Sporulation and Heat Resistance of Spores from a Clostridium sp. RKD

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Abstract
A Clostridium sp. RKD isolated from the intestine of decaying fish, showing 99% sequence identity with Clostridium tetani at a 16S rRNA level, produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E). It also showed an amplification of near‐expected size when polymerase chain reaction (PCR) was performed using group‐ and type‐specific primers for botulinum neurotoxin type B. The isolate exhibited differences with both C. tetani and Clostridium botulinum with respect to phenotypic characteristics and chemotaxo‐nomic markers. Spore production was optimized with respect to media composition and stage of growth. Time‐dependant examination of sporulation revealed 2.6% to 49.0% spores in the late stationary phase culture when grown in different broth media. A simpler method for spore production and isolation from culture grown in tryptose sulfite cycloserine (TSC)/anaerobic agar sandwich resulted in >95% sporulation, which could be purified to near homogeneity by a simple 2‐step procedure. Thermal resistance of spores revealed a biphasic inactivation at lower temperatures with D values for linear inactivation varying from 26.6, 8.0, and 4.3 min at 70 °C, 80 °C, and 90 °C, respectively. The z values of 7.86 °C and 10.47 °C were obtained in the linear and tail regions, respectively. The Weibull parameter b values at 70 °C, 80 °C, and 90 °C were 27.38, 3.55, and 0.99, respectively, with a z’ value of 13.869 °C. The shape parameter n at 70 °C, 80 °C, and 90 °C were 0.63, 0.55, 0.45, respectively. Spores produced on 2 different media (cooked meat medium [CMM] and trypticase peptone yeast‐extract glucose [TPYG] agar) exhibited differences in heat resistance. The addition of lysozyme (50 jj.g/mL) before heat treatment resulted in increased thermal resistance of spores.

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