Genotyping of French Bacillus anthracis Strains Based on 31-Loci Multi Locus VNTR Analysis: Epidemiology, Marker Evaluation, and Update of the Internet Genotype Database

Abstract

Background Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described.ResultsMLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site.ConclusionsThe present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as B. anthracis.

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

“…The MLVA-10 scheme developed in the present study was shown to be an appropriate tool to detect epidemic strains of B. cenocepacia, due to the high stability of MLVA profiles, the satisfactory discriminatory power, and the portability of the method, allowing the establishment of a Web-based database (http://mlva.u-psud.fr) (14). We suggest the use of MLVA as a first-line typing method for B. cenocepacia and further analyses of the representative MLVA profiles by means of MLST.…”

“…Therefore, MLVA is a cost-effective typing method that provides portable results, with the MLVA codes consisting of an unequivocal string of numbers that can easily be exchanged between laboratories and made accessible via internet databases (12). MLVA codes can also be deduced from wholegenome sequence data (14). An MLVA scheme has been developed to type Burkholderia pseudomallei (15,16).…”

Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia

“…MLVA genotyping was applied in order to establish whether several genotypes circulated in the epidemic zone. We initially used loci vrrA, Bams03, Bams05, Bams22, Bams44, and vntr23 derived from the MLVA7 scheme proposed by Thierry et al [27]. This set of loci allowed to genotype all isolates during one day with a high degree of reliability and proved to be very useful in conducting an epidemiological investigation, when it is required to minimize the time spent on analysis, down to hours.…”

“…Later it turned out that the same MLVA profile was found in all strains isolated in the summer of 2016 on the Yamal, regardless of the isolation place (Lake Pisyoto area, Novoportovskaya tundra, Evayakha River area) and of the institution by which the samples were analyzed (S1 table) even when using 25 loci. Querying the Bacillus anthracis v4_0 MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr [27] indicates that the Yamal strains belong to the B-clade [29]. All genotypes present in the MLVA database differed at four loci or more among the 25 loci.…”

“…MLVA was performed using primers as indicated in Thierry et al [27], but using monoplex PCR. PCR products size was evaluated using agarose gel-electrophoresis.…”