Development of a Multiple-Locus Variable-Number Tandem-Repeat Typing Scheme for Genetic Fingerprinting of Burkholderia cenocepaciaand Application to Nationwide Epidemiological Analysis

ABSTRACTOrganisms of the Burkholderia cepaciacomplex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization. B. cenocepaciaand Burkholderia multivoransaccount for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for B. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences of B. cenocepaciastrains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100 B. cenocepaciastrains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of the B. cenocepaciaIIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool in B. cenocepaciasurveillance.