Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

Abstract

Background: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing 18,000 generations.

Identification of Melioidosis Outbreak by Multilocus Variable Number Tandem Repeat Analysis

“…U'Ren et al initially described 32 VNTR loci for B. pseudomallei that had 7-28 alleles (12). Thirty of these VNTR markers were subsequently used for fi ne-scale analysis of 121 isolates of B. pseudomallei (13).…”

“…Various combinations of markers were tested by MLVA; we chose 4 markers that were highly discriminatory, enabling singlerun, 4-color analysis in a DNA sequencer. The 4 VNTR loci chosen were 2341, 389, 1788, and 933 (12). Table 1 shows the PCR primers used and the repeat region amplifi ed for each locus.…”

Molecular tracking investigation of melioidosis cases reveals regional endemicity in Hainan, China

“…Between the four clinical isolates and two colonies that were obtained from the two soil samples, a total of four and two MLVA genotypes were identified in P1 to P4 and S1 to S2, respectively. Analysis by MLST indicated that one soil (S1) and clinical colony (P1) were ST-46, as well as type (8,3,11,9) by MLVA-4, indicating that soil may have been the source of infection for this melioidosis case.…”

“…Molecular typing methods for B. pseudomallei have been successful at assigning isolates into epidemiologically-associated groups. Multilocus sequence typing (MLST) and multilocus variable number tandem repeat analysis (MLVA) provide the ability to generate robust evolutionary hypotheses that enable tracking of B. pseudomallei in epidemiological outbreaks at fine phylogenetic scale (8,9). In the present study, a molecular investigation of four indigenous cases of melioidosis, which occurred in Hainan in 2015, is described.…”

“…Clinical and environmental isolates of B. pseudomallei were evaluated by multilocus sequence typing (MLST) and 4-locus multilocus variable number tandem repeat analysis (MLVA-4) for evidence of homology between them. Analysis by MLST indicated that one environmental sample and one clinical colony were sequence type-46, as well as type (8,3,11,9) by MLVA-4. The evidence indicates a likely geographical and epidemiological association.…”