BackgroundClostridium 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.ResultsGenetic 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).ConclusionsThese 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.

Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases of infant botulism registered in Mendoza, Argentina, from 1993 to 2007. We analyzed 92 medical records of laboratory-confirmed cases and evaluated the safety and efficacy of treatment with EqBA. Forty-nine laboratory-confirmed cases of infant botulism demanding admission in intensive care units and mechanical ventilation included 31 treated with EqBA within the 5 days after the onset of signs and 18 untreated with EqBA. EqBA-treated patients had a reduction in the mean length of hospital stay of 23.9 days (P ‫؍‬ 0.0007). For infants treated with EqBA, the intensive care unit stay was shortened by 11.2 days (P ‫؍‬ 0.0036), mechanical ventilation was reduced by 11.1 days (P ‫؍‬ 0.0155), and tube feeding was reduced by 24.4 days (P ‫؍‬ 0.0001). The incidence of sepsis in EqBA-treated patients was 47.3% lower (P ‫؍‬ 0.0017) than in the untreated ones. Neither sequelae nor adverse effects attributable to EqBA were noticed, except for one infant who developed a transient erythematous rash. These results suggest that prompt treatment of infant botulism with EqBA is safe and effective and that EqBA could be considered an alternative specific treatment for infant botulism when BabyBIG is not available.

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We studied the presence of botulinum toxin-producing clostridia in 2,009 soil samples from five geographical regions of Argentina. The prevalence was 23.5%, and the distribution was not homogeneous among the regions. We observed a great multiplicity of serological types and a higher prevalence in nonvirgin soils than in virgin soils.The geographical distribution of botulinum toxin-producing clostridia (BTPC) (7) has been extensively studied in Europe, Asia, and North America (1,2,4,5,8,9,10,11,12,13,15,16,18,19,20,21,23,24,25,26). However, our knowledge concerning the presence of these anaerobes in South America is very restricted. In this study our principal aim was to examine the prevalence and distribution of BTPC in soils of Argentina.We examined 2,009 soil samples from Argentina. Because of the vast the territory and the climate and geographical variations, this country was divided into five regions ( Fig. 1 and Table 1). Soil samples were obtained and processed from 1964 until 2002. In each case the time between collection and examination was less than 2 days. Each sample was collected from a 100-cm 2 area by using a sterile metal spoon and then was transferred to a sterile receptacle and stored at room temperature until examination. Soils were classified into two categories: virgin and nonvirgin. Virgin soils were defined as soils that still were in their natural state and had not been used or changed by people. Nonvirgin soils were defined as soils that had been changed by people (cultivated, urbanized, and industrialized soils). Samples were processed by diluting 25 g of soil in 50 ml of a saline solution (0.15 M NaCl). Two aliquots were taken after 40 min of resting. One aliquot was immediately inoculated into chopped-meat medium (6). The other aliquot was subjected to a heat shock (80°C, 10 min) and later was inoculated into chopped-meat medium. After incubation for 5 days at 31°C, broth media were centrifuged at 12,000 ϫ g for 10 min at 4°C, and 0.5 ml was inoculated in duplicate intraperitoneally into mice. Mice were observed for 96 h for characteristic botulinal signs and death (14). Cultures without signs of proteolysis were treated by mixing equal volumes of the culture supernatant and 1% trypsin (1:250; Difco), followed by incubation at 37°C for 1 h. Toxic cultures were cultivated in solid media with 1.5% and 4.0% agar (0.4% meat extract, 1.0% glucose, 4.0% Proteose Peptone, 0.5% NaCl, 1.5% or 4.0% agar [pH 7.2]) and egg yolk agar (3). They were incubated for up to 24, 48, and 72 h, respectively, at 34°C in BBL jars with an atmosphere containing 80% N 2 , 10% CO 2 , and 10% H 2 . Selected colonies were transferred to chopped-meat medium and incubated for 4 days at 31°C. The presence of botulinum toxin was investigated in each of these cultures, as described previously. Toxic broth media were cultivated in solid media to ensure that the cultures were pure. Genera were identified by using gram-positive, strictly anaerobic, and sporulated bacilli. Species were not identified. Strains were cultivate...

Infant botulism is the most common form of human botulism in some countries including Argentina. However, its transmission has not been completely elucidated. In Argentina, 366 laboratory-confirmed infant botulism cases were reported between March 1982 and December 2005. The average annual incidence was 2.2 per 100,000 live births. All 108 cases diagnosed in our laboratory had botulinum toxin producing organisms in their feces, 96.3% also had botulinum toxin in feces, and 69% had detectable botulinum toxin in their serum. Biochemical tests showed uniformity among the botulinum toxin-producing clostridia isolated from infant botulism cases and soil samples. A positive relationship between presence of botulinum spores in soil and illness incidence was observed in the Northeast and West regions but not in the Central, South and Northwest regions. In the Northwest and Central regions, there was a relatively high occurrence of botulinum spores in the soil but low incidence of the disease. Type A botulinum toxin was detected in all infant botulism cases and also it was the toxin type most prevalent in the soil. Despite the presence of B and F types in the soil, there were no reports of infant botulism cases by these types in Argentina between 1982 and 2005.