Multiple-locus variable-number tandem-repeat analysis of Brucella isolates from patients in Xinjiang China

Abstract

Objective: This study is to characterize and identify the human Brucella strains in Xinjiang, China with multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme.

Methods: Brucella strains were isolated and cultured from 62 brucellosis patients. The bacteria strains were subjected to the oxidase, catalase, rapid urease, and nitrate reduction tests, and the species identification was performed using the VITEK-2 Compact system. These Brucella strains were further identified and characterized using the 16 VNTR loci in a MLVA-16 methodology.

Results: Twelve Brucella strains had been identified out of 62 patients, which were all recognized as Brucella melitensis (B. melitensis) according to the results from the VITEK-2 Compact system. Based on panel 1 (MLVA-8), these 12 Brucella isolates were clustered into three known genotypes and two new genotypes, in which 7 strains were clustered into genotype 45 (1-5-3-12-2-2-3-2), 1 strain was classified as genotype 42 (1-5-3-13-2-2-3-2), 1 stain was with genotype 62 (1-3-3-13-2-2-3-2), and the other 3 trains revealed two new genotypes, i.e., (1-5-3-12-2-3-3-2) and (1-5-3-11-2-3-3-2). Using panel 2A+2B (MLVA-16), we found that no genotypes of these strains were identical to the known genotypes, generally with differences in 2-4 loci. However, three strains shared the same genotype.

Conclusion: Brucella strains in 62 brucellosis patients from Xinjiang are all identified as B. melitensis. Based on MLVA-8, two new genotypes have been discovered. These findings might contribute to the understanding of the pathogenesis and epidemiology of brucellosis in Xinjiang, China.

Keywords: Brucella; Xinjiang; brucellosis; genotyping; multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA).

Figure 1

Brucella culture and Gram staining. Representative pictures of bacterial cultures at 48 h (A) and 72 h (B), as well as Gram staining (C) for isolate A were shown.

Figure 2

PCR analysis of the VNTRs for the Brucella strains. The 12 Brucella species were designated as isolates A to L, respectively. PCR analysis of the VNTR loci in panel 1 (A) and panel 2A+2B (B) for the Brucella strains. M, marker. Panel 1 included eight markers: Bruce 06, Bruce 08, Bruce 11, Bruce 12, Bruce 42, Bruce 43, Bruce 45, and Bruce 55. Panel 2 was composed of eight microsatellite markers: Bruce 18, bruce19, and Bruce 21 in panel 2A; and Bruce 04, Bruce 07, Bruce 09, Bruce 16, and Bruce 30 in panel 2B.

Figure 3

MLVA-based dendrogram and phylogenetic relationship analysis. Relationships among different Brucella genus were shown in the dendrogram. The length of the line represents the genetic distance.