisons of electrophoretic profiles, based on band position, were made using Bio-Rad software (FPQuest^TM software, BioRad, Ver. 4.5) and UPGMA (unweighted pair-group method with arithmetic averages). The P_i cut-off for genotype definition was 96%.

REP-PCR, used for chromosomal comparisons of V. parahaemolyticus isolates, was conducted using two primers: REP-1D, 5’-NNN RCG YCG NCA TCM GGC-3’; and REP-2D, 5’-RCG YCT TAT CMG GCC TAC-3’ (where M is A or C, R is A or G, Y is C or T, and N is any nucleotide) as reported previously (Wong and Lin, 2001 (link)). The experiment was performed followed by Mizan et al. (2017) (link), and a digital image was captured through a charge coupled device camera (Gel Doc XR system, Bio-Rad). The resulting fingerprints were analyzed using FPQuest software (Bio-Rad Laboratories, Inc., Hercules, CA, United States). Similarities between digitized profiles were counted using Pearson’s correlation, and an average linkage (unweighted pair group method with arithmetic mean, UPGMA) dendrogram was obtained.

Simpson's diversity index was calculated using an online tool (http://www.comparingpartitions.info/). Minimum spanning trees were generated based on the MLVA data using FPQuest software (Bio-Rad).

To demonstrate the clonal diversity of the analyzed S. aureus strains, they were subjected to molecular typing by pulsed-field gel electrophoresis (PFGE), using CHEF Bacterial Genomic DNA Plug Kits (Bio-Rad, Marnes-la-Coquette, France) according to the procedure described by Masiuk et al. [15 (link)]. Briefly, digestion of whole-genomic DNA was performed using the SmaI enzyme (MBI Fermentas, Ontario, Canada) according to the manufacturer’s protocol. PFGE was conducted using a CHEF DR III apparatus (Bio-Rad, Marnes-la-Coquette, France) in 2% agarose gel (DNA Gdansk, Gdansk, Poland) and 1×TBE (Tris-borate-EDTA) buffer with specific parameters of electrophoresis described by Masiuk et al. [15 (link)]. After electrophoresis, the gel was stained with ethidium bromide in a covered container for 30 min, visualized and photographed using a GelDoc-It2 Imager gel imaging system (Analytik Jena US LLC, Upland, CA, USA). S. aureus ATCC 25904 and S. aureus ATCC 6538 strains were included in the study.
The PFGE macrorestriction profiles were analyzed using FPQuest software (Bio-Rad, Marnes-la-Coquette, France). Classification of individual restriction patterns for each genetic profile was conducted using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Dice’s coefficient (2.0%). PFGE results were presented as a dendrogram.

S. Infantis isolates (n = 26), S. Manhattan isolates (n = 7), and one O-untypeable:r:1,5 isolate that were shown to carry resistance genes (bla_CMY, bla_TEM, bla_CTX-M, and/or bla_SHV) were further examined using pulsed-field gel electrophoresis (PFGE) profiling and large-plasmid profile (LPP) analysis. To compare isolates with and without resistance genes, S. Infantis isolates (n = 18) and S. Manhattan isolates (n = 18) showing susceptibility to 11 antimicrobials were also examined. Two, three, five, four, and four susceptible S. Infantis isolates were collected in 2006, 2007, 2008, 2009, and 2010, respectively. Of the 18 susceptible S. Manhattan isolates, two, one, two, three, two, two, three, and three were collected in 2003, 2004, 2005, 2006, 2007, 2008, 2009, and 2010, respectively. PFGE using BlnI restriction digestion was conducted as previously described [30 (link)]. Similarity and cluster analyses were performed using the Dice coefficients of similarity and an unweighted pair group method with average linkage, respectively, using FPQuest Software (Bio-Rad Laboratories, Hercules, CA). For LPP analysis, total DNA was treated with 2 U/ml of S1 nuclease (incubated at 37°C for 45 min), followed by PFGE separation [30 (link)].

PFGE was performed as described previously [41 (link)], with slight modifications. Briefly, the isolates were cultured in TSB supplemented with 1 μg/ml cefotaxime at 37°C overnight. After centrifugation, PFGE plugs were prepared by mixing culture pellets with 1% SeaKem Gold Agarose (Lonza, Basel, Switzerland). The plugs were digested with 30 U XbaI at 37°C overnight. The plugs were electrophoresed with a CHEF-DR III system (Biorad, Hercules, CA) in a 1% SeaKem Gold Agarose gel in 0.5× TBE buffer at 14°C and 6 V/cm for 16 h. Switching times were ramped from 2.2 to 54.2 s. Dendrographic analysis of the DNA fragments was performed using FPQuest software (Bio-rad). The Salmonella serotype Braenderup H9812 strain provided by Dr. Umeda, Osaka Institute of Public Health, was used as a DNA size standard.