Primer pairs were designed for PCR amplification and sequencing of internal portions of eight housekeeping genes (Table 4 ). Selected genes included dinB, icdA, pabB, polB, putP, trpA and trpB, previously used for phylogenetic analysis of E. coli/Shigella strains [53 (link)-55 (link)]. New PCR primers were designed in internal portions of the genes based on previously obtained sequences, in order to amplify target regions of approximately 500 – 600 bp (Table 4 ). These seven genes represent six distinct loci on the E. coli chromosome, as trpA and trpB are located in the same operon. To increase the number of loci to seven, we added gene uidA, which is used for E. coli MLST by the group of Tom Whittam [20 (link)]. Universal sequencing primer sequences were added to the 5' end of the PCR primers. All PCR products were thus sequenced using the same two sequencing primers (Table 4 ). Further details on this MLST scheme can be found at .
Nucleotide sequences were obtained using Big Dye version 3.1 chemistry on ABI 3100 or 3730 apparatuses. In order to eliminate the risk of sample mix-up, PCR and sequencing were performed using a molecular biology robot (RoboAmp 4200-PE; MWG Biotech, Courtaboeuf, France). Sequence chromatograms were edited and stored using BioNumerics version 4.5 (Applied-Maths, St. Maartens-Latem, Belgium). All nucleotides within the consensus sequence template were supported by at least two sequence chromatograms. A different allele number was given to each distinct sequence within a locus, and a distinct sequence type (ST) number was attributed to each distinct combination of alleles. Null alleles corresponding to negative PCR amplification were considered as alignment gaps in phylogenetic analyses and as allele '999' in profile-based analyses. Isolates were grouped into clonal complexes (CCs) by eBURST, if they differed at no more than 1 locus from at least one other member of the group [56 (link)]. Founder genotypes of CCs were defined as the ST of the CC with the highest number of neighboring STs (single locus variants). Nucleotide diversity was calculated using DNAsp version 4 [57 (link)]. Minimum spanning tree analysis was performed using BioNumerics version 5.10. MEGA [58 (link)] was used to draw the consensus phylogenetic tree obtained using ClonalFrame [59 (link)] after 100,000 iterations, including 50,000 burn-in.
Nucleotide sequences were obtained using Big Dye version 3.1 chemistry on ABI 3100 or 3730 apparatuses. In order to eliminate the risk of sample mix-up, PCR and sequencing were performed using a molecular biology robot (RoboAmp 4200-PE; MWG Biotech, Courtaboeuf, France). Sequence chromatograms were edited and stored using BioNumerics version 4.5 (Applied-Maths, St. Maartens-Latem, Belgium). All nucleotides within the consensus sequence template were supported by at least two sequence chromatograms. A different allele number was given to each distinct sequence within a locus, and a distinct sequence type (ST) number was attributed to each distinct combination of alleles. Null alleles corresponding to negative PCR amplification were considered as alignment gaps in phylogenetic analyses and as allele '999' in profile-based analyses. Isolates were grouped into clonal complexes (CCs) by eBURST, if they differed at no more than 1 locus from at least one other member of the group [56 (link)]. Founder genotypes of CCs were defined as the ST of the CC with the highest number of neighboring STs (single locus variants). Nucleotide diversity was calculated using DNAsp version 4 [57 (link)]. Minimum spanning tree analysis was performed using BioNumerics version 5.10. MEGA [58 (link)] was used to draw the consensus phylogenetic tree obtained using ClonalFrame [59 (link)] after 100,000 iterations, including 50,000 burn-in.
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