Examination of Clostridium botulinum Subtype B7 Isolates

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious neuroparalytic disease that can result in death if not treated. Four naturally occurring forms of... [ view full abstract ]

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious neuroparalytic disease that can result in death if not treated. Four naturally occurring forms of botulism have been described: foodborne botulism, caused by pre-formed BoNT; infant botulism; wound botulism; and adult intestinal colonization.  Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of one year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. 

There are seven known serotypes of C. botulinum (A-G) characterized by the ability of specific antitoxins to neutralize BoNTs.  The molecular era has allowed researchers to further narrow these into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene.  One of the most recently recognized subtypes for BoNT/B is subtype BoNT/B7, reported by Kalb et al through mass spectrometry analysis of strain Bac-04-07755 isolated from an infant botulism case in New York.  Kalb et al reported a second isolate with the BoNT/B7 subtype -- NCTC 3807 from an unknown source in Germany.  To date these are the only two of this subtype referenced in the literature.

We were able to identify, through whole genome sequencing, five BoNT/B7 isolates among our collection, including a patient isolate and an epidemiologically-linked isolate from an open can of infant formula.  In addition to the five isolates identified in the CDC strain collection, the New York State Department of Health (NY DOH) provided the sequence for strain Bac-04-07755. The CDC isolates were subjected to 200bp shotgun sequencing using the Ion Torrent Personal Genome Machine and Ion Chef automated preparation robot.  The NY DOH isolate was sequenced with the Nextera XT library preparation kit and a 500 cycle MiSeq cartridge.  Average nucleotide identity (ANI) and high quality single nucleotide polymorphism (hqSNP) analysis (LYVE SET 1.1.4f) showed a high degree of homology between all 6, though four clades were identified by hqSNP.  The epidemiologically linked isolates differed from each other by only 2-6 hqSNPs while this clade was separated from the other isolates by 95-119 hqSNPs.  Isolates with no epidemiological link that were separated in time and geography were separated from the epidemiologically linked infant botulism case and infant formula isolate, indicating that hqSNPs can be a useful tool for analyzing closely related strains in an outbreak situation.

Acknowledgements

Thanks to the NYS DOH Wadsworth Center Applied Genomic Technologies Core for performing shotgun sequencing on BAC-04-07755.