Case report: Aberrant fecal microbiota composition of an infant diagnosed with prolonged intestinal botulism.

BACKGROUND: Intestinal botulism is primarily reported in small babies as a condition known as infant botulism. The condition results from the ingestion of environmental or foodborne spores of botulinum neurotoxin (BoNT) producing Clostridia, usually Clostridium botulinum, and subsequent spore germination into active botulinum neurotoxinogenic cultures in the gut. It is generally considered that small babies are susceptible to C. botulinum colonization because of their immature gut microbiota. Yet, it is poorly understood which host factors contribute to the clinical outcome of intestinal botulism. We previously reported a case of infant botulism where the infant recovered clinically in six weeks but continued to secrete C. botulinum cells and/or BoNT in the feces for seven months. CASE PRESENTATION: To further understand the microbial ecology behind this exceptionally long-lasting botulinum neurotoxinogenic colonization, we characterized the infant fecal microbiota using 16S rRNA gene amplicon sequencing over the course of disease and recovery. C. botulinum could be detected in the infant fecal samples at low levels through the acute phase of the disease and three months after recovery. Overall, we observed a temporal delay in the maturation of the infant fecal microbiota associated with a persistently high-level bifidobacterial population and a low level of Lachnospiraceae, Bacteroidaceae and Ruminococcaceae compared to healthy infants over time. CONCLUSION: This study brings novel insights into the infant fecal composition associated with intestinal botulism and provides a basis for a more systematic analysis of the gut microbiota of infants diagnosed with botulism. A better understanding of the gut microbial ecology associated with infant botulism may support the development of prophylactic strategies against this life-threatening disease in small babies.

A Novel Prophage-like Insertion Element within yabG Triggers Early Entry into Sporulation in Clostridium botulinum.

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence.

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures.

IMPORTANCE: Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in Clostridium botulinum type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of C. botulinum. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption.

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard.

Genomic and Phenotypic Characterization of Clostridium botulinum Isolates from an Infant Botulism Case Suggests Adaptation Signatures to the Gut.

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum.

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Comparative Genome Analysis and Spore Heat Resistance Assay Reveal a New Component to Population Structure and Genome Epidemiology Within Clostridium perfringens Enterotoxin-Carrying Isolates.

Clostridium perfringens causes a variety of human and animal enteric diseases including food poisoning, antibiotic-associated diarrhea, and necrotic enteritis. Yet, the reservoirs of enteropathogenic enterotoxin-producing strains remain unknown. We conducted a genomic comparison of 290 strains and a heat resistance phenotyping of 30 C. perfringens strains to elucidate the population structure and ecology of this pathogen. C. perfringens genomes shared a conserved genetic backbone with more than half of the genes of an average genome conserved in >95% of strains. The cpe-carrying isolates were found to share genetic context: the cpe-carrying plasmids had different distribution patterns within the genetic lineages and the estimated pan genome of cpe-carrying isolates had a larger core genome and a smaller accessory genome compared to that of 290 strains. We characterize cpe-negative strains related to chromosomal cpe-carrying strains elucidating the origin of these strains and disclose two distinct groups of chromosomal cpe-carrying strains with different virulence characteristics, spore heat resistance properties, and, presumably, ecological niche. Finally, an antibiotic-associated diarrhea isolate carrying two copies of the enterotoxin cpe gene and the associated genetic lineage with the potential for the emergence of similar strains are outlined. With C. perfringens as an example, implications of input genome quality for pan genome analysis are discussed. Our study furthers the understanding of genome epidemiology and population structure of enteropathogenic C. perfringens and brings new insight into this important pathogen and its reservoirs.

Mobile Elements Harboring Heavy Metal and Bacitracin Resistance Genes Are Common among Listeria monocytogenes Strains Persisting on Dairy Farms.

Listeria monocytogenes is a foodborne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes, and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements (MGEs) of Listeria strains inhabiting dairy farms. We performed a single-nucleotide polymorphism (SNP)-based phylogenomic analysis to identify 14 monophyletic clades of L. monocytogenes persistent on the farms for ≥6 months. We found that prophages and other mobile genetic elements were, on average, more numerous among isolates in persistent than nonpersistent clades, and we demonstrated that resistance genes against bacitracin, arsenic, and cadmium were significantly more prevalent among isolates in persistent than nonpersistent clades. We identified a diversity of mobile elements among the 250 farm isolates, including three novel plasmids, three novel transposons, and a novel prophage harboring cadmium resistance genes. Several of the mobile elements we identified in Listeria were identical to the mobile elements of enterococci, which is indicative of recent transfer between these genera. Through a genome-wide association study, we discovered that three putative defense systems against invading prophages and plasmids were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements that may spread to the food industry. IMPORTANCE Animal-derived raw materials are an important source of L. monocytogenes in the food industry. Knowledge of the factors contributing to the pathogen's transmission and persistence on farms is essential for designing effective strategies against the spread of the pathogen from farm to fork. An increasing body of evidence suggests that mobile genetic elements support the adaptation and persistence of L. monocytogenes in the food industry, as these elements contribute to the dissemination of genes encoding favorable phenotypes, such as resilience against biocides. Understanding of the role of farms as a potential reservoir of these elements is needed for managing the transmission of mobile elements across the food chain. Because L. monocytogenes coinhabits the farm ecosystem with a diversity of other bacterial species, it is important to assess the degree to which genetic elements are exchanged between Listeria and other species, as such exchanges may contribute to the rise of novel resistance phenotypes.

Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing.

Clostridium botulinum group III is the anaerobic Gram-positive bacterium producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol for obtaining high-molecular-weight DNA from C. botulinum group III is described.

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research.

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt "bookmark" sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and "bookmark"-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique "watermark" through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.

Phage lysin that specifically eliminates Clostridium botulinum Group I cells.

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment.

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Genomic Epidemiology and Phenotyping Reveal on-Farm Persistence and Cold Adaptation of Raw Milk Outbreak-Associated Yersinia pseudotuberculosis.

Packaged raw milk contaminated with Yersinia pseudotuberculosis mediated a large yersiniosis outbreak in southern Finland in 2014. The outbreak was traced back to a single dairy farm in southern Finland. Here we explore risk factors leading to the outbreak through epidemiologic investigation of the outbreak farm and through genomic and phenotypic characterization of the farm's outbreak and non-outbreak associated Y. pseudotuberculosis strains. We show that the outbreak strain persisted on the farm throughout the 7-month study, whereas the non-outbreak strains occurred sporadically. Phylogenomic analysis illustrated that the outbreak strain was related to previously published genomes of wild animal isolates from Finland, implying that wild animals were a potential source of the outbreak strain to the farm. We observed allelic differences between the farm's outbreak and non-outbreak strains in several genes associated with virulence, stress response and biofilm formation, and found that the outbreak strain formed biofilm in vitro and maintained better growth fitness during cold stress than the non-outbreak strains. Finally, we demonstrate the rapid growth of the outbreak strain in packaged raw milk during refrigerated storage. This study provides insight of the risk factors leading to the Y. pseudotuberculosis outbreak, highlights the importance of pest control to avoid the spread of pathogens from wild to domestic animals, and demonstrates that the cold chain is insufficient as the sole risk management strategy to control Y. pseudotuberculosis risk associated with raw drinking milk.

Longitudinal Study of Shiga Toxin-Producing Escherichia coli and Campylobacter jejuni on Finnish Dairy Farms and in Raw Milk.

Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni are notable health hazards associated with the consumption of raw milk. These bacteria may colonize the intestines of asymptomatic cattle and enter bulk tank milk via fecal contamination during milking. We studied the frequency of STEC O157:H7 and C. jejuni contamination in tank milk (n = 785) and the in-line milk filters of milking machines (n = 631) versus the frequency of isolation from cattle feces (n = 257) on three Finnish dairy farms for 1 year. Despite simultaneous isolation of STEC O157:H7 (17%) or C. jejuni (53%) from cattle, these bacteria were rarely isolated from milk filters (2% or <1%, respectively) and milk (0%). As revealed by phylogenomics, one STEC O157:H7 strain at a time was detected on each farm and persisted for ≤12 months despite rigorous hygienic measures. C. jejuni strains of a generalist sequence type (ST-883 and ST-1080) persisted in the herds for ≥11 months, and several other C. jejuni types were detected sporadically. The stx gene carried by STEC was detected more frequently from milk filters (37%) than from milk (7%), suggesting that milk filters are more suitable sampling targets for monitoring than milk. A questionnaire of on-farm practices suggested lower stx contamination of milk when major cleansing in the barn, culling, or pasturing of dairy cows was applied, while a higher average outdoor temperature was associated with higher stx contamination. Because pathogen contamination occurred despite good hygiene and because pathogen detection from milk and milk filters proved challenging, we recommend heat treatment for raw milk before consumption.IMPORTANCE The increased popularity of raw milk consumption has created demand for relaxing legislation, despite the risk of contamination by pathogenic bacteria, notably STEC and C. jejuni However, the epidemiology of these milk-borne pathogens on the herd level is still poorly understood, and data are lacking on the frequency of milk contamination on farms with cattle shedding these bacteria in their feces. This study suggests (i) that STEC contamination in milk can be reduced, but not prevented, by on-farm hygienic measures while fecal shedding is observable, (ii) that milk filters are more suitable sampling targets for monitoring than milk although pathogen detection from both sample matrices may be challenging, and (iii) that STEC and C. jejuni genotypes may persist in cattle herds for several months. The results can be utilized in developing and targeting pathogen monitoring and risk management on the farm level and contributed to the revision of Finnish legislation in 2017.

Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species.

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria.