Computational protocol: Multilocus Sequence Typing and Further Genetic Characterization of the Enigmatic Pathogen, Staphylococcus hominis

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Protocol publication

[…] In staphylococci, resistance to novobiocin can occur by point mutations in gyrB, which encodes the target of novobiocin, DNA gyrase B –. Full-length gyrB sequences were obtained by primer walking from a diverse set of six novobiocin-sensitive and six novobiocin-resistant isolates. The sequences of both DNA strands of gyrB were assembled, edited, and aligned using Lasergene software v7.2.1 (DNAStar, Madison, USA). Nonsynonymous mutations in gyrB were compared with the novobiocin susceptibilities to identify candidate resistance mutations. A fragment of gyrB containing the only identified candidate resistance mutation was subsequently amplified by PCR in all isolates using the same conditions as used for multilocus sequence typing (described below). These gyrB amplicons were subjected to single nucleotide polymorphism (SNP) typing on a Luminex 200 instrument (Millipore, Billerica, USA), according to the protocol outlined in . PCR primer information is listed in , and Luminex results for all isolates are listed in .Resistance to oxacillin in staphylococci occurs by acquisition of the Staphylococcal Chromosomal Cassette mec (SCCmec) genetic element . The SCCmec element was typed using several PCR assays that score the presence of mecA class A, class B, and class C, and ccrAB1, ccrAB2, ccrAB3, and ccrC gene complexes [41–43; additional information on the classification of these elements is available at:]. The presence of the mecA gene was scored using a separate PCR assay . Three additional PCR assays, including those of Kondo et al. , Oliveira et al. , and a modified version of the Oliveira method developed here, were used to score the presence of ccrAB4. The modified Oliveira method consisted of decreasing the annealing temperature to 52°C, increasing the number of PCR cycles to 35, and increasing the final elongation time to 10 min. To confirm the specificity of the modified Oliveira method for amplifying ccrAB4, amplicons from eight isolates were purified and sequenced on both DNA strands. Alignments of the translated ccrAB4 sequences along with a variety of reference sequences were made with MUSCLE v3.7 and curated with Gblocks v0.91b , using default settings. A maximum likelihood tree was constructed from the curated alignments using PhyML under a WAG model of amino acid substitution . [...] Nucleotide sequences for each MLST locus were aligned using MUSCLE v3.7 software . The number of polymorphic sites, nucleotide diversity per site (π), and allelic diversity (Hd) were calculated for each locus using DnaSP v5.10 software . ST diversity was measured by Simpson’s index , . Multilocus linkage disequilibrium was measured by the standardized index of association (IAs), using LIAN v3.5 software .ClonalFrame v1.2 software was used to estimate population-scaled mutation and recombination parameters. The nucleotide sequence alignments of each MLST locus were input as individual blocks, using the sequences from single representatives of each ST. ClonalFrame was run five times, and each run used a Monte Carlo Markov Chain of 500,000 iterations, discarding the first 250,000 iterations as burn-in and saving every 100th iteration thereafter. The mixing and convergence of the runs were judged to be satisfactory based on inspections with the program's built-in tools. Recombination tract length could not be reliably estimated, so it was fixed to 1000 bp to allow comparisons of other parameter estimates with those of S. aureus and S. epidermidis . The other parameter estimates represent the averages of the five runs.To further test the role of recombination in generating allelic variation, the pairwise homoplasy index (PHI) test , implemented in SplitsTree v4.0 software , was calculated for each locus and for a concatenate of loci. A genetic algorithm for recombination detection (GARD) was used to identify the number and location of recombination breakpoints within each locus . The concatenated nucleotide sequences were also analyzed for reticulate structure by the neighbor-net algorithm implemented in SplitsTree. […]

Pipeline specifications

Software tools MUSCLE, Gblocks, PhyML, DnaSP, LIAN, ClonalFrame, SplitsTree, GARD
Applications Phylogenetics, Population genetic analysis, Nucleotide sequence alignment
Organisms Staphylococcus hominis, Homo sapiens, Mycoplasma hominis
Diseases Pierre Robin Syndrome
Chemicals Novobiocin, Oxacillin