Intraspecies biodiversity of the genetically homologous species Brucella microti

Abstract

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.

Fig 1

Identification of Brucella microti using the Bruce-ladder multiplex PCR. The B. microti strains showed a consistent amplicon pattern comprising seven fragments also found in B. suis (1,682 bp, 1,071 bp, 794 bp, 587 bp, 450 bp, 272 bp, and 152 bp) and an additional, B. microti-specific 510-bp amplicon (indicated by the arrow). Lane 1, B. microti type strain CCM 4915; lane 2, strain CCM 4916; lane 3, field isolate IBM 257; lane 4, field isolate IBM 284; lanes 5 and 6, field isolate BMS 10, presented with a rough and a smooth phenotype, respectively; lane 7, field isolate F 303 Mi; lane 8, field isolate FK 210908; lane 9, field isolate FH 2208; lane 10, field isolate FW 70608; lane 11, field isolate BMS 17; lane 12, field isolate BMS 20. A 1-kb Plus DNA ladder (Invitrogen Ltd.) was used as molecular weight (MW) markers. The B. ceti NCTC 12891 and B. microti CCM 4915^T reference strains were used as positive controls for all possible amplicons in the Bruce-ladder PCR, and distilled water was used as a negative control.

Fig 2

Schematic multiple nucleotide sequence alignment of the omp2a and omp2b genes of Brucella strains. Nucleotide sequences are represented by rectangles divided into boxes of 30 nucleotides. The B. microti omp2b gene sequence was used as a reference. B. microti omp2a-specific nucleotides are shaded. The numbers in the corresponding boxes indicate the number of omp2a-specific nucleotides present in the sequence considered. Numbers in parentheses represent insertions and deletions. Numbers in boldface indicate nucleotide differences that are not due to gene conversion. *, there is a premature stop codon in omp2a of B. ovis 63/290.

Fig 3

Condensed dendrogram of clustered MLVA-16 genotypes of Brucella spp. A total of 344 Brucella isolates revealed 340 different genotypes. The bars reflect the percentages of divergence. The cluster of the B. microti isolates and closely related species, such as B. neotomae, B. inopinata, and B. suis bv. 5, are presented in more detail. Two atypical isolates are separately marked.

Fig 3

Condensed dendrogram of clustered MLVA-16 genotypes of Brucella spp. A total of 344 Brucella isolates revealed 340 different genotypes. The bars reflect the percentages of divergence. The cluster of the B. microti isolates and closely related species, such as B. neotomae, B. inopinata, and B. suis bv. 5, are presented in more detail. Two atypical isolates are separately marked.

Fig 4

Dendrogram of clustered MLVA-16 genotypes (panels 1 and 2). The 11 B. microti isolates were clustered into 11 different genotypes based on the differences in the numbers of repeat units at 16 VNTR loci. Key, DNA batch.

Fig 5

Maximum-parsimony analysis of 11 B. microti isolates and the closely related strains B. neotomae 5K33, B. inopinata BO1, and B. suis bv. 5 (513 and ELT80), based on MLVA-16 data. Species are distinguished by different colors.