Computational protocol: Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry

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Protocol publication

[…] Molecular typing was performed by modified GTS analysis []. The pvpA, gapA, and lp (MGA_0319) partial gene sequences were amplified using primers pvpA 4F/3R, gapA 3F/4R, and lp 1F/1R described previously []. However, the mgc2 gene was amplified using primers mgc2 2F/2R, previously described by Garcia et al., [], resulting in an amplicon of about 300 bp. Amplified gene fragments of the respective genes were sequenced as described above.The sequences obtained from each corresponding forward and reverse primer were assembled using the SeqMan program (Lasergene, DNASTAR) and the extremities showing single strand sequences, as well as aberrant sequences, were trimmed. All sequences obtained for each gene were aligned using Clustal V (Megalign, Lasergene, DNASTAR, Madison, Wisconsin, USA) and trimmed to the same size for diversity analysis. Phylogenetic trees for individual genes were constructed from the Clustal V alignments by the neighbor-joining method and 1000 bootstrap replicate analysis using the MEGA 5 software [,]. In contrast with the previously published GTS analysis, in which mgc2 and pvpA gene fragments were 584 bp and 455 bp-long, respectively [], mgc2 and pvpA sequences were 300 bp and 700 bp-long, respectively (sizes corresponding to the M. gallisepticum Rlow genome []). The different sequences obtained for each gene fragment were assigned different allele numbers, designated by Arabic numerals (1, 2, 3, etc; data not shown). The GTS types, based on the allelic profiles of the four genes, were designated by Roman numerals (I, II, III, etc) (Table ).The four gene sequences corresponding to each of the GTS types identified amongst the 51 strains analysed, were concatenated head-to-tail for diversity analysis using Darwin 5.0 [] available at []. A distance tree was constructed using the neighbor-joining algorithm with the "simple matching" option (no correction applied to dissimilarities). The "pairwise gap block correction" option was selected with a minimal length for gap blocks of one nucleotide. This implied that all consecutive gaps, starting from one nucleotide, were considered as a single event. A bootstrap analysis with 1000 replicates was performed to test the stability on randomly chosen sets of positions.Sequences of 50 M. gallisepticum isolates and 1 Israeli reference strain were submitted to GenBank under the following accession numbers: gapA, JN102573-102623; MGA_0319, JN102624-102674; pvpA, JN113291-113341; mgc2, JN 13342-113392. […]

Pipeline specifications

Software tools MEGA, DARwin
Application Phylogenetics
Organisms Mycoplasma gallisepticum
Chemicals Tylosin, Macrolides, Fluoroquinolones