The E. coli phylogroups including A, B1, B2, C, D, E, F and Escherichia cryptic clade I were characterised by the Clermont phylo-typing scheme [66 (link)]. Genetic relatedness was illustrated by a dendrogram construction based on rep-PCR fingerprint analysis. The repetitive regions were amplified by 1 µM REP-1 primer (5′-GCGCCGICATCAGGC-3′) and 1 µM REP-2 primer (5′-ACGTCTTATCAGGCCTAC-3′) in 50 µL of PCR reaction mixture [67 (link)]. The DNA fingerprint patterns were illustrated by 1.5% agarose gel electrophoresis, and the dendrogram was constructed using the Bionumeric software (Applied Maths, Sint-Martens-Latem, Belgium) with 1% position tolerance. Isolates presenting more than 70% similarity were categorised in the same cluster. Representative isolates of identical phylogroups and rep-PCR patterns that contained each blaCTX-M variants and the strains having blaNDM-5 or mcr-3 genes were selected for MLST. The STs were identified by the Achtmann MLST scheme by comparing them to sequences and alleic profiles on the Enterobase website (https://enterobase.warwick.ac.uk/species/ecoli/ ) (accessed on 15 March 2021) [13 (link)].
Bionumeric software
Manufactured by bioMérieux
Sourced in Belgium
Bionumeric software is a data management solution developed by bioMérieux. It is designed to facilitate the organization and analysis of data generated from various laboratory equipment and processes.
Automatically generated - may contain errors
5 protocols using bionumeric software
1
Characterization of E. coli Phylogroups
2
Molecular Typing of Streptococcus suis
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This method was modified from Son et al. [10 (link)]. For each S. suis serotype 2 strain, two independent extractions of DNA were performed to verify the reproducibility of patterns. Briefly, the cell suspension was prepared from overnight Todd-Hewitt Broth (Difco) culture and mixed with 1% agarose to form the agarose plugs. The cells were lysed with lysozyme and then treated with proteinase. The DNA was digested with SmaI and its fragments were resolved by PFGE within 1% electrophoresis grade agarose gel using a CHEF-DR II system (Bio-Rad). The gels were stained with ethidium bromide and photographed under a UV light. The cluster analysis was provided by Bionumeric software (Applied Maths) and transformed into an agglomerative cluster using the unweighted pair group method with arithmetic averages (UPGA) (based on Dice coefficients).
3
Analyzing Antibiotic Resistance Patterns
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Bionumeric software (v.7.0, Applied Maths, Biomeriux, USA) was used to analyze antibiotic resistance pattern of the isolates used in the study and dendrogram was generated using UPGMA algorithm inbuilt in the software. Pearson correlation coefficient of OMPs and in-vitro adhesion and invasion frequency were generated using XLSTAT software (v. 2017, www.xlstat.com/en/ ). Analyzed matrix was then plotted in biplot to assess the association between attributes and the pathogenic potential was derived from the biplot generated from principal component analysis. GraphPad prism v. 7.0 was used to generate graphs from in-vitro infection assays.
4
Genotyping Enterococcus faecalis Using MLST and PFGE
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MLST was performed as described [26 (link)] using the web-accessible database https://pubmlst.org/organisms/enterococcus-faecalis (last accessed 18th October 2021) to establish alleles and sequence types (STs) [27 (link)]. STs were grouped into CCs by the comparative eBURST analysis performed against the whole E. faecalis MLST database (https://pubmlst.org ; last accessed 31st March 2020). The MLST data for three isolates (6210/09, 6432/09 and 6878/09) were reported previously [28 ] and used in the current analyses. Pulsed-field gel electrophoresis (PFGE) was performed according to de Lancastre et al. [29 (link)] for agarose plugs preparation, followed by the procedure of Clark et al. [30 (link)] for total genomic DNA purification. Purified DNA in plugs was digested with the SmaI restriction enzyme (Fermentas, Vilnius, Lithuania). Electrophoresis was performed at 14 °C for 22 h with pulse time 1–30 s at 6 V/cm2 in 0.5 × TBE buffer. Lambda PFG Ladder (New England Biolabs, Ipswich, MA) was used as a DNA size standard. The Dice similarity coefficient (1% optimization, 1% tolerance, 1% tolerance change) and the unweighted pair-group mean arithmetic method (UPGMA) in the Bionumeric software (Applied Maths, Kortrijk, Belgium) were used to analyse PFGE-banding patterns, with the 85% similarity cut-off value defining a PFGE type (PT).
5
Clonal Analysis of A. baumannii Isolates
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The IC of A. baumannii isolates was identified by tri-locus
sequence-based typing (3LST), specifically amplifying ompA,
csuE and blaOXA-51-like [38 (link), 42 (link)]. The STs
were identified by Pasteur’s MLST scheme, which analyzed internal sequence of
cpn60, fusA, gltA,
pyrG, recA, rplB and
rpoB to differentiate clones of the isolates. New STs and alleles were
assigned by submission to the curator (www.pubmlst.org). Repetitive extragenic palindromic
element-PCR (rep-PCR) was performed in 50-μL PCR reaction with 1 μM of
REP-1 and REP-2 primers [40 (link)]. The PCR products were
run in 1% Tris-acetate EDTA agarose gel electrophoresis to illustrate DNA fingerprint
patterns. To illustrate clonal relationships, a dendrogram was constructed by UPGMA with
1% position tolerance, using the Bionumeric Software (Applied Maths, Sint-Martens-Latem,
Belgium).
sequence-based typing (3LST), specifically amplifying ompA,
csuE and blaOXA-51-like [38 (link), 42 (link)]. The STs
were identified by Pasteur’s MLST scheme, which analyzed internal sequence of
cpn60, fusA, gltA,
pyrG, recA, rplB and
rpoB to differentiate clones of the isolates. New STs and alleles were
assigned by submission to the curator (www.pubmlst.org). Repetitive extragenic palindromic
element-PCR (rep-PCR) was performed in 50-μL PCR reaction with 1 μM of
REP-1 and REP-2 primers [40 (link)]. The PCR products were
run in 1% Tris-acetate EDTA agarose gel electrophoresis to illustrate DNA fingerprint
patterns. To illustrate clonal relationships, a dendrogram was constructed by UPGMA with
1% position tolerance, using the Bionumeric Software (Applied Maths, Sint-Martens-Latem,
Belgium).
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