Isolation and Characterization of a Unique Phage Carrying Strain of Clostridium botulinum from Carrot Juice

Introduction: Clostridium botulinum is an important foodborne pathogen capable of forming thermally resistant endospores and producing deadly botulinum neurotoxins (BoNTs).  In 2006, this pathogen was responsible for an international outbreak of botulism attributed to the consumption of commercially prepared carrot juice, resulting in six cases including one death.  From this outbreak, the CDC isolated two genetically distinct strains of C. botulinum namely, CDC51303 and CDC51348 from incriminated carrot juice bottles.  

Purpose: The purpose of this study was to isolate additional strains of C. botulinum that may have contributed to the high quantities of BoNT detected in the adulterated product.  

Methods: Additional carrot juice bottles retrieved from the facility during the outbreak were used to plate out and obtain toxin-producing isolates which were subsequently analyzed using pulsed-field gel electrophoresis (PFGE) and Southern hybridization analysis.  

Results: None of these additional toxigenic isolates tested exhibited a restriction banding pattern similar to strain CDC51348. PFGE of XhoI and SmaI digested DNA samples showed that isolates CJ4-1 and CJ10-1 shared an identical pulsotype to strain CDC51303. Although CJ5-1 exhibited an identical PFGE pattern to CDC51303 when SmaI was used as the restriction enzyme, this strain displayed a unique pulsotype when PFGE was performed using DNA digested with XhoI. CJ4-1 and CJ5-1 were selected for further analysis using a focused DNA microarray. This analysis revealed several phage related genes present in CJ5-1, but which were absent in CJ4-1. Southern hybridization analysis of digested and non-digested DNA of CJ4-1 and CJ5-1 using probes specific for each phage gene suggested their chromosomal rather than extrachomosomal location.

Significance: The acquisition or loss of bacteriophages has been demonstrated among strains of C. botulinum in a food, clinical or environmental sample. This phenomenon presents a challenge for the timely, accurate identification of an outbreak strain during a botulism outbreak investigation.