Germination of spores from Clostridium botulinum B-aphis and Ba410

Abstract

The germination of spores fromn Clostridium botulinum B-aphis and Ba410 was examined. In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl. In a defined medium (CTB [D. B. Rowley and F. Feeherry, J. Bacteriol. 104:1151-1157, 1970]), the parent strain B-aphis germinated at a rate of 0.77% min-' in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germninated at rates of 0.16% min-' in CTB and 0.04% min-' in CTB containing 2% NaCl. L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains. When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl. Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl. L-Alanineand L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine. Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful. Differences in the germination kinetics of both strains could not be explained by ultrastructural differences. Transmission electron micrographs revealed striking similarities between the strains. Complete inhibition of botulinal toxigenesis requires NaCl levels of 5% for type E strains (2, 31) and up to 8 and 10o for type A and proteolytic B strains (1, 26) under otherwise optimal conditions. Lower salt concentrations are, however, inhibitory when used with another inhibitor (10, 32). Complex interactions among pH, salt, and nitrite are responsible Relatively little work has been done on the influence of NaCl on clostridial spore germination (2, 4, 26). While examining the interaction of pH and salt on botulinal physiology (22, 24), we isolated and characterized a salt-tolerant variant of Clostridium botulinum B-aphis. This variant, strain Ba410, differs from the parent strain in salt sensitivity, * Corresponding author. t Publication D-10112-1-85 of the New Jersey Agricultural Experiment Station.

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

“…botulinum type A and B (strains 62A, B-aphis and Ba410) induced by L L-alanine/thioglycollate/ NaHCO 3 and by L L-cysteine/thioglycollate/NaHCO 3 . Neither D D-alanine nor D D-cysteine acted as germinants in the presence of thioglycollate/NaHCO 3 (Rowley and Feeherry 1970;Montville et al 1985). The present study has con®rmed previous ®ndings (Ando 1971(Ando , 1974) that D D-lactate is able to replace L L-lactate in germination of spores of non-proteolytic Cl.…”

“…botulinum type A and B (strains 62A, 190, B‐aphis, Ba410) was triggered by a defined three‐component mixture comprising L ‐alanine (or L ‐cysteine)/ L ‐lactate (or sodium thioglycollate or a number of alpha‐hydroxy acids)/NaHCO 3 in buffer at a neutral pH. All three components were necessary for germination (Rowley and Feeherry 1970; Ando 1974; Smoot and Pierson 1982; Montville et al . 1985).…”

Apertures in the Clostridium sporogenes spore coat and exosporium align to facilitate emergence of the vegetative cell

“…1 b,c,d). This morphological arrangement appears to be conserved in C. pasteurianum , C. botulinum , C. sporogenes and Clostridium novyi ( Mackey and Morris, 1972a; Montville et al., 1985; Panessa-Warren et al., 1994; Plomp et al., 2007 ). Moreover, the exosporium appearance observed here differs from that of C. difficile which is often described as less organised and lacking a clear gap/inner coat space observed in other species ( Paredes-Sabja et al., 2014 ).…”

“…The exosporium covers the entire spore and has a rough textured appearance. Several species of Clostridium have an exosporium including C. botulinum Groups I and II ( Lund and Peck, 2000; Montville et al., 1985; Stevenson and Vaughn, 1972; Takumi et al., 1979 ), C. sporogenes ( Panessa-Warren et al., 1994, 1997 ), C. pasteurianum ( Mackey and Morris, 1972a,b ), C. novyi ( Plomp et al., 2007 ) and C. difficile ( Panessa-Warren et al., 1997 ). The function of the exosporium is poorly understood, and is reported to provide a barrier to large molecules (e.g.…”

Use of the direct epifluorescent filter technique for the enumeration of bacterial spores

“…Like sporulation, spore germination is a complex phenomenon usually triggered by a variety of environmental stimuli and related to the age and origin of the individual spore (Gould 1970;Smoot & Pierson 1982;Montville et al 1985). If the staining of spores is similar to vegetative cells, it is possible that the orangefluorescing spores in the DEFT have germinated (either naturally or due to the heat shock) and that the green spores are ungerminated, dormant or non-viable.…”