Computational protocol: Outbreak with clonally related isolates of Corynebacterium ulcerans in a group of water rats

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Protocol publication

[…] MLST was performed according to Bolt et al. []. Briefly, seven housekeeping genes atpA, dnaE, dnaK, fusA, leuA, odha, and rpoB were amplified and both strands of the PCR products were sequenced by Eurofins MWG (Ebersberg, Germany) using the amplification primers. Primer pairs for leuA (leuAulcf CGTTCACTTCTACAATTC and leuAulcr GCCGTGGTCAGTTTTCAT) and dnaK (dnaKulcf ACTTGGGTGGCGGAACCT and dnaKulcr TGGTAAAGGTCTCAGAA) were modified to improve amplification of C. ulcerans leuA and dnaK genes. Sequence analysis was done with Lasergene (DNASTAR, Madison, USA). Partial MLST was further performed for seven C. ulcerans strains from game animals [] and from humans [], which were also included in FT-IR analysis. Additional housekeeping gene sequences were obtained from available whole genome sequences of C. ulcerans human isolates 809 [] [Accession-No. CP0022790] and 0102 [] [AP012284] and of canine isolate BR-AD22 [] [CP002791]. For calculating a phylogenetic tree of the concatenated sequences of atpA, dnaK, fusA, rpoB, and odhA genes we used RAxML (Randomized Axelerated Maximum Likelihood) 8 []. This is a program for sequential and parallel Maximum Likelihood based inference of large phylogenetic trees. In detail, we used the General Time Reversible model of nucleotide substitution under the Gamma model or rate heterogeneity. A total number of 100 bootstrap replicates with random seed were calculated and visualization of the tree was performed with Dendroscope 3 []. […]

Pipeline specifications

Software tools RAxML, Dendroscope
Applications Phylogenetics, WGS analysis
Organisms Corynebacterium ulcerans, Homo sapiens, Rattus norvegicus, Bos taurus, Canis lupus familiaris, Felis catus
Diseases Diphtheria, Infection, Lymphadenitis, Skin Ulcer