Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections

Abstract

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.

FIG. 1

Genetic relationships among 222 strains of B. cereus and B. thuringiensis from this study and a previous study of soil isolates (18). The dendrogram was generated by the average-linkage method of clustering from a matrix of coefficients of genetic distance based on 13 enzyme loci. Isolates are placed on the same branch when the genetic distance is less than 0.1. Numbers in the dendrogram indicate ETs in this study as listed in Table 1. Symbols indicate strains isolated from patients (▴), moss (soil sample) (●), other soil samples (○), and dairies (■). Arrows indicate reference strains: a, B. cereus ATCC 10987; b, B. cereus ATCC 4342; c, B. cereus type strain ATCC 14579.

FIG. 2

PFGE of DNAs from three B. cereus and B. thuringiensis strains of ETs 24 and 25 digested with AscI (lanes 2 to 4) and NotI (lanes 7 to 9). Lanes: 1 and 6, lambda concatemers; 2 and 7, AH 817; 3 and 8, AH 831; 4 and 7, B. cereus ATCC 4342; 5 and 8, S. cerevisiae chromosomes. The electrophoresis was run as described for Fig. 3.

FIG. 3

PFGE of undigested DNAs from B. cereus and B. thuringiensis. (A) The electrophoresis was run on a Beckman apparatus with 4-s pulses for 10 min at 170 mA and 30-s pulses for 20 h at 150 mA. (B) Hybridization of extrachromosomal DNA of 300 kb from AH 818. Lanes 1 to 7 and 10 to 13 are of ETs 24 and 25. Lanes: 1, AH 825; 2, AH 827; 3, AH 828; 4, AH 831; 5, AH 829; 6 AH 826; 7, AH 818; 8, S. cerevisiae chromosomes; 9, lambda concatemers; 10, AH 817; 11, AH 819; 12, AH 823; 13, AH 818; 14, AH 812 (ETs 22 and 23); 15, AH 810 (ET 21); 16, AH 814 (ET 41); 17, S. cerevisiae chromosomes; 18, lambda concatemers; 19, AH 725 (ET 41); 20, AH 814 (ET 41); 21, AH 718 (ET 41); 22, AH 811 (ET 40); 23, AH 815 (ET 39); 24, AH 601 (ET 44); 25, AH 818; 26, S. cerevisiae chromosomes; 27, lambda concatemers; 28, AH 407 (ET 6); 29, AH 406 (ET 7); 30, AH 610 (ET 1); 31, AH 612 (ET 5); 32, AH 608 (ET 31); 33, AH 818; 34, AH 722 (ET 34). Isolate AH 818 is used as a control in lanes 13, 25, and 33. Symbols indicate strains isolated from patients (▴) or dairies (■).