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Redalyc.Phenotypic Characterization of Clostridium Botulinum Strains Revista Argentina de Microbiología ISSN: 0325-7541 [email protected] Asociación Argentina de Microbiología Argentina Sagua, M. D.; Lúquez, C.; Barzola, C. P.; Bianco, M. I.; Fernández, R. A. Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in Argentina Revista Argentina de Microbiología, vol. 41, núm. 3, julio-septiembre, 2009, pp. 141-147 Asociación Argentina de Microbiología Buenos Aires, Argentina Available in: http://www.redalyc.org/articulo.oa?id=213016782004 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Phenotypic characterization of Clostridium botulinum strainsISSN 0325-7541 141 ARTÍCULO ORIGINAL Revista Argentina de Microbiología (2009) 41: 141-147 Phenotypic characterization of Clostridium botulinum strains isolated from infant botulism cases in Argentina M. D. SAGUA*, C. LÚQUEZ1, C. P. BARZOLA, M. I. BIANCO, R. A. FERNÁNDEZ Área Microbiología, Departamento de Patología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo. Centro Universitario, Parque General San Martín S/N (5500) Mendoza, Argentina *Correspondence. E-mail: [email protected] ABSTRACT Infant botulism is the most common form of human botulism; however, its transmission has not been completely explained yet. Some of the most recognized potential sources of Clostridium botulinum spores are the soil, dust, honey and medicinal herbs. In Argentina, 456 cases of infant botulism were reported between 1982 and 2007. C. botulinum type A was identified in 455 of these cases whereas type B was identified in just one case. However, in Argentina, types A, B, E, F, G, and Af have been isolated from environmental sources. It is not clearly known if strains isolated from infant botulism cases have different characteristics from strains isolated from other sources. During this study, 46 C. botulinum strains isolated from infant botulism cases and from environmental sources were typified according to phenotypic characteristics. Biochemical tests, antimicrobial activity, and haemagglutinin-negative botulinum neurotoxin production showed uniformity among all these strains. Despite the variability observed in the botulinum neurotoxin’s binding to cellular receptors, no correlation was found between these patterns and the source of the botulinum neurotoxin. However, an apparent geographical clustering was observed, since strains isolated from Argentina had similar characteristics to those isolated from Italy and Japan, but different to those isolated from the United States. Key words: infant botulism; phenotypic characterization; Clostridium botulinum RESUMEN Caracterización fenotípica de cepas de Clostridium botulinum aisladas de casos de botulismo del lactante en Argentina. El botulismo del lactante es la forma más común del botulismo humano; sin embargo, su forma de transmisión no ha sido totalmente explicada. El suelo, el polvo ambiental, la miel y algunas hierbas medicinales son potenciales fuentes de esporas de Clostridium botulinum. Entre 1982 y 2007 se informaron en Argentina 456 casos de botulismo del lactante, 455 casos debidos al serotipo A y uno al serotipo B. Sin embargo, los serotipos A, B, E, F, G y Af han sido aislados de suelos y otras fuentes en Argentina. No se conoce si las cepas aisladas de casos de botulismo del lactante poseen características diferentes de las cepas aisladas de otras fuentes. Durante este estudio se caracterizaron 46 cepas de C. botulinum. Las pruebas bioquímicas y de sensibilidad a los antimicrobianos y la producción de neurotoxina botulínica hemaglutinina-negativa mostraron uniformidad entre estas cepas. A pesar de la variabilidad observada respecto de la unión de la neurotoxina a receptores celulares, no se observó una correlación entre estos patrones de unión y la fuente de aislamiento. Sin embargo, se observó una aparente agrupación geográ- fica, ya que las cepas aisladas en Argentina tuvieron características similares a las observadas en las cepas aisladas en Italia y Japón, pero diferentes de las que se registraron en las cepas aisladas en los Estados Unidos. Palabras clave: botulismo del lactante, caracterización fenotípica, Clostridium botulinum INTRODUCTION acetylcholine release, producing a descending, symmet- ric, flaccid paralysis. Severely ill patients are at risk of Clostridium botulinum is the principal causal agent of respiratory failure and require hospitalization for up to two infant botulism, a severe neurological disease that affects months. Meticulous supportive and respiratory care in an infants under one year of age. In addition, some unusual intensive-care setting is the indicated case manage- neurotoxigenic Clostridium baratii and Clostridium ment (1). butyricum strains have also been isolated from infant C. botulinum produces seven botulinum neurotoxins botulism cases (2, 14, 25, 26). Ingested botulinum spores designated A through G, but only BoNT types A, B, E, germinate, colonize the large intestine and produce the and F have been reported as the cause of botulism in botulinum neurotoxin (BoNT) in situ. The absorbed BoNT humans. Types C and D produce disease in animals, and is carried to peripheral cholinergic synapses and prevents type G has been isolated from soil and from cases of un- explained deaths in Switzerland (12, 36, 37). C. botuli- 1Present address: Enteric Diseases Laboratory Branch, Centers for num strains are usually described as belonging to one of Disease Control and Prevention, Atlanta, Georgia, USA four different groups (groups I, II, III, and IV) based on 142 Revista Argentina de Microbiología (2009) 41: 141-147 metabolic characteristics (16). The composition of these MATERIALS AND METHODS groups are as follows: group I for type A strains and type B and F proteolytic strains; group II for type E strains and Bacterial isolates Origin and serotypes of the 46 C. botulinum strains used type B and F non-proteolytic strains; group III for type C during this study are described in Table 1. All strains were and D strains and group IV for type G strains. Based on its obtained from the laboratory collection of Área Microbiología, phenotypic and genetic traits, it has been proposed that Facultad de Ciencias Médicas, Universidad Nacional de Cuyo group IV would be named Clostridium argentinense (39). (Mendoza, Argentina). The molecular masses of all types of BoNTs are ap- Toxin production proximately 150 kDa, but in fluid cultures and contami- Strains were cultivated in chopped-meat medium (CMM) and nated foods, these toxins exist as part of large complexes incubated at 31 °C for 18 h. An aliquot was cultured in a dialysis (15). The smallest complex (12S, 300 kDa) consists of bag immersed in toxin production medium (38) and was incubated for 6 days at 32 °C. After incubation, the contents of the dialysis the BoNT and a non toxic-non haemagglutinin compo- bags were centrifuged at 12,000 g for 10 min at 4 °C and serially nent (NTNH). A larger complex (16S, 500 kDa) is formed diluted twofold in buffered solution, and 0.5 ml of each dilution by the conjugation of the 12S toxin with components hav- was intraperitoneally inoculated into six mice. Deaths were recorded for 96 h, and 50% lethal doses (LD ) were calcula- ing haemagglutinin activity (HA). The largest complex 50 ted (32). (19S, 900 kDa) is a dimer of the 16S toxin. Types A, B, C, D, and G can include HA in their complex, but types E and Serologic typing F produce only the 12S complex. It is thought that nontoxic Serologic typing was carried out by quantitative neutralization components of the complexes could protect the neurotoxin tests on toxic cultures using toxin levels of 20, 200, and 2,000 LD (13). from the acidity and proteases in the gastrointestinal tract 50 (40). In addition, it has been suggested that the HA com- Neurotoxin purification ponents could play a critical role in the permeation of the Toxins at high levels of toxicity were purified by a precipitation BoNT into the intestinal epithelium (28). methodology described by Das Gupta & Sathyamoorthy (9). In Argentina, 456 infant botulism cases were reported Determination of haemagglutinin activity between 1982 and 2007, and the average annual inci- Production of haemagglutinin-positive neurotoxins (HA- dence of infant botulism (2.2 per 100,000 live births) (23) positive BoNT) was detected by a microtitration method using was similar to its incidence in the United States (1.9 per multiwell plates (16). In addition, we studied the HA activity in 100,000 live births) (5). Although infant botulism is the different media: peptone-yeast extract-trypticase (PYT) (1.0% proteose peptone, 1.0% yeast extract, 2.0% trypticase peptone, most common form of human botulism, its transmission 1.0% glucose, 0.075% cysteine, pH 7.2); PYT supplemented with has not been completely explained, and the most recog- green beans, CMM, brain-heart infusion broth and peptone-yeast nized potential sources of C. botulinum spores are the extract-glucose (2% peptone, 1% yeast extract, 1% glucose, soil, dust, honey and medicinal herbs. In Argentina, 99.8% 0.05% cysteine, 4% saline solution, pH 7.2). of the infant botulism cases was caused by C. botulinum Binding to cellular receptors type A, and 0.2% of them was caused by type B. How- BoNT’s binding to glycosphingolipids was analyzed by thin ever, types A, B, F, G, and Af
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