SNaPBcen : a Novel and Practical Tool for Genotyping Burkholderia cenocepacia

Abstract

c Burkholderia cenocepacia is the most prevalent and feared member of the Burkholderia cepacia complex in lung infections of cystic fibrosis (CF). Genotyping and monitoring of long-term colonization are critical at clinical units; however, the differentiation of specific lineages performed by multilocus sequence typing (MLST) is still limited to a small number of isolates due to the high cost and time-consuming procedure. The aim of this study was to optimize a protocol (the SNaPBcen assay) for extensive bacterial population studies. The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the presence of multiple clonal variants in patients chronically colonized with B. cenocepacia. Main phylogenetic subgroups IIIA, IIIB, and IIIC of B. cenocepacia could be separated by the Gl94R polymorphism included in the panel. The SNaPBcen assay proved to be a rapid and robust alternative to the standard MLST for B. cenocepacia, allowing the simultaneous analysis of multiple polymorphisms following amplification and mini-sequencing reactions.

Identification and analysis of genomic islands in Burkholderia cenocepacia AU 1054 with emphasis on pathogenicity islands

“…By using recA gene sequence analysis and multilocus sequence typing, B. cenocepacia may be subdivided into four phylogenetic clusters, IIIA to IIID [7]. However, almost all clinically relevant isolates belong to the IIIA and IIIB groups [8, 9]. Epidemiological studies showed that strains ET-12 and several other epidemic dominant in Canada and Europe are part of the IIIA subgroup [10].…”

SNaPBceBcon : a Practical Tool for Identification and Genotyping of Burkholderia cepacia and Burkholderia contaminans

“…The flexibility of the method allows the modification and addition of extra markers in case they prove to be relevant for the genotyping of specific populations (30). In this work, 7 extra markers (At66F, At248F, At359F, At443F, Gl313F, Gy218F, and Ph318F) were defined for B. cepacia and B. contaminans genotyping, in addition to 11 other markers previously defined for B. cenocepacia (13), suggesting that a set of well-characterized markers can be used to genotype Bcc species. The proposed methodology, the SNaPBceBcon assay, was reproducible, practical, and capable of distinguishing the species B. cepacia and B. contaminans and efficiently detected the MLST profiles in our collection of isolates.…”

“…Before the primer annealing, touchdown PCR with three successive cycles (total of nine cycles) at increasing temperatures was performed. The strategy used for development of the SNaPBceBcon assay was similar to that previously described for B. cenocepacia (13); the primers are presented in Table 2. The SNaPBceBcon assay was carried out in a final volume of 5 l, as previously described (29).…”

“…Multilocus sequence typing (MLST) has also been shown to be excellent for both species identification and strain differentiation (12), but its applicability is limited due to its high cost and time-consuming procedure. An alternative methodology, the SNaPBcen assay, which facilitates large-scale analysis of bacterial isolates, had been proposed for the identification and genotyping of B. cenocepacia (13).…”

“…The recA PCR RFLP analysis was also performed by amplification of the entire recA gene (28), amplicons were digested with HaeIII (Amersham Biosciences) restriction endonuclease, and the restriction fragments were separated by electrophoresis in 2% (wt/vol) agarose gels. The MLST fragments were amplified in B. cepacia and B. contaminans isolates by a multiplex reaction, as previously described (13). The PCR conditions were slightly modified accordingly: denaturation for 15 min at 95°C; 26 cycles with denaturation for 1 min at 95°C, primer annealing for 30 s at 57°C (recA, atpD, gltB, lepA, phaC, and trpB) or 60°C (gyrB), and extension for 2 min at 72°C; and final extension for 10 min at 72°C.…”