Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.

BACKGROUND: Clostridium botulinum is a diverse group of bacteria characterized by the production of botulinum neurotoxin. Botulinum neurotoxins are classified into serotypes (BoNT/A-G), which are produced by six species/Groups of Clostridia, but the genetic background of the bacteria remains poorly understood. The purpose of this study was to use comparative genomics to provide insights into the genetic diversity and evolutionary history of bacteria that produce the potent botulinum neurotoxin. RESULTS: Comparative genomic analyses of over 170 Clostridia genomes, including our draft genome assemblies for 59 newly sequenced Clostridia strains from six continents and publicly available genomic data, provided in-depth insights into the diversity and distribution of BoNT-producing bacteria. These newly sequenced strains included Group I and II strains that express BoNT/A,/B,/E, or/F as well as bivalent strains. BoNT-producing Clostridia and closely related Clostridia species were delineated with a variety of methods including 16S rRNA gene, concatenated marker genes, core genome and concatenated multi-locus sequencing typing (MLST) gene phylogenies that related whole genome sequenced strains to publicly available strains and sequence types. These analyses illustrated the phylogenetic diversity in each Group and the diversity of genomic backgrounds that express the same toxin type or subtype. Comparisons of the botulinum neurotoxin genes did not identify novel toxin types or variants. CONCLUSIONS: This study represents one of the most comprehensive analyses of whole genome sequence data for Group I and II BoNT-producing strains. Read data and draft genome assemblies generated for 59 isolates will be a resource to the research community. Core genome phylogenies proved to be a powerful tool for differentiating BoNT-producing strains and can provide a framework for the study of these bacteria. Comparative genomic analyses of Clostridia species illustrate the diversity of botulinum-neurotoxin-producing strains and the plasticity of the genomic backgrounds in which bont genes are found.

Improved Cardiorespiratory Fitness Is Associated with Increased Cortical Thickness in Mild Cognitive Impairment.

Cortical atrophy is a biomarker of Alzheimer's disease (AD) that correlates with clinical symptoms. This study examined changes in cortical thickness from before to after an exercise intervention in mild cognitive impairment (MCI) and healthy elders. Thirty physically inactive older adults (14 MCI, 16 healthy controls) underwent MRI before and after participating in a 12-week moderate intensity walking intervention. Participants were between the ages of 61 and 88. Change in cardiorespiratory fitness was assessed using residualized scores of the peak rate of oxygen consumption (V̇O2peak) from pre- to post-intervention. Structural magnetic resonance images were processed using FreeSurfer v5.1.0. V̇O2peak increased an average of 8.49%, which was comparable between MCI and healthy elders. Overall, cortical thickness was stable except for a significant decrease in the right fusiform gyrus in both groups. However, improvement in cardiorespiratory fitness due to the intervention (V̇O2peak) was positively correlated with cortical thickness change in the bilateral insula, precentral gyri, precuneus, posterior cingulate, and inferior and superior frontal cortices. Moreover, MCI participants exhibited stronger positive correlations compared to healthy elders in the left insula and superior temporal gyrus. A 12-week moderate intensity walking intervention led to significantly improved fitness in both MCI and healthy elders. Improved V̇O2peak was associated with widespread increased cortical thickness, which was similar between MCI and healthy elders. Thus, regular exercise may be an especially beneficial intervention to counteract cortical atrophy in all risk groups, and may provide protection against future cognitive decline in both healthy elders and MCI.

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

A two-stage multiplex method for quantitative analysis of botulinum neurotoxins type A, B, E, and F by MALDI-TOF mass spectrometry.

In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.

Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.

Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical characterization of the Group classification within the species.

Real-time PCR assays that detect genes for botulinum neurotoxin A-G subtypes.

The role of Real-Time PCR assays for surveillance and rapid screening for pathogens is garnering more and more attention because of its versatility and ease of adoption. The goal of this study was to design, test, and evaluate Real-Time TaqMan PCR assays for the detection of botulinum neurotoxin (bont/A-G) genes from currently recognized BoNT subtypes. Assays were computationally designed and then laboratory tested for sensitivity and specificity using DNA preparations containing bont genes from 82 target toxin subtypes, including nine bivalent toxin types; 31 strains representing other clostridial species; and an extensive panel that consisted of DNA from a diverse set of prokaryotic (bacterial) and eukaryotic (fungal, protozoan, plant, and animal) species. In addition to laboratory testing, the assays were computationally evaluated using in silico analysis for their ability to detect bont gene sequences from recently identified toxin subtypes. Seventeen specific assays (two for each of the bont/C, bont/D, bont/E, and bont/G subtypes and three for each of the bont/A, bont/B, and bont/F subtypes) were designed and evaluated for their ability to detect bont genes encoding multiple subtypes from all seven serotypes. These assays could provide an additional tool for the detection of botulinum neurotoxins in clinical, environmental and food samples that can complement other existing methods used in clinical diagnostics, regulatory, public health, and research laboratories.

Navigating the Complexities Involving the Identification of Botulinum Neurotoxins (BoNTs) and the Taxonomy of BoNT-Producing Clostridia.

Botulinum neurotoxins are a varied group of protein toxins that share similar structures and modes of activity. They include at least seven serotypes and over forty subtypes that are produced by seven different clostridial species. These bacterial species are not limited strictly to BoNT-producers as neuro-toxigenic and non-neuro-toxigenic members have been identified within each species. The nomenclature surrounding these toxins and associated bacteria has been evolving as new isolations and discoveries have arisen, resulting in challenges in diagnostic reporting, epidemiology and food safety studies, and in the application of therapeutic products. An understanding of the intricacies regarding the nomenclature of BoNTs and BoNT-producing clostridia is crucial for communication that allows for accurate reporting of information that is pertinent to each situation.

The Value of Advance Care Planning for Spokespersons of Patients With Advanced Illness.

CONTEXT: Advance Care Planning (ACP) has fallen under scrutiny primarily because research has not consistently demonstrated patient-focused benefits. OBJECTIVES: To better understand how spokespersons regard, engage with, and find value in ACP during decision-making for their loved ones. METHODS: This qualitative analysis was part of a randomized controlled trial involving spokespersons of patients with advanced illness who had completed ACP. After making a medical decision on behalf of their loved one (or that loved one's death), semi-structured interviews explored spokespersons' experience of decision-making and if (and how) ACP played a role. Thematic analysis was conducted on interview transcripts. RESULTS: From 120 interviews, five themes emerged: 1) Written advance directives (ADs) helped increase spokespersons' confidence that decisions were aligned with patient wishes (serving as a physical reminder of previous discussions and increasing clarity during decision-making and family conflict); 2) Iterative discussions involving ACP facilitated "In the moment" decision-making; 3) ADs and ACP conversations helped spokespersons feel more prepared for future decisions; 4) Spokespersons sometimes felt there was "no choice" regarding their loved one's medical care; and 5) Regrets and second-guessing were the most common negative emotions experienced by spokespersons. CONCLUSION: Considering the recent debate about the utility of ACP and ADs, this analysis highlights the value of ACP for spokespersons involved in surrogate decision-making. Reframing the goals of ACP in terms of their benefit for spokespersons (and identifying appropriate outcome measures) may provide additional perspective on the utility of ACP.

De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics.

Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required.

Does Caregiver Participation in Advance Care Planning Using a Decision Support Tool Together With Patients Reduce Caregiver Strain, Burden and Anxiety Over Time? A Post-Hoc Analysis of a Randomized Controlled Trial.

CONTEXT: Surrogate decision makers experience significant amounts of anxiety, burden, and strain in their role as caregivers and decision makers for loved ones. OBJECTIVES: To investigate longitudinally whether surrogate decision makers engaging in ACP together with their loved one reduces perceived anxiety, burden, and strain felt by surrogate decision makers. METHODS: Post-hoc analysis of a randomized controlled trial evaluating caregivers' perceived self-efficacy to serve as surrogate decision makers. The trial employed a 2×2 study design of patient/caregiver dyads who engaged in advance care planning (ACP) using a standard living will form vs "Making Your Wishes Known" (MYWK), and having the patient engage in ACP alone vs together with the family caregiver. Surrogates completed validated survey instruments surveys longitudinally to compare levels of anxiety, burden, and strain. RESULTS: 246 of 285 dyads completed the measures. No significant reductions in anxiety, burden, or strain were found longitudinally in surrogate decision makers using MYWK together with loved one's vs other control groups. Increases in strain and anxiety were seen across all study groups and increases in burden across 2/4 study groups. Strain and burden increased most in the MYWK Together arm (▴ = +2.22 and ▴ = +1.91 respectively). CONCLUSION: Family caregivers who engaged in ACP together with patients using the decision support tool MYWK did not experience less strain, burden, or anxiety longitudinally compared to other study arms. These results may help inform the design of future studies and interventions that promote caregivers' involvement in ACP interventions.

What Surrogates Understand (and Don't Understand) About Patients' Wishes After Engaging Advance Care Planning: A Qualitative Analysis.

BACKGROUND: The goal of advance care planning (ACP) is to improve end-of-life decision-making for patients and their spokespersons, but multiple studies have failed to show substantial or consistent benefit from ACP. Understanding how and why ACP under-performs in the setting of complex medical decision-making is key to optimizing current, or designing new, ACP interventions. AIM: To explore how ACP did or did not contribute to a spokespersons' understanding of patient wishes after engaging in ACP. DESIGN: Thematic analysis of 200 purposively sampled interviews from a randomized control trial of an ACP decision aid. SETTING/PARTICIPANTS: 200 dyads consisting of patients 18 years or older with advanced serious illness and their spokesperson at 2 tertiary care centers in Hershey, PA and Boston, MA. Participants were interviewed 1 month after completing ACP. RESULTS: ACP helped participants: 1) express clear end-of-life wishes, 2) clarify values, and 3) recognize challenges associated with applying those wishes in complex situations. Shortcomings of ACP included 1) unknown prognostic information or quality-of-life outcomes to inform decision-making, 2) skepticism about patients' wishes, and 3) complicated emotions impacting end-of-life discussions. CONCLUSIONS: Helping patients and their spokespersons better anticipate decision-making in the face of prognostic and informational uncertainty as well as the emotional complexities of making medical decisions may improve the efficacy of ACP interventions.

Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins.

Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing.

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.

Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies.

BACKGROUND: Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. RESULTS: In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. CONCLUSIONS: In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H(c))] expressed in Pichia pastoris.

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H(c))], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H(c)) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H(c)) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H(c))/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H(c))/kg WCW at the pilot scale. The purified rBoNTC(H(c)) was stable for at least 3 months at 5 and -80°C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H(c)) was intact.

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum, Clostridium sporogenes, and Clostridium argentinense.

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements.

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

The gene CBO0515 from Clostridium botulinum strain Hall A encodes the rare enzyme N5-(carboxyethyl) ornithine synthase, EC 1.5.1.24.

Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme N(5)-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum.