Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China.

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events.

Analysis of the core genome and pangenome of Clostridium butyricum.

Clostridium butyricum is an anaerobic bacterium that inhabits broad niches. Clostridium butyricum is known for its production of butyrate, 1,3-propanediol, and hydrogen. This study aimed to present a comparative pangenome analysis of 24 strains isolated from different niches. We sequenced and annotated the genome of C. butyricum 3-3 isolated from the Chinese baijiu ecosystem. The pangenome of C. butyricum was open. The core genome, accessory genome, and strain-specific genes comprised 1011, 4543, and 1473 genes, respectively. In the core genome, Carbohydrate metabolism was the largest category, and genes in the biosynthetic pathway of butyrate and glycerol metabolism were conserved (in the core or soft-core genome). Furthermore, the 1,3-propanediol operon existed in 20 strains. In the accessory genome, numerous mobile genetic elements belonging to the Replication, recombination, and repair (L) category were identified. In addition, genome islands were identified in all 24 strains, ranging from 2 (strain KNU-L09) to 53 (strain SU1), and phage sequences were found in 17 of the 24 strains. This study provides an important genomic framework that could pave the way for the exploration of C. butyricum and future studies on the genetic diversification of C. butyricum.

Clostridium butyricum MIYAIRI 588 Increases the Lifespan and Multiple-Stress Resistance of Caenorhabditis elegans.

Clostridium butyricum MIYAIRI 588 (CBM 588), one of the probiotic bacterial strains used for humans and domestic animals, has been reported to exert a variety of beneficial health effects. The effect of this probiotic on lifespan, however, is unknown. In the present study, we investigated the effect of CBM 588 on lifespan and multiple-stress resistance using Caenorhabditis elegans as a model animal. When adult C. elegans were fed a standard diet of Escherichia coli OP50 or CBM 588, the lifespan of the animals fed CBM 588 was significantly longer than that of animals fed OP50. In addition, the animals fed CBM588 exhibited higher locomotion at every age tested. Moreover, the worms fed CBM 588 were more resistant to certain stressors, including infections with pathogenic bacteria, UV irradiation, and the metal stressor Cu2+. CBM 588 failed to extend the lifespan of the daf-2/insulin-like receptor, daf-16/FOXO and skn-1/Nrf2 mutants. In conclusion, CBM 588 extends the lifespan of C. elegans probably through regulation of the insulin/IGF-1 signaling (IIS) pathway and the Nrf2 transcription factor, and CBM 588 improves resistance to several stressors in C. elegans.

Comparative phenotypic analysis of "Clostridium neonatale" and Clostridium butyricum isolates from neonates.

"Clostridium neonatale" was recently described as a new species within the Cluster I of the Clostridium genus sensu stricto. In this study, we characterized "C. neonatale" isolates (n = 42) and compared their phenotypic properties with those of Clostridium butyricum (n = 26), a close related species. Strains isolated from fecal samples of healthy neonates were tested for different phenotypic characteristics. Compared to C. butyricum, "C. neonatale" showed a significant higher surface hydrophobicity (p = 0.0047), exopolysaccharide production (p = 0.0069), aero-tolerance (p = 0.0222) and viability at 30 °C (p = 0.0006). A lower swimming ability (p = 0.0146) and tolerance against bile (0.3%) (p = 0.0494), acid (pH 4.5) (p < 0.0001), osmolarity (NaCl 5%, p = 0.0188) and temperature at 50 °C (p = 0.0013) characterized "C. neonatale" strains. Our results showed that "C. neonatale" behaves very differently from C. butyricum and suggests specific responses to environmental changes. Besides it is the first study on clinical isolates for these two anaerobic members of the newborns' gut microbiota and broadens our knowledge about their phenotypic traits.

Multi-spacer typing as an effective method to distinguish the clonal lineage of Clostridium butyricum strains isolated from stool samples during a series of necrotizing enterocolitis cases.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease with high morbidity and mortality that predominantly affects preterm neonates during outbreaks. In a previous study, the present authors identified 15 Clostridium butyricum isolates from stool samples during a series of NEC cases involving four neonatal intensive care units. A clonal lineage of these strains was observed by in-silico multi-locus sequence typing. AIM: To confirm the previous findings by sequencing a larger number of C. butyricum genomes and using other genotyping approaches. METHODS: The previously isolated 15 C. butyricum strains were characterized and compared with 17 other commensal and environmental C. butyricum strains using whole-genome sequencing (WGS). In addition, the clustering was analysed using multi-spacer sequence typing (MST). FINDINGS: The core genome of C. butyricum was composed of 1251 genes, and its pan-genome consisted of 12,628 genes with high variability between strains. It was possible to distinguish the clonal lineage of strains from a series of NEC cases, forming three clades with geographical clustering. The results obtained using WGS and MST approaches were congruent. CONCLUSION: MST is a fast, cheap and effective genotyping method for investigating NEC outbreaks associated with C. butyricum.

Complete genome sequence of Clostridium butyricum JKY6D1 isolated from the pit mud of a Chinese flavor liquor-making factory.

Clostridium butyricum is an important fragrance-producing bacterium in the traditional Chinese flavor liquor-making industry. Here the complete genome sequence of C. butyricum JKY6D1 isolated from the pit mud of a Chinese flavor liquor-making factory is presented. The genome is 4,618,327bp with the GC content of 28.74% and a plasmid of 8060bp. This is the first complete genome sequence of C. butyricum strains available so far.

Laboratory Investigation of the First Case of Botulism Caused by Clostridium butyricum Type E Toxin in the United States.

We report here the laboratory investigation of the first known case of botulism in the United States caused by Clostridium butyricum type E. This investigation demonstrates the importance of extensive microbiological examination of specimens, which resulted in the isolation of this organism.

Infant botulism due to C. butyricum type E toxin: a novel environmental association with pet terrapins.

We describe two cases of infant botulism due to Clostridium butyricum producing botulinum type E neurotoxin (BoNT/E) and a previously unreported environmental source. The infants presented at age 11 days with poor feeding and lethargy, hypotonia, dilated pupils and absent reflexes. Faecal samples were positive for C. butyricum BoNT/E. The infants recovered after treatment including botulism immune globulin intravenous (BIG-IV). C. butyricum BoNT/E was isolated from water from tanks housing pet 'yellow-bellied' terrapins (Trachemys scripta scripta): in case A the terrapins were in the infant's home; in case B a relative fed the terrapin prior to holding and feeding the infant when both visited another relative. C. butyricum isolates from the infants and the respective terrapin tank waters were indistinguishable by molecular typing. Review of a case of C. butyricum BoNT/E botulism in the UK found that there was a pet terrapin where the infant was living. It is concluded that the C. butyricum-producing BoNT type E in these cases of infant botulism most likely originated from pet terrapins. These findings reinforce public health advice that reptiles, including terrapins, are not suitable pets for children aged <5 years, and highlight the importance of hand washing after handling these pets.

Biotechnological potential of Clostridium butyricum bacteria.

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

Biotechnological potential of Clostridium butyricum bacteria

In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties.

Structure and genetic content of the megaplasmids of neurotoxigenic clostridium butyricum type E strains from Italy.

We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the CRISPR-cas system remains an open question.

The potential of bacteria isolated from ruminal contents of seaweed-eating North Ronaldsay sheep to hydrolyse seaweed components and produce methane by anaerobic digestion in vitro.

The production of methane biofuel from seaweeds is limited by the hydrolysis of polysaccharides. The rumen microbiota of seaweed-eating North Ronaldsay sheep was studied for polysaccharidic bacterial isolates degrading brown-seaweed polysaccharides. Only nine isolates out of 65 utilized >90% of the polysaccharide they were isolated on. The nine isolates (eight Prevotella spp. and one Clostridium butyricum) utilized whole Laminaria hyperborea extract and a range of seaweed polysaccharides, including alginate (seven out of nine isolates), laminarin and carboxymethylcellulose (eight out of nine isolates); while two out of nine isolates additionally hydrolysed fucoidan to some extent. Crude enzyme extracts from three of the isolates studied further had diverse glycosidases and polysaccharidase activities; particularly against laminarin and alginate (two isolates were shown to have alginate lyase activity) and notably fucoidan and carageenan (one isolate). In serial culture rumen microbiota hydrolysed a range of seaweed polysaccharides (fucoidan to a notably lesser degree) and homogenates of L. hyperborea, mixed Fucus spp. and Ascophyllum nodosum to produce methane and acetate. The rumen microbiota and isolates represent potential adjunct organisms or enzymes which may improve hydrolysis of seaweed components and thus improve the efficiency of seaweed anaerobic digestion for methane biofuel production.

Identification of Clostridium beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum isolated from silage, raw milk and hard cheese by a multiplex PCR assay.

Late blowing, caused by the outgrowth of clostridial spores present in raw milk and originating from silage, can create considerable product loss, especially in the production of hard and semi-hard cheeses. The conventional method for the isolation of Clostridium spp. from cheeses with late-blowing symptoms is very complicated and the identification of isolates is problematic. The aim of this work was the development of a multiplex PCR method for the detection of the main dairy-related clostridia such as: Cl. beijerinckii, Cl. butyricum, Cl. sporogenes, Cl. tyrobutyricum. Samples derived from silage, raw milk and hard cheese were analysed by the most probable number (MPN) enumeration. Forty-four bacterial strains isolated from gas positive tubes were used to check the reliability of the multiplex PCR assay. The specificity of the primers was tested by individually analysing each primer pair and the primer pair combined in the multiplex PCR. It was interesting to note that the samples not identified by the multiplex PCR assay were amplified by V2-V3 16S rRNA primer pair and the sequencing revealed the aligned 16S rRNA sequences to be Paenibacillus and Bacillus spp. This new molecular assay provides a simple promising alternative to traditional microbiological methods for a rapid, sensitive detection of clostridia in dairy products.

Screening of environmental samples for bacteria producing 1,3-propanediol from glycerol.

Twenty nine environmental samples were screened for the presence of anaerobic microorganisms fermenting glycerol with 1,3-propanediol as a final product. Seven samples were then selected for the next step of our research and eight bacteria strains were cultured anaerobically. Seven of them produced 1,3-propanediol with a yield of 0.47-0.58. Six of the the isolated microorganisms were then classified as Clostridium butyricum (four strains), C. lituseburense (one strain), and C. sartagoforme (one strain). We suggest that of all these strains C. butyricum 2CR371.5 is the best 1,3-propanediol producer as producing no lactate as a by-product and growing well on a glycerol-containing medium.

Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions.

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.