Characterization of Clostridium Baratii Type F Strains Responsible for an Outbreak of Botulism Linked to Beef Meat Consumption in France

Abstract

Introduction: A second botulism outbreak due to Clostridium baratii occurred in France in August 2015 and included three patients who had their meal in a restaurant the same day. We report the characterization of C. baratii isolates including whole genome sequencing (WGS).

Methods: Four C. baratii isolates collected in August 2015 from the outbreak 2 were analysed for toxin production and typing as well as for genetic characterization. WGS was done using using the NEBNext Ultra DNA Library Prep kit for Illumina (New England Biolabs) and sequenced on MiSeq machine (Illumina) in paired-end reads of 250 bases. The phylogenetic tree was generated based on the UPGMA method with genetic distances computed by using the Kimura two-parameter model. Evolutionary analyses were conducted in Bionumerics (V.6.6 Applied Maths).

Results: Three C. baratii isolates for patient's stools and one isolate from meat produced botulinum neurotoxin (BoNT) type F and retained a bont/F7 gene in OrfX cluster. All isolates were identical according to the WGS. However, phylogeny of the core genome showed that the four C. baratii strains were distantly related to that of the previous C. baratii outbreak in France in 2014 and from the other C. baratii strains reported in databanks.

Discussion: The fact that the strains isolated from the patients and meat samples were genetically identical supports that the meat used for the Bolognese sauce was responsible for this second botulism outbreak in France. These isolates were unrelated to that from the first C. baratii outbreak in France in 2014 indicating a distinct source of contamination. WGS provided robust determination of genetic relatedness and information regarding BoNT typing and toxin gene locus genomic localization.

Fig 1. Colonies and morphology of C. baratii 694-15. (A) Colonies on sheep blood TGY agar surrounded by hemolysis halo. Phase contrast microscopy (magnification 1000x) of culture in TGY broth in the exponential growth phase (B), stationary phase (C), and sporulation phase (D).

Fig 2. Phylogenetic relatedness of BoNT/F7 nucleotide sequences and deduced proteins. The dendograms were constructed using the UPGMA method. The scale bar indicates similarity values. The numbers next to each node indicate the bootstrap values (n=100). Strains noted * were isolated from the same botulism outbreak. Genbank accesion numbers are: IBCA03-0045, JX847735; CDC32356, GU213234; CDC35112, GU213233, CDC51192; GU213232; CDC59837, GU213231; Sullivan, CP006905; Orange, HM746655; CDC51267, GU213235; ATCC 43756, X68262.

Fig 3. Genomic environment of BoNT/F7 toxin clusterThe sequence between the AraC family transcriptional regulator and ABC transporter substrate-binding protein genes of C. baratii strain Sullivan was aligned with contigs carrying the BoNT/F7 operon of strains 796-15 and 771-14. The conserved regions (≥ 99% identity) are indicated in red.

Fig 4. Phylogenetic tree of seven C. baratii isolates. Scale bar represents 0.005 nucleotide substitutions per character. Confidence supports at branches were estimated by a bootstrap procedure (500 replicates). Rooting was performed by using 
the genome sequence of C. perfringens strain SM101 as an outgroup (not shown).