LETTERS to the EDITOR

Infant botulism due to Clostridium botulinum type C toxin

Beekeeping industry is developing day by day in the world and accordingly, there is an increase in the variety of beekeeping products. One of these products is pollen which is the main source of proteins necessary for bees to complete their growth and development and also an excellent functional food ingredient for humans. In this study, it is aimed to determine the presence of Clostridium botulinum and its toxin types in the pollen samples obtained from seven different regions of Turkey. A total of 200 pollen samples collected from Black Sea Region (n:51), Mediterranean Region (n: 32), Central Anatolia (n: 38), Aegean Region (n: 14), Eastern Anatolia (n: 41), Southeastern Anatolia (n:8) and Marmara Region (n:16) were used as material. The conventional method determined by the Food and Drug Administration was used for isolation. Real Time PCR technique was used for identification and determination of toxin types. As a result, C. botulinum was detected only in one (0.5%) of the analyzed samples (supplied from the Black Sea region) and the toxin type was determined as Type A.

Botulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers.

BoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days.

Despite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action.

BoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs.

BoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

Botulism type C was suspected in a 46-year old man after consumption of sick poultry from a flock where botulism type C was confirmed. The patient developed characteristic signs of botulism, but investigation of biological samples did not confirm the presence of Clostridium botulinum or botulinum toxin. Despite having classical botulism symptoms, the man recovered very quickly. This raises the question of botulism transmission to humans by ingestion of contaminated poultry.

Two small molecular weight inhibitors, compounds CB7969312 and CB7967495, that displayed inhibition of botulinum neurotoxin serotype A in a previous study, were evaluated for inhibition of botulinum neurotoxin serotypes B, C, E, and F. The small molecular weight inhibitors were assessed by molecular modeling, UPLC-based peptide cleavage assay; and an ex vivo assay, the mouse phrenic nerve – hemidiaphragm assay (MPNHDA). While both compounds were inhibitors of botulinum neurotoxin (BoNT) serotypes B, C, and F in the MPNHDA, compound CB7969312 was effective at lower molar concentrations than compound CB7967495. However, compound CB7967495 was significantly more effective at preventing BoNTE intoxication than compound CB7969312. In the UPLC-based peptide cleavage assay, CB7969312 was also more effective against LcC. Both compounds inhibited BoNTE, but not BoNTF, LcE, or LcF in the UPLC-based peptide cleavage assay. Molecular modeling studies predicted that both compounds would be effective inhibitors of BoNTs B, C, E, and F. But CB7967495 was predicted to be a more effective inhibitor of the four serotypes (B, C, E, and F) than CB7969312. This is the first report of a small molecular weight compound that inhibits serotypes B, C, E, and F in the ex vivo assay.