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Letters in Applied Microbiology, Volume 36, Issue 1, Page 41-45, January 2003

Variability in  spore germination response  by strains of proteolytic  Clostridium botulinum  types A, B  and F

F. Alberto, V. Broussolle, D.R. Mason, F. Carlin and M.W. Peck

ABSTRACT

Aims: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum.

Methods and Results: An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus.

Conclusions: Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species.

Significance and Impact of the Study: The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.

INTRODUCTION

The strictly anaerobic bacteria Cl. botulinum is responsible for foodborne botulism, a severe form of food poisoning. Spores of Cl. botulinum are heat resistant and, if not inactivated during processing, can germinate under favourable conditions and lead to growth and production of the deadly Cl. botulinum neurotoxin(Lund and Peck 2000). Germination is therefore a key event in the growth process. Germination is triggered by an external signal, generally an organic molecule, called germinant (Moir et al. 1994). The physiological and molecular mechanism of spore germination, in this species, is imperfectly established. Previous works done on single strains of proteolytic Cl. botulinum in different test conditions have shown contradictory results (Rowley and Feeherry 1970; Ando 1974; Smoot and Pierson 1982; Foegeding and Busta 1983; Montville et al. 1985; Chaibi et al. 1996). Moreover, the work done on spores of nonproteolytic Cl. botulinum (Plowman and Peck 2002) can not be transposed to proteolytic Cl. botulinum because of great metabolic and phylogenetic differences between the two groups (Lund and Peck 2000). The aim of this work is to determine common features of variability in spore germination of a range of proteolytic Cl. botulinum strains, tested in the same experimental conditions.

MATERIALS AND METHODS

Bacteria

Five type A strains (Eyemouth, 62A, 97A, NCTC 7272 and HallA [ATCC 3502]), four type B strains (B6, 213B, NCIB 4301 and Beans) and one type F strain (Langeland) of proteolytic Clostridium botulinum were used in the experiments. All strains were obtained from the Institute of Food Research collection.

Spore production

Modified Anellis broth (Gaze and Brown 1988) was inoculated with 1 ml of an overnight culture of Cl. botulinum in PYGS broth and incubated at 30 °C. The spores were harvested after 4-10 d, cleaned and highly purified according to Plowman and Peck (2002). Suspensions contained more than 99% spores. Dilutions were made in water to give suspensions of 2  10⁹ spores ml ¹.

Preparation of germinant solutions

The tested germination solutions were amino acids (l-alanine, d-alanine, l-asparagine, l-serine, l-valine, l-histidine and glycine, all at 100 mmol l ¹), sugars (d-glucose, d-fructose, at 10 mmol l ¹), salts (KCl, KHCO₃ and NaCl, all at 50 mmol l ¹), l-lactate (50 mmol l ¹) and inosine (100 mmol l ¹), in potassium phosphate buffer (100 mmol l ¹, pH 7·0) with NaHCO₃ (50 mmol l ¹) unless otherwise specified. Tris-HCl buffer (500 mmol l ¹, pH 7·4) was used to test the effect of the AGFK mixture (l-asparagine, d-glucose, d-fructose, KCl). d-Cycloserine was used at 20 mmol ¹. All the chemicals used were from Sigma (Sigma-Aldrich Company Ltd, Dorset, UK). Filter-sterilized solutions of potential germinants were prepared using anaerobic distilled water in an anaerobic cabinet (Mark 3 Whitley anaerobic cabinet, Don Whitley Ltd, Shipley, UK) containing CO₂ : H₂ : N₂ (5 : 10 : 85) (v/v), and stored at 4 °C until use.

Germination measurement

Germination was followed by change in optical density at 600 nm (O.D.₆₀₀) of spore suspensions in solutions of germinants. The change in O.D.₆₀₀ was measured using a Bioscreen C analyser system (Labsystems, Uxbridge, UK) in an anaerobic cabinet containing CO₂ : H₂ : N₂ (5 : 10 : 85) (v/v). Each well contained 300 µl of germinant solution and 100 µl of heat-activated (80 °C for 10 min) spore suspensions (2  10⁸ spores). Three replicate wells were tested for each combination of germinants. The microplates were incubated, without shaking, for 10 h at 30 °C (or 15 °C) with the O.D.₆₀₀ of each well recorded every 2 min. The initial O.D.₆₀₀ was between 0·5 and 0·8. The percentage fall in O.D.₆₀₀ was obtained using the formula:

FIGURES

RESULTS AND DISCUSSION

Measurement of fall in O.D.₆₀₀ using a Bioscreen system provided a reliable measure of germination of spores of proteolytic Cl. botulinum during the 10-h experimental period. Microscopical observations confirmed that no spore germination occurred in phosphate buffer alone. A linear relationship (R ² = 0·92) was established between the percentage of germinated spores determined by phase-contrast microscopy and fall in O.D.₆₀₀, although falls in O.D.₆₀₀ < 10% cannot be considered significant. This automated turbidometric system has been used previously to study germination of spores of nonproteolytic Cl. botulinum (Plowman and Peck 2002) and B. subtilis (Romick and Tharrington 1997).

For all 10 strains of proteolytic Cl. botulinum tested, germination was significantly higher [Tukey's Honest Significant Difference Test (P < 0·001)] in l-alanine and in l-alanine/l-lactate than in inosine or the AGFK (asparagine, glucose, fructose, KCl) mixture (Table 1). The strains showed some variability in their response to l-alanine, with almost all spores of strain 213B germinating, but only about 20% of spores of strains HallA and NCIB 4301 germinating. Germination of spores of 97A, HallA, NCIB 4301 was tested in l-alanine with addition of d-cycloserine, inhibitor of the l-alanine racemase. No increase in the final extent of germination was found (data not shown). In contrast, with Bacillus spores(Titball and Manchee 1987), germination of proteolytic Cl. botulinum spores is not influenced by l-alanine racemase activity. Previous work indicated that spores of two type A strains were germinated by l-alanine alone, whereas four type A or B strains required additional components to be present (Rowley and Feeherry 1970; Ando 1974; Smoot and Pierson 1982; Foegeding and Busta 1983; Montville et al. 1985; Chaibi et al. 1996). Spores of nonproteolytic Cl. botulinum were not germinated by l-alanine alone (Plowman and Peck 2002). Addition of l-lactate significantly increased (P < 0·001) the extent of germination of most of the tested strains and particularly those showing a low level of germination in presence of l-alanine alone, for example strain NCIB 4301 (Table 1). Addition of l-lactate also increased the initial rate of germination for most strains (data not shown). l-lactate was not itself an effective germinant, but acted in a synergistic manner with l-alanine, as shown previously with spores of proteolytic Cl. botulinum types A and B (Ando 1974; Foegeding and Busta 1983). However, high concentrations of l-lactate may inhibit germination of proteolytic Cl. botulinum (Houtsma et al. 1994).

Germination was negligible in inosine (except for strain Langeland) and in AGFK mixture (except for strains NCTC 7272 and Langeland). Inosine and the AGFK mixture have been shown to be effective germinants for spores of B. subtilis (Corfe et al. 1994) or B. cereus (Clements and Moir 1998), but not for nonproteolytic Cl. botulinum (Plowman and Peck 2002).

When tested in the presence of l-lactate, l-valine, glycine and l-serine stimulated germination of spores of proteolytic Cl. botulinum, but were not as effective as l-alanine (Table 2), whereas germination with l-histidine was significantly lower (P < 0·001). l-alanine, glycine and l-valine are hydrophobic amino acids with a short side chain (l-serine is less hydrophobic than the others amino acids tested, but it is very close to glycine on a polarity scale), whereas l-histidine is a hydrophilic amino acid. These findings are consistent with those made in B. subtilis, where the observation that l-alanine-induced germination is inhibited by hydrophobic compounds indicates the hydrophobic nature of the l-alanine receptor (Yasuda-Yasaki et al. 1978).

Spores of three strains of proteolytic Cl. botulinum did not germinate in the presence of d-alanine, and l-alanine-induced germination was inhibited by d-alanine (Rowley and Feeherry 1970; Montville et al. 1985). Germination of spores of three strains of nonproteolytic Cl. botulinum was not triggered by d-alanine, and germination induced by l-alanine/l-lactate was not inhibited by d-alanine (Plowman and Peck 2002). In the present study, tests on proteolytic Cl. botulinum demonstrated: (i) an absence of germination in the presence of d-alanine (100 mmol l ¹) alone; and (ii) a significant (P < 0·001) inhibitory effect of d-alanine on l-alanine-induced germination, with the effect dependent on the tested strain (P < 0·001). For two strains (Beans and NCIB 4301), addition of d-alanine (100 mmol l ¹) reduced germination triggered by 100 mmol l ¹ l-alanine by 20%, and by 10 mmol l ¹ l-alanine by 50%. The other tested strains, however, were affected to a lesser extent.

The effect of cations and anions on l-alanine-induced germination was tested in potassium phosphate buffer. Whereas there was little difference between KHCO₃ and NaHCO₃, germination was significantly lower (P < 0·001) in the presence of NaCl for most of the tested strains (Table 3), indicating the importance of the anionic composition. The importance of bicarbonate/CO₂ in promoting germination of clostridial spores, but inhibiting germination of spores of B. cereus, has been documented previously (Wynne and Foster 1948; Enfors and Molin 1978; Plowman and Peck 2002).

Germination in the presence of l-alanine and l-alanine/l-lactate was tested at 15 °C (Fig. 1), close to the minimum growth temperature for proteolytic Cl. botulinum (Lund and Peck 2000). For all strains, the initial rate of germination was more rapid at 30 °C than 15 °C (data not shown), and after 10 h incubation, the O.D.₆₀₀ fall was markedly higher at 30 °C than 15 °C (P < 0·001). A strong variability of germination, in sensitivity to temperature, exists between the tested strains. For some strains (e.g. Eyemouth, B6) germination after 10 h at 15 °C was negligible, but some other strains seem unaffected (e.g. NCTC 7272, Langeland). This confirms the effect of temperature on germination kinetics, shown previously on germination lag period (Billon et al. 1997; Plowman and Peck 2002). As suggested by Johnstone (1994), temperature may influence the conformation of germinant receptors and be important for the activity of germination lytic enzymes.

In conclusion, the organization of the germination system in proteolytic Group I Cl. botulinum show specific characteristics when compared to non-proteolytic Group II strains and other spore-forming bacteria. The differences in sensitivity to germinants observed on the tested strains could be exploited for a better understanding of the germination mechanism in proteolytic Cl. botulinum.

ACKNOWLEDGEMENTS

The authors would like to thank Dr S. Stringer, Dr M.G. Grotte, and Mr F. Gauillard for helpful assistance in the data treatment. MWP and DRM are grateful to the BBSRC for funding.

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