Comparative genomic hybridization analysis of two predominant Nordic group I (proteolytic) Clostridium botulinum type B clusters

Abstract

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.

FIG. 1.

Sites of isolation of Finnish group I C. botulinum type B strains. Cluster BI strains have been marked with normal type, and cluster BII strains have been marked with boldface type.

FIG. 2.

Dendrogram of 32 Nordic group I C. botulinum type B strains and ATCC 3502 based on CGH analysis with ATCC 3502 microarrays. The two clusters have been marked as clusters BI and BII. The scale describes the Euclidean distance between two strains, i.e., the square root of a sum of squared gene-wise differences. Since the trinary GACK method gives values of −1, 0, and 1 for gene absent/highly divergent, unreliable labeling, and gene present, respectively, the squared difference of one gene present in one strain and absent in another strain equals [1 − (−1)]^2̂ = 4. Thus, a Euclidean distance of 20 corresponds to 100 genes missing in one strain and present in another strain, and a Euclidean distance of 40 corresponds to 400 genes missing in one strain and present in another strain.

FIG. 3.

Phylogram of the botB sequences from Nordic cluster BI strain M-18/3 and cluster BII strain KV-39/1 and 27 previously published botB sequences. Both Nordic strains clustered together with strains previously identified as belonging to subtype B2 (11).

FIG. 4.

Growth of cluster BI strains (M-1/3, P-120/6, and DA-58/1) (marked with open symbols) and cluster BII strains (KH-318/1, KS-44/10, and M-43/15) (marked with solid symbols) in the presence of 0 to 0.1 mM sodium arsenite (diamonds, 0 mM; squares, 0.05 mM; triangles, 0.1 mM) (A) or 0 to 5 mM cadmium chloride (diamonds, 0 mM; triangles, 5 mM) (B). Each curve represents the means of five (A) or four (B) replicate measurements of the three individual strains. OD₆₀₀, optical density at 600 nm.