Investigation of Bacteria from Spoiled Bottled Salad Dressing Leading to Gas Explosion

Background: In 2020, an incident involving spoiled salad dressing from a commercial source occurred. Upon opening the bottle, the contents exploded from gas that seemed to have fermented inside the bottle. For safety concerns, we sought to investigate the bacteria from the salad dressing in order to notify the company that made the product and relevant authorities. Methods: Anaerobic and carbon dioxide culture methods were used. To determine species of colonies, MALDI-TOF-MS and 16S rRNA whole sequencing were performed. Results: There were no colonies grown in anaerobic condition; however, we obtained three colonies from the carbon dioxide atmosphere. We determined the first colony as Alkalihalobacillus clausii (Bacillus clausii), the second as Bacillus spp. such as B. australimaris, B. safensis or B. safensis subsp. osmophilus and the third as B. paralicheniformis. Phylogenic tree analysis using the16S rRNA sequence revealed these colonies to be in a proximity of known gas-producing species. The NCBI database search revealed that a key gas production pathway gene, pyruvate formate-lyase (pfl), of which the gene product catalyzes pyruvate to formate conversion, exists in B. paralicheniformis. Formate dehydrogenase (FdhH) produces CO2 from formate that the coding gene fdhF positive bacteria can participate in gas production when formate is present in the culture. And we found fdhF from A. clausii, B. australimaris/B. safensis and B. paralicheniformis. Furthermore, under butanediol producing pathway, genes coding two enzymes involved in CO2 production, namely als and ald, existed in B. australimaris/B. safensis and B paralicheniformis, whereas A. clausii possessed als. Conclusion: Candidate species A. clausii, B. australimaris/B. safensis and B. paralicheniformis from spoiled salad dressing were thought to produce CO2 gas each from their own enzymes, or in combination, which caused the explosion upon opening. The endospore forming nature of Bacillus should alert us to be cautious when considering food producing process regulations where we need to thoroughly heat any product during manufacture in order to inactivate any bacteria as there is the possibility of this type of dangerous occurrence.