Reference MLSA typing Fragments from five housekeeping
genes argH (argininosuccinate lyase), cya (adenylate cyclase), murC (UDP N-acetylmuramate-L-Ala ligase, pta (phosphate acetyltransferase) and purH (phoshoribosylminoimiazolcarboxylase ATPase subunit) were amplified using the sets of primers as previously described (21). The sequences of each one of these five housekeeping genes retrieved from 48 M. Mocetinostat abscessus sequenced genomes, were also included in the MLSA analysis (Additional file 1). MST analysis Sequences of the whole intergenic spacers were extracted from the reference M. abscessus CIP104536T (ATCC19977) genome (GenBank accession CU458896.1) using the perl script selleck products software and a total of 8 spacers with a 200-700-bp sequence size were further used in analysis. For each of these 8 spacers, specific PCR primers were designed using Primer3 software v 0.4.0 ( http://frodo.wi.mit.edu/primer3) and tested in silico for specificity using BLAST software ( http://www.ncbi.nlm.nih.gov).
The PCR conditions were first optimized using DNA extracted from the reference M. abscessus, “M. bolletii” and “M. massiliense” isolates before analysis of DNA extracted from the 17 clinical isolates (Table 1). The PCR amplifications were performed in a 50 μl PCR mixture containing 5 μl 10x buffer (Qiagen, Courtaboeuf, France), 200 mM each dNTP, 1.5 mM MgCl2, 1.25 U HotStarTaq polymerase (Qiagen), 1 μl each primer (10 pM), 33 μl nuclease-free water and 5 μl DNA template. The amplification program consisted of an initial 15 min denaturation step at 95°C followed by 40 cycles of 30 s at 95°C, 30 s at 60°C and 1 min at 72°C; the amplification was completed by a final Selleck NVP-HSP990 5-min elongation step at 72°C. Negative controls consisting of PCR mixture without DNA template were included in each PCR run. The products were visualized by gel electrophoresis, purified using a MultiScreen PCR filter plate (Millipore, Molsheim, learn more France) and sequenced in both directions using the
BigDye Terminator sequencing kit (Applied Biosystems, Villebon-sur-Yvette, France), as previously described [27]. The sequences were edited using the ChromasPro software (version 1.42; Technelysium Pty Ltd), aligned using Clustal W (MEGA 5 software) and compared with the reference M. abscessus ATCC 19977 sequences (GenBank accession CU458896.1). For MST and MLSA discrimination power was calculated using the Hunter-Gaston Index [31]: where D is the numerical index of discrimination, N is the total number of isolates in the sample population, s is the total number of different types and nj is the number of isolates belonging to the jth type. Phylogenetic analysis Phylogenetic trees were constructed based on rpoB gene, concatenated MLSA genes, concatenated spacers and MST spacer n°2 sequences using the neighbor–joining method with Kimura’s two-parameter (K2P) distance correction model with 1000 bootstrap replications in the MEGA version 5 software package [32].