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19 protocols using fpquest software

1

PCR Melting Profile Analysis

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The polymerase chain reaction melting profile (PCR MP) procedure was performed as described by Krawczyk et al. [14 (link)]. A dendrogram was generated with the Dice Similarity Coefficient (DSC) with a 1% band tolerance setting, and using the unweighted pair group method with arithmetic mean (UPGMA) (FPQuestTM software, BioRad, Ver. 4.5).
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2

Genomic Diversity Analysis of E. coli

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The polymerase chain reaction melting profiles (PCR MP) procedure was used to assess the genomic diversity of the isolated E. coli. The PCR MP was carried out as described in Krawczyk et al. [38 (link)]. Amplified genomic DNA fragments were separated using polyacrylamide gel electrophoresis (6%) in 1×TBE buffer and stained with ethidium bromide. Images of the gels were analyzed and archived using the Versa Doc Imaging System version 1000 (Bio-Rad Laboratories, Hercules, USA). Dendrogram was generated by the Dice Coefficient (DC) with UPGMA method and with a setting of 1% band tolerance (FPQuest TM software, BioRad; version 4.5).
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3

Genotyping and Molecular Characterization of Vancomycin-Resistant Enterococcus faecium Isolates

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Genotyping was carried out for 19 isolates, including 18 LZDREF isolates collected in the period from 2013 to 2017 and one LRVREF E. faecium strain isolated from the same hospital but in 2003 (control strain K2003). Molecular characterization of clonal identity of all isolates was performed by means of PCR melting profiles (PCR MP) method as has been described previously [9 (link)] with the following modification. We used the EcoRI restriction enzyme at the step of digestion of total genomic DNA instead of the Hind III. All sequences of oligonucleotides for adapters and primers are included in S1 Table. The reaction profile with the denaturation temperature in the PCR cycles was the same as described in the work of Krawczyk et al. [9 (link)]. Polyacrylamide gel electrophoresis (6%) in 1xTBE buffer for the separation of DNA fragments was prepared and then was stained with ethidium bromide. Subsequently, FPQuest TM software (BioRad; version 4.5) for comparative analysis of the DNA patterns was used. Similarities between genetic fingerprints were calculated using Dice band-based similarity coefficient (SD) with UPGMA method.
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4

PCR Fingerprinting Protocol Validation

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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 (FPQuestTM software, BioRad, Ver. 4.5) and UPGMA (unweighted pair-group method with arithmetic averages). The Pi cut-off for genotype definition was 96%.
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5

Pulsed-Field Gel Electrophoresis for Bacterial DNA Analysis

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Agarose-embedded plugs containing intact bacterial DNA were prepared using a CHEF (Contour-Clamped Homogenous Electric Fields) Mapper Plug Kit (Bio-Rad Laboratories, Hemel Hempstead, UK) according to the manufacturer's instructions. Following overnight digestion of the plugs with Xbal at 37 8C, PFGE was performed on a CHEF Mapper XA PFGE System (Bio-Rad Laboratories) according to the PulseNet standardised laboratory protocol [9] . The electrophoresis conditions were optimised for separation of the 30-600 kb fragments in a 1% agarose gel with 0.5Â TBE [Tris-borate-ethylene diamine tetra-acetic acid (EDTA)] buffer and a voltage of 6 V/cm for 19 h, with an initial switch time of 2.2 s and final switch time of 54.2 s. A dendrogram was constructed using the Dice coefficient with an optimisation of 1% and a tolerance of 1% in band position difference (FPQuest TM Software; Bio-Rad Laboratories). The clonal relationship among isolates was determined according to the criteria of Tenover et al. [10] . Clonal groups were assigned based on a similarity of !80% ( 3 band difference in restriction profile).
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6

Chromosomal Comparison of V. parahaemolyticus Using REP-PCR

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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.
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7

Microbial Diversity Analysis using MLVA

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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).
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8

Molecular Typing of S. aureus Isolates

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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.
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9

Molecular Profiling of Salmonella Isolates

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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 (blaCMY, blaTEM, blaCTX-M, and/or blaSHV) 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)].
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10

Pulsed-Field Gel Electrophoresis for Bacterial Strain Typing

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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.
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