Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

OBJECTIVE: To assess the consequences of infant botulism that result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." STUDY DESIGN: Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. RESULTS: Thirty bivalent cases of infant botulism occurred in the 45 years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n = 22), Bf (n = 7), and Ab (n = 1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. CONCLUSIONS: Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

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.

Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

Infant botulism in Costa Rica: first report from Central America.

INTRODUCTION: Clostridium botulinum is known to cause descending paralysis in infants throughout the world. METHODOLOGY: The subject of this study was a three-month-old Costa Rican boy who was hospitalized because of poor suction and feeding, hypotonia, and constipation. Clinical history and physical examination findings suggested infant botulism. Samples were sent to the Winnipeg Public Health Laboratory, where Clostridium botulinum toxin A was identified by PCR and culture from the stools, making this the first report of infant botulism in Central America. CONCLUSIONS: Although infant botulism is a known disease, the limitations in identifying it in Central America contributes to the misdiagnosis and under-reporting of this disease.

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype.

BACKGROUND: Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon. RESULTS: Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B). CONCLUSIONS: These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Genes that encode botulism neurotoxins A, B, E and F in Neotropical bee honey identified with the Polymerase Chain Reaction

Honey can be used for the treatment of wounds, sores and skin burns, but it might be contaminated with Clostridium botulinum spores. In order to evaluate Costa Rican raw honey samples, the detection of neurotoxin gene sequences (corresponding to the bacterium) C. botulinum A, B, E and F was done with the polymerase chain reaction. A total of 64 raw honey samples, coming from different Costa Rican sites were analyzed. Reference C. botulinum strains type A (ATCC 19397), type B (ATCC 7949), type E (ATCC 17786) and type F (ATCC 25764) were used as templates for testing the effectivity of the method. The process consisted in culturing the honey samples in prereduced triptose-peptone-glucose-yeast extract media (TGPY)for 5 days. After this, the bacteria lysate obtained was used for PCR. The amplicons, product of the reaction, were visualized using agarose gel 2%. From the 64 honey samples analyzed, none produced positive results in the PCR, since no amplicons were obtained. Even though, all the reference C. botulinum strains used as controls were visualized and showed the effectivity of the extraction method and of the PCR used. The results obtained show promising therapeutic uses for honey from Costa Rica, but further evaluations shall be done in order to be sure of the safety of the product.

La miel de abeja es un producto que podría ser utilizado en el tratamiento de heridas, abrasiones y quemaduras de piel; no obstante, podría estar contaminada con esporas de C. botulinum. Con el fin de evaluar muestras de miel de origen costarricense, se detectó las secuencias de genes productores de neurotoxina correspondientes a C. botulinum tipos A, B, E y F utilizando la técnica de PCR (reacción de polimerasa en cadena). 64 diferentes muestras de miel, provenientes de diversos sitios costarricenses, fueron analizadas. Con el fin de evaluar la efectividad del método, se utilizó cepas de referencia tipos A (ATCC 19397), B (ATCC 7949),E (ATCC 17786)y F (ATCC 25764). El procedimiento consistió en cultivar las muestras de miel en caldo triptona peptona glucosa levadura prerreducido, por cinco días. Luego de esto, se obtuvo un lisado bacteriano, el cual fue utilizado para la técnica de PCR. Los amplicones producto de la reacción fueron visualizados utilizando geles de azarosa al 2%.De las 64 muestras de miel analizadas, ninguna produjo resultados positivos en el PCR. No obstante, todas las cepas de referencia usadas como controles produjeron bandas, que demuestran la efectividad del método de extracción utilizado y de la técnica en sí. Los resultados obtenidos demuestran la ausencia de contaminación por C.botulinum de la miel de abeja de origen costarricense, no obstante,deben realizarse evaluaciones adicionales para garantizar la seguridad del producto.

Genes that encode botulism neurotoxins A, B, E and F in neotropical bee honey identified with the polymerase chain reaction.

Honey can be used for the treatment of wounds, sores and skin bums, but it might be contaminated with Clostridium botulinum spores. In order to evaluate Costa Rican raw honey samples, the detection of neurotoxin gene sequences (corresponding to the bacterium) C. botulinum A, B, E and F was done with the polymerase chain reaction. A total of 64 raw honey samples, coming from different Costa Rican sites were analyzed. Reference C. botulinum strains type A (ATCC 19397), type B (ATCC 7949), type E (ATCC 17786) and type F (ATCC 25764) were used as templates for testing the effectivity of the method. The process consisted in culturing the honey samples in prereduced triptose-peptone-glucose-yeast extract media (TGPY) for 5 days. After this, the bacteria lysate obtained was used for PCR. The amplicons, product of the reaction, were visualized using agarose gel 2%. From the 64 honey samples analyzed, none produced positive results in the PCR, since no amplicons were obtained. Even though, all the reference C. botulinum strains used as controls were visualized and showed the effectivity of the extraction method and of the PCR used. The results obtained show promising therapeutic uses for honey from Costa Rica, but further evaluations shall be done in order to be sure of the safety of the product.

Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B.

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes.

Nucleotide sequence of the gene coding for Clostridium botulinum (Clostridium argentinense) type G neurotoxin: genealogical comparison with other clostridial neurotoxins.

The neurotoxin gene from Clostridium botulinum type G was cloned as a series of overlapping DNA fragments generated using polymerase chain reaction (PCR) technology and primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 5'-end of the gene was obtained using a primer based on a conserved region of the nontoxic-nonhaemagglutinin gene lying upstream of the toxin gene. Translation of the nucleotide sequence derived from the cloned PCR fragments demonstrated that the gene encodes a protein of 1297 amino acid residues (rmm 149, 147). Comparative alignment of the determined BoNT/G sequence with those of other clostridial neurotoxins revealed highest sequence relatedness (approx. 58% amino acid identity) with BoNT/B of proteolytic and non-proteolytic C. botulinum. Tetanus toxin (TeTx) and other BoNT types revealed lower levels of relatedness with BoNT/G (approximate range 35-42% amino acid identity).

Multilocus enzyme electrophoresis of Clostridium argentinense (Clostridium botulinum toxin type G) and phenotypically similar asaccharolytic clostridia.

Twenty-three strains of Clostridium argentinense, C. subterminale, C. hastiforme, and other phenotypically similar asaccharolytic clostridia recently placed in seven DNA hybridization groups were compared by multilocus enzyme electrophoresis. The three nontoxigenic strains of C. argentinense were most closely related to the toxigenic strains of this species. All nine toxigenic strains of C. argentinense belonging to a single DNA hybridization group had identical enzyme types on the basis of nine enzymes. All other strains except for two derived from the type strain of C. subterminale were differentiable. Overall, there was excellent agreement between DNA relatedness and multilocus enzyme electrophoresis results.

Crecimiento de Clostridium botulinum en medios con ajo (Allium sativum) / Growth of Clostridium botulinum in media with garlic (Allium sativum)

El efecto del ajo sobre el crecimiento y toxinogénesis de Clostridium botilinum fue estudiado utilizando como sustrato el jugo obtenido por previsión y filtración de dientes de las variedades "blanco" y "colorado", y dientes cortados en trozos. El pH de ambos varió, según los lotes, entre 5,7 y 6,0. Los diluyentes de los medios con ajo fueron caldo peptona-glucosa-extracto de levadura, o agua destilada. C. botulinum a 110 fue sembrado en diluciones del jugo y en los medios con trozos e incubado en atmósferas aireada y anaeróbica, 15 d a 37-C. botulinum creció y produjo toxina en los diferents sustratos, en concentraciones variables (desde vestigios hasta 5.000 DL50/ml) pero menores que el control (10.000 DL50/ml). Mientras no se amplíen los resultados preliminares obtenido en este trabajo, la industria conservera deberá tener en cuenta que para su preparación como conserva acuosa en envase hermético o aireado, el ajo deberá ser considerado un producto de acidez baja apto para el crecimiento y producción de toxina de C. botulinum, no esterilizable por el calor, por lo que el contenido deberá ser acidificado hasta un pH equilibrado de 4,5 o ,menos, de acuerdo a las regulamentaciones generales vigentes para el tratamiento de alimentos conservados de acidez baja

Crecimiento de Clostridium botulinum en medios con ajo (Allium sativum) / Growth of Clostridium botulinum in media with garlic (Allium sativum)

El efecto del ajo sobre el crecimiento y toxinogénesis de Clostridium botilinum fue estudiado utilizando como sustrato el jugo obtenido por previsión y filtración de dientes de las variedades "blanco" y "colorado", y dientes cortados en trozos. El pH de ambos varió, según los lotes, entre 5,7 y 6,0. Los diluyentes de los medios con ajo fueron caldo peptona-glucosa-extracto de levadura, o agua destilada. C. botulinum a 110 fue sembrado en diluciones del jugo y en los medios con trozos e incubado en atmósferas aireada y anaeróbica, 15 d a 37-C. botulinum creció y produjo toxina en los diferents sustratos, en concentraciones variables (desde vestigios hasta 5.000 DL50/ml) pero menores que el control (10.000 DL50/ml). Mientras no se amplíen los resultados preliminares obtenido en este trabajo, la industria conservera deberá tener en cuenta que para su preparación como conserva acuosa en envase hermético o aireado, el ajo deberá ser considerado un producto de acidez baja apto para el crecimiento y producción de toxina de C. botulinum, no esterilizable por el calor, por lo que el contenido deberá ser acidificado hasta un pH equilibrado de 4,5 o ,menos, de acuerdo a las regulamentaciones generales vigentes para el tratamiento de alimentos conservados de acidez baja (AU)