Phylogenetic analysis of Clostridium botulinum type A by multi-locus sequence typing

Abstract

The genus Clostridium comprises a heterogeneous group of organisms for which the phylogeny and evolutionary relationships are poorly understood. The elucidation of these evolutionary relationships necessitates the use of experimental methods that can distinguish Clostridium lineages that are time and cost effective, and can be accurately and reproducibly employed in different laboratories. Multi-locus sequence typing (MLST) has been successfully used as a reproducible and discriminating system in the study of eukaryotic and prokaryotic evolutionary biology, and for strain typing of various bacteria. In this study, MLST was applied to evaluate the evolutionary lineages in the serotype A group of Clostridium botulinum. C. botulinum type A has recently been shown to produce multiple subtypes, suggesting that it is not monophyletic as previously reported, but comprises distinct lineages. For MLST analysis, we initially evaluated 14 housekeeping genes (gapdh, tuf, sod, oppB, hsp60, dnaE, aroE, pta, 23S rDNA, aceK, rpoB, 16S rDNA, mdh and recA) for amplification and sequence analysis. In the first phase of the analysis, 30 C. botulinum type A strains producing botulinum neurotoxin subtypes A1-A4 were examined. Results of this pilot study suggested that seven of the genes (mdh, aceK, rpoB, aroE, hsp60, oppB and recA) could be used for elucidation of evolutionary lineages and strain typing. These seven housekeeping genes were successfully applied for the elucidation of lineages for 73 C. botulinum type A strains, which resulted in 24 distinct sequence types. This strategy should be applicable to phylogenetic studies and typing of other C. botulinum serotypes and Clostridium species.

Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

“…In particular, C. sporogenes appears to be related to group I (10). So far, several genotyping methods have been applied to C. botulinum strains for taxonomic, molecular epidemiology, and forensic purposes: pulsed-field gel electrophoresis (PFGE) (9,18), amplified fragment length polymorphism (AFLP) (10,14,17), multilocus sequence typing (MLST) (13), and whole-genome DNA microarrays (2). The clustering of populations using these genotyping methods is only partially congruent with BoNT gene lineage, suggesting that BoNT genes have evolved separately from the bacterial chromosome through horizontal genetic transfer events (2,4,8,10,11).…”

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia

“…Seven-loci multiple locus sequence typing (MLST) (35) was performed in silico, by uploading de novo-assembled contig files to the PubMLST Clostridium botulinum query page (http://pubmlst.org/cbotulinum/). The MLST allele sequences obtained from NSW1_A2 were then downloaded and concatenated to create an MLST pseudomolecule (see Table S1 in the supplemental material).…”

“…Emerging evidence suggests that the genotyping of strains can help to link clustered cases and identify potential shared sources of infections (34)(35)(36)(37)(38). Unfortunately, the resolution of standard typing methods and the frequent absence of environmental samples often make it difficult to conclusively link cases or identify environmental reservoirs.…”

“…Some BoNT/A strains also carry a silent serotype B toxin, with the presence of the inactive toxin indicated in parentheses [e.g., BoNT/A(B)] (2). BoNT is produced by neurotoxin gene complexes that are located on mobile genetic elements (MGE) which can move horizontally between strains with diverse genetic backgrounds, making the bont gene or BoNT complex sequences unreliable targets for phylogenetic comparisons (35,49,50).…”

“…Some BoNT/A strains also carry a silent serotype B toxin, with the presence of the inactive toxin indicated in parentheses [e.g., BoNT/A(B)] (2). BoNT is produced by neurotoxin gene complexes that are located on mobile genetic elements (MGE) which can move horizontally between strains with diverse genetic backgrounds, making the bont gene or BoNT complex sequences unreliable targets for phylogenetic comparisons (35,49,50).The largest epidemiological studies of infant botulism have been carried out in Japan, where 31 cases have been reported since 1986 (4), and in California in the United States, where 978 cases have been reported since 1976, with 20 to 50 cases reported each year (34). Dabritz et al applied amplified fragment length polymorphism (AFLP) analysis to genotype isolates and BoNT-specific real-time PCR to characterize BoNT subtypes.…”