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

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.

Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes.

In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements.

A vanG-type locus in Clostridium argentinense.

OBJECTIVES: The objective was to study a new vanG-type locus in Clostridium argentinense vanGCar and to determine its impact on glycopeptide susceptibility of the host. METHODS: The whole genome of C. argentinense NCIB 10714 was sequenced using Illumina single-reads sequencing technology. The presence of vanGCar in seven C. argentinense strains was tested by PCR and its expression was tested by quantitative RT-PCR (qRT-PCR). Glycopeptide susceptibility was determined by the Etest procedure. RESULTS: The vanGCar locus contained four genes encoding a carboxypeptidase, a d-alanine:d-serine ligase, a serine transporter and a serine racemase, and was present in the seven C. argentinense studied. An AraC-type transcriptional regulator was found upstream from the genes. C. argentinense NCIB 10714 was susceptible to vancomycin and to teicoplanin. qRT-PCR experiments revealed that vanGCar was not expressed without or with induction by a subinhibitory concentration of vancomycin. CONCLUSIONS: The new vanGCar locus was cryptic in C. argentinense and intrinsic to this species. Emergence of vancomycin resistance in C. argentinense due to decryptification of the vanGCar gene cluster could occur.

An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham.

An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.

Eisenbergiella tayi gen. nov., sp. nov., isolated from human blood.

A catalase-positive, rod-shaped, non-proteolytic, non-motile, anaerobic bacterial strain, designated B086562(T), was isolated from a blood culture of an 84-year-old male patient in Israel. According to 16S rRNA gene sequence phylogeny, this strain has no known close relatives among recognized bacteria but should be placed within the family Lachnospiraceae. The most closely related recognized bacteria were from the 'Clostridium clostridioforme group': C. clostridioforme (92.4%) and Clostridium bolteae (92.3%). The isolate produced butyrate, lactate, acetate and succinate as major metabolic end products. The major fatty acids were C16:0 and C18:1 cis 9 DMA and the DNA G+C content was 46.0 mol%. On the basis of the phenotypic properties and phylogenetic distinctiveness, the blood isolate represents a novel species of a new genus in the family Lachnospiraceae, for which the name Eisenbergiella tayi gen. nov., sp. nov. is proposed. The type strain of Eisenbergiella tayi is B086562(T) ( = LMG 27400(T) = DSM 26961(T) = ATCC BAA-2558(T)).

First case of intra-abdominal infection with Clostridium disporicum.

We describe a case of post-operative intra-abdominal infection with Clostridium disporicum, a gram-positive anaerobic rod rarely isolated in clinical specimens. Its typical doubled-spored form, when present, facilitates its recognition. Phenotype-based identification methods used here were not reliable and a 16S rRNA sequence analysis was needed.