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33 protocols using gelcompar 2 software

1

PFGE Analysis of Salmonella Serotype Braenderup

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After digestion with Xba I and Apa I endonucleases, the DNA was separated using a CHEF-DR II system (BioRad, Birmingham, UK) [42 (link),43 (link)]. The Salmonella serotype Braenderup strain (H9812) was included in each PFGE gel as an internal control. Computer analysis of PFGE profiles was done using the Gel Compar II software, v.6.6.11 (Applied Maths, Inc.; Sint-Martens-Latem, Belgium) after visual inspection using the criteria of Tenover [44 (link)]. The Dice coefficients were calculated and were then transformed into an agglomerative cluster by the unweighted pair group method with arithmetic average (UPGMA).
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2

Clonality Analysis of MRSA and Klebsiella

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The clonality of MRSA and meropenem-resistant isolates of Klebsiella pneumoniae detected at the Department of Anesthesiology and Intensive Care Medicine was assessed with pulsed-field gel electrophoresis (PFGE). Bacterial DNA extracted with a technique described by Husičková et al. [19 (link)] was digested by the XbaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 37 °C in Klebsiella pneumoniae isolates and by the SmaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 25 °C in Staphylococcus aureus strains. The obtained DNA fragments were separated by PFGE on 1.2% agarose gel for 24 h at 6 V/cm and pulse times of 2–35 s for both Klebsiella pneumoniae and Staphylococcus aureus strains. Subsequently, the gel was stained with ethidium bromide. The resulting restriction profiles were analyzed with the GelCompar II software (Applied Maths, Kortrijk, Belgium) using the Dice coefficient (1.2%) for comparing similarity and unweighted pair group method with arithmetic means for cluster analysis. The results were interpreted according to criteria described by Tenover et al. [20 (link)].
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3

Amplification and Fingerprinting of Microbial Communities

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Total community DNA was used as the template for amplification of the partial 16S rRNA gene fragment using Taq DNA polymerase (Bioline, Lückenwalde, Germany) with primer F968 with a GC clamp attached to the 5′ end and universal bacterial primer R1401.1b. For ITS1 amplification, primers EF4/ITS4 were used; this PCR was followed by a second amplification with primers ITS1f-GCITS2. Primer sequences, the reactions mixtures, and cycling conditions have been described (Pereira e Silva et al., 2012 (link)). The DGGE was performed in 6 % (w/v) polyacrylamide gels with 45–65 % and 20–50 % denaturant gradients for bacterial and fungal communities, respectively (100 % denaturant is defined as 7.0 M urea with 40 % deionized formamide). Electrophoresis was carried out at 100 V and 75 mA, for 16 h at 60 °C. The gels were subsequently stained for 40 min in 0.5 % TAE buffer with SYBR gold (final concentration 0.5 μg/L) (Invitrogen, Breda, the Netherlands) (Fig. S1 in the Supplementary Material). Gel images were digitized using Imagemaster VDS (Amersham Biosciences, Buckinghamshire, UK). The DGGE patterns were then transformed to a band-matching table using GelCompar II software (Applied Maths, Sint Martens Latem, Belgium).
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4

Multilocus Sequence Typing and PFGE Analysis

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The sequences of seven loci (adk, fumC, gyrB, icd, mdh, purA, and recA) were amplified using PCR, and STs were determined by sequence alignment using the Achtman scheme available at https://pubmlst.org/escherichia/. Genetic relatedness among identified isolates were analyzed using the PFGE method as described previously (31 (link)). The similarity index was calculated with the Dice coefficient and dendrogram constructed by the UPGMA (unweighted pair group method using average linkages) algorithm using GelCompar II software (Applied Maths, Belgium).
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5

Standardized PFGE Analysis of B. pertussis

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PFGE analysis was performed according to references [31 (link), 32 (link)] with some modifications. The procedure stated in brief: B. pertussis genomic DNA from 18 isolates in a Seakem Gold Agarose-gel plug was extracted from the bacteria with lysis buffer containing 1 % (w/v) sarcosyl and 100 μg/mL proteinase K and the plug incubated in a shaker bath at 56 °C for 16 h, and finally washed with 1X TE buffer six times at 50 °C in shaker bath. Genomic DNA was digested with the restriction enzyme XbaI at 37 °C for 6 h. Finally, electrophoresis was performed in a CHEF Mapper XA (Bio-Rad) apparatus for 23 h, with an initial pulse of 5 s and a final pulse of 35 s. The analysis and comparison of PFGE patterns were performed with Gel Compar II software, version 5.1 (Applied Maths), on the basis of the banding pattern of Salmonella Branderup (H9812) DNA as the normalization standard.
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6

ESBL-Producing Isolates Genotyping by PFGE

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All isolates producing ESBL detected by phenotype tests (ceftazidime and/or cefepime-resistant isolates) were typed by PFGE according to the Seifert et al. 2005 protocol [26 (link)] with modifications, using the SpeI enzyme (Thermo Scientific). Electrophoresis was performed by using a CHEF DR II system (BioRad). The migration conditions were as follows: switch angle 120, voltage 6 V/cm, temperature 12°C, and a two-blocks program with a total run time of 26 h (the first-block 1-20 s for 20 h and the second-block 15-40 s for an additional 6 h). The genomic DNA of Salmonella serotype Braenderup strain (H9812) was digested with XbaI (Thermo Scientific) [27 (link)] and the Lambda-DNA Ladder PFG Marker (New England BioLabs) were used as the DNA molecular-weight markers. PFGE patterns were analysed using GelCompar II software (Applied Maths, Belgium) with the Dice coefficient and clustering by UPGMA with 1% tolerance. According to the recommendation of Tenover et al. [28 (link)], the isolates were clustered in the PFGE pulsotypes (PTs).
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7

Extracting S-Layer Proteins from Lactobacillus

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A slightly modified method by Uroić et al. [58 (link)] was used to extract S-layer proteins from the cell surfaces of selected LAB cells isolated from human milk. Lactobacillus cells were grown in MRS broth to an OD620 nm of 2.0, washed and treated with Laemmli buffer. Samples were boiled and subjected to Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), as described by Kos et al. [59 (link)]. Electrophoretic separation of proteins was performed at a constant voltage of 100 V, along with the ProSieve QuadColor Protein Marker 4.6–315 kDa (Lonza, Morristown, NJ, USA). Gel images were characterised using the HP Scanjet 3800 Photo Scanner, and the obtained electrophoretic profiles were analysed using the GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium) and depicted in the form of a dendrogram. Identification of the extracted S-layer proteins was achieved, according to Banić et al. [53 (link)]. Briefly, protein bands corresponding to a molecular size in the range of 45–55 kDa were excised from the SDS-PAGE gel, trypsinised and subjected to an LC-MS combined with a Mascot database search.
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8

Molecular Typing of Bacterial Isolates

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Typing by randomly amplified polymorphic DNA (RAPD) analysis was performed according to the protocol published by Deschaght et al.60 (link) using the primer RAPD4 (5′-AAGACGCCGT-3′). Briefly, two microliters of the DNA template were added to 12.5 μL multiplex mastermix (MyTaq™HM Mix, Bioline®, MA, USA), 1 μL primer (10 pmol), and 9.5 μL H2O. PCR cycles of initial incubation at 94 °C for 15 min followed by cycling for 40 times at 94 °C for 1 min, 37 °C for 1 min, and a final elongation at 72 °C for 2 min was performed.
ERIC typing was carried out using the primer ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) using a similar PCR program to that of the RAPD method except for an extension time of 8 min59 (link).
RAPD and ERIC fragments were visualized by 1.5% w/v agarose gel electrophoresis and results were analyzed using GelCompar II software (Version 6.6.11, Applied Maths, Kortrijk, Belgium). The patterns were normalized with bands of the marker and bands that were consistently present in all patterns. Computer-assisted analyses implemented in this study were performed according to the manufacturer’s instructions.
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9

Genotyping B. pertussis Isolates via PFGE

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Pulsed-field gel electrophoresis (PFGE) of B pertussis isolates, including the control isolate Salmonella Braenderup H9812 (kindly provided by the Minnesota Department of Health), was performed using XbaI restriction enzyme (Roche Applied Science) as previously described.12 ,13 The gel image was captured on a Gel Doc XR system (Bio-Rad Laboratories, Inc). Analysis of PFGE patterns was performed with GelCompar II software (Applied Maths). Similarity coefficients were calculated using the Dice algorithm.
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10

Genetic Relatedness Analysis of Bacterial Strains

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We performed pulsed-field gel electrophoresis (PFGE) for the Tokyo, Tochigi, and Tottori strains to determine their genetic relatedness. DNA was digested with SmaI. We used the CHEF Mapper Pulsed Field Electrophoresis Systems (Bio-Rad Laboratories, https:///www.bio-rad.com) for electrophoresis. We analyzed resulting photographic images by using the GelCompar II software (Applied Maths, http://www.applied-maths.com). Isolates with a PFGE fingerprint similarity >80% were clustered into the same PFGE cluster type (15 (link)).
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