Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Journal of Applied Microbiology, 2002 vol. 92, No. 4, pp. 681-694

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

J. Plowman & M.W. Peck

Aims: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F.

Methods and Results: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l ¹ of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5·5-8·0, germination occurred at 1-40°C, but not at 50°C, and was optimal at 20-25°C.

Conclusions: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum.

Significance and Impact of the Study: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.

INTRODUCTION

Clostridium botulinum is a group of four physiologically and phylogenetically distinct clostridia that share the common feature of producing the extremely potent botulinum neurotoxin (Lund and Peck 2000). Two members of this group, proteolytic Cl. botulinum (Group I) and non-proteolytic Cl. botulinum (Group II), are responsible for food-borne botulism, an intoxication in which consumption of food containing as little as 30 ng of pre-formed neurotoxin can result in severe illness. While foodborne botulism is relatively rare, the severity of the disease, the high cost of treatment, and the high economic impact of outbreaks, ensures that the prevention of outbreaks remains a major aim of regulators and industry.

Non-proteolytic Cl. botulinum is a particular hazard for the safe production of minimally heat-processed refrigerated foods (Peck 1997; Carlin et al. 2000). The heat treatment applied to these foods will result in the elimination of vegetative cells, but not bacterial spores (Peck 1997) and consequently, the hazard presented by non-proteolytic Cl. botulinum will be initially in the form of spores. Growth from spores comprises a number of distinct steps, the first of which involves the triggering of spore germination, and can be achieved by nutrient, non-nutrient, enzymatic and physical stimuli (Gould and Dring 1972). Nutrient germinants for spores of Bacillus species include L-alanine, the AGFK mixture (L-asparagine, glucose, fructose and potassium ions) and inosine (Johnstone 1994; Moir et al. 1994; Clements and Moir 1998). The germinants activate the germination receptor committing the spore to undergo an irreversible reaction, leading to loss of heat resistance and hydrolysis of peptidoglycan in the spore cortex (Johnstone 1994). Spore germination in clostridial species is less studied than in Bacillus species and frequently involves combinations of nutrient germinants (Gould and Dring 1972). Limited information indicates that spores of non-proteolytic Cl. botulinum type E strain Iwanai respond to a combination of nutrient germinants (Ando and Iida 1970; Ando 1971, 1974). Lysozyme brings about enzymatic germination of spores of non-proteolytic Cl. botulinum (Peck et al. 1993).

Established methods for studying germination of bacterial spores include use of a spectrophotometer to measure the associated decrease in optical density, and direct microscopic observation of loss of refractility using phase contrast optics. Both techniques are time consuming, rather tedious and use a large number of spores. The advent of automated instruments (e.g. Bioscreen) has enabled optical density data to be acquired more efficiently. Automated measurement of optical density has been used widely to study growth of micro-organisms and, more recently, to study the L-alanine trigger of Bacillus subtilis spore germination (Romick and Tharrington 1997). The aim of the present study was to develop the use of an automated turbidometric system to characterize germination requirements of spores of three strains of non-proteolytic Cl. botulinum, in order to increase understanding of germination and to provide a basis for improved control and detection of these spores in foods.

MATERIALS AND METHODS

Preparation of spore suspensions

Non-proteolytic Cl. botulinum type B (strain Eklund 17B), type E (strain Beluga) and type F (strain Craig 610) were maintained as described previously (Peck et al. 1992). Spores of non-proteolytic Cl. botulinum were produced on a two-phase meat medium (Peck et al. 1992). Free spores, together with sporulating cells, vegetative cells and debris, were harvested by centrifugation (15 000  g , 4°C, 15 min) and cleaned using discontinuous density gradient centrifugation (Kihm et al. 1988). The pellets were washed four to six times in 50 ml cold sterile glass-distilled water (SGDW), with centrifugation gradually reduced from 8000 to 2000  g . After each centrifugation the top layer of the pellet, consisting predominantly of vegetative cells, was discarded and the bottom layer resuspended. Pellets were finally resuspended in 5 ml SGDW, layered onto 15 ml 50% (v/v) aqueous solution of sodium/meglumine diatrizoate (Urografin 370, Schering, Germany) (Tamir and Gilvarg 1966) and centrifuged (35 000  g , 4°C, 40 min). The top layers containing debris, vegetative cells and germinated spores were removed with a Pasteur pipette, while the pellet, consisting predominately of phase-bright spores, was resuspended in 50 ml SGDW. This procedure occasionally caused spores to clump and when this occurred, the clumps were dispersed by sonication (Peck et al. 1992). Pellets were then washed a further six times in 50 ml SGDW (2000  g , 4°C, 15 min), resuspended in 5 ml SGDW and stored at 1-2°C. Spores were enumerated on peptone, yeast extract, glucose, starch (PYGS) medium (Stringer et al. 1999), with purity checked by plating onto VL blood agar and Reinforced Clostridial Medium containing 5% (w/v) skim milk (Peck et al. 1992). Clean spore suspensions were diluted to a final concentration of 2  10⁸ ml ¹ and stored at 1°C. Microscopic examination confirmed that the suspensions consisted of > 95% phase-bright spores. For anaerobic tests, spores were diluted in deoxygenated SGDW and stored at 1°C in sealed vials under a headspace of H₂ : N₂ (10 :  90, v/v).

Spores of B. subtilis (strain 168) were produced on plates of nutrient agar supplemented with MnSO₄ (4 mg l ¹) (Gould 1971). Plates were spread with 1 ml of a 6 h culture in Tryptone Soya Broth (Oxoid) and incubated at 37°C for 4 days. Growth was scraped from the agar surface and suspended in SGDW. Spores were washed, cleaned and stored as described for spores of non-proteolytic Cl. botulinum.

Preparation of solutions of potential germinants

Solutions were prepared in distilled water, sterilized by filtration (0·22 m, Millipore) and stored at 4°C until required. Solutions of cysteine and of sodium bicarbonate (NaHCO₃) were prepared freshly, since cysteine precipitated when refrigerated and the pH of NaHCO₃ solution changed during storage. Potassium phosphate (500 mmol l ¹, pH 7·0) and Tris/HCl (500 mmol l ¹, pH 7·4) buffers were sterilized by autoclaving and stored at room temperature. The three buffering systems used to test the effect of pH on spore germination (sodium acetate [500 mmol l ¹, pH 4·6-6·0], sodium phosphate [500 mmol l ¹, pH 6·5-7·7] and sodium carbonate/bicarbonate [500 mmol l ¹, pH 8·0-10·0]) were filter sterilized and stored at 4°C. Anaerobic solutions were prepared under CO₂ : H₂ : N₂ (5 : 10 : 85, v/v) using strict anaerobic technique (Holdeman et al. 1977), typically in deoxygenated water in an anaerobic cabinet (Don Whitley, Shipley, UK), and then filter sterilized.

Assessment of spore germination

Germination of both unheated and heat-activated spores was studied. Immediately before use, spores were heat-activated at 60°C for 15 min. Unheated and heat-activated spores were then equilibrated for 1 h at the temperature of the experiment (except when germination was tested at 1°C, when spores were held on ice). Germination of spore suspensions was measured by fall in optical density (O.D.) at 600 nm and monitored automatically using a Bioscreen C analyser system (Labsystems, Basingstoke, UK). Tests were at 20°C except in experiments to examine the effect of temperature, when the Bioscreen system was placed inside a refrigerated workstation (Don Whitley). The temperature was within 0·1°C of the target temperature throughout all experiments. Test solutions were prepared immediately before use by mixing individual components in 1 ml sterile microfuge tubes. Duplicate 100 l volumes were dispensed aseptically into honeycomb microplates (Labsystems), which were equilibrated for 30 min at the temperature of the experiment in the Bioscreen system. After this time, 100 l pre-incubated spore suspension were added to each well. For tests of germination under anaerobic conditions, all manipulations were performed in an anaerobic cabinet (Don Whitley) containing CO₂ : H₂ : N₂ (5 : 10 : 85). Inoculated microplates were incubated for up to 20 h with continuous shaking between measurements, with the O.D. of each well recorded automatically at appropriate intervals. Each experiment was performed twice. The extent of germination was expressed in terms of measured O.D. as a percentage of the initial O.D. (typically, initial O.D.=0·5). The rate of germination was determined from the steepest part of the curve and expressed as the maximum percentage fall in O.D. min ¹. At the end of each experiment, the proportion of germinated (phase-dark) spores was estimated by visual assessment of 200 spores in a minimum of four fields using phase-contrast microscopy.

RESULTS

Utility of an automated turbidity system to evaluate spore germination

The experimental procedure, using the Bioscreen system, proved to be highly efficient in determining the effect of a large number of potential germinants on spores of three strains of non-proteolytic Cl. botulinum. Spore germination measured using the Bioscreen system correlated well with direct counts of phase-dark spores by phase-contrast microscopy (Fig. 1). Preliminary results indicated a strong germination response ( 90% germination within 6 h) with L-alanine (100 mmol l ¹), L-lactate (50 mmol l ¹) and NaHCO₃ (50 mmol l ¹) in phosphate buffer (100 mmol l ¹, pH 7·0), and this mixture was included as a positive control in subsequent tests. A suspension of spores in phosphate buffer without addition served as a negative control.

Germination in aerobic and anaerobic atmospheres

It was more convenient to assess spore germination under aerobic conditions rather than anaerobic conditions. In order to establish whether spore germination was similar under aerobic and anaerobic conditions, the response of spores of strain Eklund 17B to seven germinant mixtures was tested (Fig. 2). Aerobic incubation did not inhibit the response to germinants. Under both aerobic and anaerobic incubation, heat activation increased the rate of germination. Spores of strain Eklund 17B were not germinated by L-alanine/NaHCO₃ or the AGFK mixture (L-asparagine, glucose, fructose and potassium ions) under aerobic or anaerobic conditions (Fig. 2). From these observations it was concluded that germination of spores of non-proteolytic Cl. botulinum could be studied adequately under aerobic conditions.

Effect of individual compounds on spore germination

A variety of chemicals (including: amino acids, carboxylic acids, sugars, sugar phosphates and purine ribosides, as detailed in Figs 3 and 4) were screened for their individual effect on germination of spores of non-proteolytic Cl. botulinum types B (strain Eklund 17B), E (strain Beluga) and F (strain Craig 610). Most of the compounds tested have been reported as germinants or co-germinants for spores of species of Clostridium or Bacillus. Each was tested at two concentrations (10 mmol l ¹ and 20-100 mmol l ¹, depending on solubility) in phosphate buffer (pH 7·0, 100 mmol l ¹) containing NaHCO₃ (50 mmol l ¹) on both unheated and heat-activated spore suspensions of each strain. In all cases, germination was negligible (< 10% germination) over a 20 h period (data not shown).

Germination with amino acids (50 mmol l ¹) and additional compounds

The ability of an amino acid (L-alanine, L-cysteine or L-serine) combined with another compound to trigger germination of spores of non-proteolytic Cl. botulinum types B, E or F was assessed at 20°C for up to 20 h in phosphate buffer (pH 7·0, 100 mmol l ¹) containing NaHCO₃ (50 mmol l ¹). When germination occurred, it proceeded rapidly, with little further germination after 6 h. Heat activation increased the initial germination rate, and increased or had no effect on the final extent of germination. L-cysteine combined with L-lactate produced the most rapid initial rate of germination of heat-activated spores (69% of initial O.D., aproximately 60% germination after 30 min). Germination triggered by L-alanine/L-lactate was slower (79% of initial O.D., approximately 40% germination after 30 min). Of the 27 combinations of germinants tested on spores of strains Eklund 17B and Craig 610, L-lactate was the most effective in combination with each of the amino acids (60% of initial O.D.,  90% germination after 6 h). Adenosine and inosine were partially effective in combination with L-alanine, particularly with heat-activated spores (approximately 70% of initial O.D., 70-80% germination), but had less effect combined with L-cysteine and L-serine. Sugar phosphates stimulated slight germination of heat-activated spores when combined with L-alanine, but none of the sugars or other carboxylic acids promoted germination (approximately 90% of initial O.D.). Several amino acids triggered germination in combination with L-alanine (Fig. 3). The most effective were L-cysteine, L-isoleucine and L-valine (68-75% of initial O.D., 50-70% germination). Heat activation had a relatively large effect on the extent of germination triggered by pairs of amino acids (Fig. 3). Limited tests with spores of strain Beluga gave a similar response to spores of strains Eklund 17B and Craig 610 (data not shown).

Germination with L-lactate (50 mmol l ¹) or inosine (10 mmol l ¹) and additional compounds

Several amino acids (glycine, L-cysteine, L-norvaline, L-serine, L-threonine, L-valine, L-alanine) in combination with L-lactate induced germination of  60% spores of strain Eklund 17B ( 70% of initial O.D. after 6 h) (Fig. 4). Spores of strain Beluga and, to a lesser extent, strain Craig 610 were also germinated by a number of amino acids in combination with L-lactate. Heat activation often increased both the initial rate and the overall extent of germination, particularly for spores of strain Beluga (Fig. 4). Sugar phosphates combined with L-lactate were ineffective, but adenosine and inosine in combination with L-lactate triggered germination of spores of strains Eklund 17B and Craig 610 (65-80% of initial O.D., 30-75% germination). When amino acids were combined with inosine, only L-alanine promoted substantial germination of all three spore suspensions (60-80% of initial O.D., 40-80% germination). L-cysteine and L-serine promoted some germination, but the extent varied between strains (Fig. 4).

Combinations of germinants that gave  30% germination in basal medium containing phosphate buffer (pH 7·0, 100 mmol l ¹) and NaHCO₃ (50 mmol l ¹) in 6 h at 20°C are shown in Table 1. Three combinations gave  90% germination, a further 12 combinations gave  60% germination and a further nine combinations gave  30% germination. Spores germinated optimally ( 90% germination) in a defined mixture containing L-lactate plus L-alanine or L-cysteine or L-serine.

Characterization of the L-alanine/ L-lactate germination system

To characterize further the L-alanine/L-lactate system, suspensions of unheated and heat-activated spores were germinated in combinations of L-lactate, L-alanine, NaHCO₃ and sodium thioglycollate (Fig. 5). Germination was dependent on the presence of L-alanine and L-lactate, although DL-lactate or D-lactate could replace L-lactate, with D-lactate giving lower germination of unheated spores (results not shown). D-alanine (100 mmol l ¹) had no effect on germination; it was unable to replace L-alanine and did not inhibit germination in the presence of L-alanine (10 mmol l ¹) for any of the three strains tested (data for strain Craig 610 are in Fig. 6).

Optimal concentrations of L-alanine and L-lactate were  5 mmol l ¹ and  25 mmol l ¹, respectively. Sodium thioglycollate and NaHCO₃ were non-essential components that increased the rate and overall extent of germination, particularly when added together and to unheated spores (Fig. 5). The optimum concentrations of NaHCO₃ and sodium thioglycollate were  10 mmol l ¹ and  20 mmol l ¹, respectively. Addition of glucose (50 mmol l ¹) had no effect on the rate or extent of germination (results not shown). There was less germination in PYGS medium than in the defined system (Fig. 5). Similar results to those obtained with strain Eklund 17B (Fig. 5) were also obtained with spores of strains Beluga and Craig 610, although slight germination (510% as judged by phase microscopy) of heat-activated spores of Beluga was observed with L-alanine (50 mmol l ¹) and NaHCO₃ (50 mmol l ¹).

Heat activation at 60°C for 15 min was not essential for germination in any of the conditions tested, but it increased the germination rate (particularly in non-optimal conditions) and reduced the effect of the non-essential co-germinants, NaHCO₃ and sodium thioglycollate (Fig. 5). Spores heat-activated at 70°C for 15 min germinated less well (approximately 80% of initial O.D. after 4 h) than spores heat-activated at 60°C for 15 min (approximately 55% of initial O.D. after 4 h). Germination was greatest over a pH range of 5·5-8·0 ( 90% of spores germinated, as judged microscopically, after 4 h), lower at pH 5·3 and 8·9 (50% of spores germinated after 4 h); only 10% of spores germinated in 4 h at pH 9·9, and no germination was observed at pH 5·0 or less in acetate buffer after 4 h.

Effect of L-asparagine/glucose/fructose/potassium ion mixture on spore germination

While spores of B. subtilis (strain 168) were germinated by a four factor system comprising L-asparagine/glucose/fructose/potassium ions (AGFK mixture) (data not shown), spores of all three strains of non-proteolytic Cl. botulinum failed to respond. The response for spores of strain Eklund 17B is shown in Figs 7 and 8. Replacement of L-asparagine in the AGFK system with either L-alanine (10 mmol l ¹) or L-lactate (50 mmol l ¹) or NaHCO₃ (50 mmol l ¹) did not trigger spore germination. When L-alanine and L-lactate were added together to the AGFK system, spore germination proceeded similarly to that observed when they were present on their own (Fig. 7). The AGFK system also failed to trigger germination under anaerobic conditions (Fig. 2).

Effect of temperature on kinetics of spore germination

Germination of unheated and heat-activated spores of non-proteolytic Cl. botulinum strain Eklund 17B was assessed at 1 to 50°C in PYGS medium and potential germinant mixtures. After incubation for 20 h, spore germination was detected at 1-40°C, but not at 50°C. For most mixtures, the maximum germination rate was recorded at 35°C (Fig. 8). A mixture of L-alanine/L-lactate/NaHCO₃/sodium thioglycollate gave the most rapid germination at the majority of temperatures. For all mixtures, the greatest extent of germination occurred at 20-25°C (Fig. 8). Heat activation increased both the range of incubation temperature and the number of mixtures at which maximum germination occurred. Weak germinant mixtures (e.g. L-alanine/inosine/NaHCO₃, L-alanine/L-valine/NaHCO₃) triggered incomplete germination at 10-35°C, and no germination outside this temperature range. Three test mixtures (L-alanine/NaHCO₃, AGFK, L-alanine/GFK) did not trigger germination at any temperature tested. Germination in PYGS medium was different from that in the defined media in that a lag period was always observed before the O.D. started to fall (particularly for unheated spore suspensions), and germination was most rapid at 20°C.

FIGURES

Fig. 1 Comparison of methods for measuring germination of spores of non-proteolytic Clostridium botuli...

Fig. 2 Aerobic (a, b) and anaerobic (c, d) germination of spores of non-proteolytic Clostridium botuli...

Fig. 3 Effect of L-alanine (a,d), L-cysteine (b,e) and L-serine (c) in combination with other compound...

Fig. 4 Effect of L-lactate (a, c, e) and inosine (b, d, f) in combination with amino acids on the exte...

Fig. 5 Characterization of L-alanine/L-lactate-induced germination of spores of non-proteolytic Clostr...

Fig. 6 Effect of D-alanine on germination of heat-activated spores of non-proteolytic Clostridium botu...

Fig. 7 Effect of AGFK mixture (L-asparagine[L-asn], glucose[G], fructose[F], potassium ions[K]) on ger...

Fig. 8 Effect of temperature on germination of spores of non-proteolytic Clostridium botulinum type B ...

DISCUSSION

Growth from bacterial spores occurs through a number of steps that are distinguishable but poorly understood. The first of these steps is germination. As the safety margin of minimally heat-processed refrigerated foods is small, it is imperative that spore germination is well understood for non-proteolytic Cl. botulinum, the pathogen of most concern for the safety of these foods. The use of the automated turbidometric method described here to investigate germination of spores of non-proteolytic Cl. botulinum has enabled testing of a large number of potential germinants, singly and in combination, and determination of the effect of aerobic/anaerobic conditions, temperature and pH. This has provided more extensive and systematic information than was available previously about the germination of spores of non-proteolytic Cl. botulinum. Germination of spores of non-proteolytic Cl. botulinum types B, E and F was triggered by a combination of factors. In phosphate buffer (100 mmol l ¹, pH 7·0) containing NaHCO₃ (50 mmol l ¹), three pairs of germinants (L-alanine/L-lactate, L-serine/L-lactate and L-cysteine/L-lactate, all at 50 mmol l ¹) were the most effective, inducing germination of  90% of spores after 6 h at 20°C. Other combinations of factors were less effective germinants. All the compounds tested individually (amino acids, carboxylic acids, purine ribosides, sugar phosphates and sugars) in phosphate buffer plus NaHCO₃ were ineffective, inducing germination of < 10% of spores. Well studied spore germination triggers in Bacillus species, including L-alanine, AGFK mixture and inosine, were ineffective with spores of non-proteolytic Cl. botulinum types B, E and F.

Although combinations of nutrient germinants are required generally to trigger germination of clostridial spores, some workers have reported single germinants to be effective (Gould and Dring 1972; Sarathchandra et al. 1976). Germination of spores of non-proteolytic Cl. botulinum type E (strain D8) was triggered by single amino acids (e.g. glycine, 2 mmol l ¹, pH 7·0) (Ward and Carroll 1966). A high concentration (100 mmol l ¹) of single amino acids (e.g. L-alanine, L-cysteine, L-serine, L-threonine) induced germination of spores of non-proteolytic Cl. botulinum type E (strain Iwanai) at pH 9·0 in the presence of NaHCO₃ (Ando 1971). At neutral pH, a situation more relevant to food, germination by a single amino acid was moderate, but multi-component mixtures triggered extensive germination (Ando and Iida 1970). Subsequent work confirmed that while L-alanine (25 mmol l ¹)/NaHCO₃ (60 mmol l ¹) in phosphate buffer (40 mmol l ¹, pH 6·8) had no effect, further addition to the mixture of glucose, galactose, maltose, glucose-6-phosphate, fructose-1,6-diphosphate, L-lactate, D-lactate, DL-lactate, guanosine or inosine at 2-20 mmol l ¹ induced germination of spores of strain Iwanai (Ando 1971). A number of alpha-hydroxy acids were also effective (Ando 1974). Germination of spores of a non-toxigenic strain of non-proteolytic Cl. botulinum was triggered by L-alanine (100 mmol l ¹)/DL-lactate (100 mmol l ¹)/glucose (90 mmol l ¹)/NaHCO₃ (12 mmol l ¹)/sodium thioglycollate (2 mmol l ¹) in potassium phosphate (pH 7·0) (Peck et al. 1995). In the present study, germination was more rapid in some of the defined mixtures than in PYGS medium, indicating that the lag time of growth in PYGS medium may be reduced by addition of appropriate germinants. This may facilitate detection of viable spores. While germination of spores of strains Eklund 17B, Beluga and Craig 610 showed similarities to that of strain Iwanai, there were differences. In the present study, single germinants were not effective, sugars (including glucose) failed to trigger germination in combination with other factors, while the effectiveness of a number of germination mixtures was demonstrated for the first time.

Generally, spores of other neurotoxigenic clostridia also required combinations of germinants. Germination of spores of 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)/NaHCO₃ 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). Other reports indicate that spores of proteolytic Cl. botulinum were germinated by L-alanine/NaHCO₃ in Tris-hydrochloride buffer (pH 7·0), L-alanine in citrate-phosphate buffer (pH 7·0) and buffered glucose solution (pH 7·0)(Wynne et al. 1954; Foegeding and Busta 1983; Chaibi et al. 1996). Spores of Cl. botulinum Group IV type G germinated optimally in a defined three-component system of L-cysteine/L-lactate/NaHCO₃ in phosphate buffer (pH 7·0). All three components were necessary for germination (Takeshi et al. 1988). Spores of a neurotoxigenic strain of Cl. butyricum were also germinated by multi-component mixtures (e.g. L-alanine/L-lactate/glucose/NaHCO₃) (Takeshi et al. 1991).

While L-alanine and L-lactate were essential germinants, NaHCO₃, sodium thioglycollate and heat shock were non-essential components that increased the rate and extent of germination of spores of non-proteolytic Cl. botulinum. When present together, L-alanine and L-lactate germinated 55% of unheated spores in 4 h at 20°C. This was increased to 70-80% by addition of NaHCO₃ or sodium thioglycollate, and to  90% by addition of NaHCO₃ and sodium thioglycollate. More than 90% of heat-shocked spores were germinated by L-alanine/L-lactate in phosphate buffer in 4 h at 20°C. The finding that NaHCO₃ acted as a non-essential component for germination of spores of non-proteolytic Cl. botulinum (especially unheated spores) agrees with similar observations made with spores of other clostridial species, although there are reports that NaHCO₃ may also act as an essential component for germination (see above). In tests with a laboratory medium, small concentrations of CO₂ (e.g. 5%) reduced the time to turbidity from spores of non-proteolytic Cl. botulinum (Gibson et al. 2000; Fernandez et al. 2001). Germination of spores of proteolytic Cl. botulinum, Cl. sporogenes and Cl. perfringens was also promoted by CO₂, while germination of B. cereus spores was inhibited (Wynne and Foster 1948; Enfors and Molin 1978). Indeed, CO₂ is often added to media to stimulate growth of clostridia. Sodium thioglycollate also acted as a non-essential component for germination of unheated spores of non-proteolytic Cl. botulinum, with only a marginal effect on heat-shocked spores and no effect under anaerobic conditions. Other tests indicated an inconsistent effect of sodium thioglycollate on germination of clostridial spores (Rowley and Feeherry 1970; Barker and Wolf 1971; Ando 1973a). The additive effect of NaHCO₃ and sodium thioglycollate suggests that they may have a separate site of action. A similar proposal was made for the effect of non-essential components (L-lactate, L-phenylalanine and L-arginine) on L-alanine/sodium ions-induced germination of Cl. bifermentans (Bright and Johnstone 1987). Activation by sub-lethal heat shock was not required for germination, but increased the rate and extent of germination of spores of non-proteolytic Cl. botulinum, and alleviated the effect of the non-essential germinants (NaHCO₃ and sodium thioglycollate). It has been proposed that heat shock leads to a conformational change in the germination receptor, making it more responsive to germinants (Johnstone 1994).

Germination of clostridial spores is generally less susceptible to oxygen inhibition than subsequent outgrowth (Sperber 1982). Spores of non-proteolytic Cl. botulinum type B germinated similarly under aerobic and anaerobic conditions (the present study), and germination of spores of non-proteolytic Cl. botulinum type E was independent of redox potential (Ando and Iida 1970). Spores of proteolytic Cl. botulinum type A also germinated under aerobic and anaerobic conditions independently of redox potential (Ando 1973a). In all three cases, germination occurred under conditions where subsequent outgrowth could not follow.

The temperature range for germination of spores of Bacillus and Clostridium species is frequently wider than that for growth (Smoot and Pierson 1982). Germination of spores of non-proteolytic Cl. botulinum has been reported at 1-40°C, but not 50°C, for the type B strain Eklund 17B (present study), 20-40°C for the type E strain Iwanai (Ando and Iida 1970), 5-45°C for the type E strain Minnesota (Strasdine 1967), 2-50°C for the type E strain VH (Grecz and Arvay 1982) and 10-30°C for the non-toxigenic strain NCIB 4270 (Evans et al. 1997). Germination outside the growth range has been reported for spores of three strains of non-proteolytic Cl. botulinum (present study; Strasdine 1967; Grecz and Arvay 1982), two strains of proteolytic Cl. botulinum (Rowley and Feeherry 1970; Ando 1973a) and one strain of Cl. botulinum Group IV type G (Takeshi et al. 1988). In the present study, spores of non-proteolytic Cl. botulinum strain Eklund 17B germinated most rapidly at 35°C, while the highest extent of germination was at 20-25°C. The optimum temperature for germination (in terms of rate and extent) of spores of other strains of non-proteolytic Cl. botulinum has been reported as 9°C for strain VH (Grecz and Arvay 1982), 30°C for strains Minnesota and NCIB 4270 (Strasdine 1967; Evans et al. 1997), and 37°C for strain Iwanai (Ando and Iida 1970). The difference in temperature optima may reflect strain differences or a response to different germinants. For example, spores of Cl. bifermentans germinated optimally at 37°C in the presence of L-alanine/sodium ions, but addition of L-phenylalanine/L-arginine/L-lactate raised this optimum to 53°C (Waites and Wyatt 1974). The optimum temperature for germination of spores of other neurotoxigenic clostridia has been reported as 30-37°C for proteolytic Cl. botulinum type A (Rowley and Feeherry 1970; Ando 1973a) and 37-45°C for Cl. botulinum Group IV type G (Takeshi et al. 1988).

The optimum pH for germination of spores of strain Eklund 17B in L-alanine/L-lactate/NaHCO₃/sodium thioglycollate in the present study was in the range 5·5-8·0. Some germination occurred at pH 5·3 but none was detected at pH 5·0; however, inhibition of germination at this pH may have been partly due to undissociated acetic acid in the buffer. There appear to be no reports in the literature of growth and toxin formation by non-proteolytic Cl. botulinum at a pH lower than 5·0 (Lund and Peck 2000). The optimum pH for germination of spores of non-proteolytic Cl. botulinum type E strain Iwanai in a complex medium was pH 6·6 (Ando and Iida 1970), in L-alanine/L-lactate/NaHCO₃, pH 7·2 (Ando 1971), and for the type E strain Minnesota in a complex medium, pH 7·0 (Strasdine 1967). While there is a single report of a high pH germination system for spores of strain Iwanai in the presence of high concentrations of L-alanine (> 100 mmol l ¹)/NaHCO₃ (60 mmol l ¹) (Ando 1971), this system does not appear to be present in the three strains tested in the present study. Germination of spores of other neurotoxigenic clostridia has also been reported as optimal at a neutral pH. In response to different germinants, spores of proteolytic Cl. botulinum germinated optimally at pH 6·5-7·5, but not below pH 4·8 or above pH 9·2(Rowley and Feeherry 1970; Winarno et al. 1971; Ando 1973a); strain differences were also identified (Blocher and Busta 1985). A predictive model of the effect of temperature (15-30°C), pH (5·5-6·5) and NaCl (0·5-4·0%) on germination of spores of strain 56A in Brain Heart Infusion Broth has been published (Chea et al. 2000).

D-alanine is a competitive inhibitor of L-alanine-induced germination of spores of B. subtilis (Johnstone 1994). In the present study, addition of D-alanine did not inhibit germination of spores of non-proteolytic Cl. botulinum types B, E and F in L-alanine/L-lactate/NaHCO₃ (pH 7·0), even when added at 10 times the concentration of L-alanine, and neither was D-alanine able to replace L-alanine. In a previous study with non-proteolytic Cl. botulinum type E (strain Iwanai), a threefold excess of D-alanine (compared with L-alanine) had no effect on germination induced by L-alanine/L-lactate/NaHCO₃ (pH 6·8) or L-alanine/inosine/NaHCO₃ (pH 6·8), but did inhibit germination induced by L-alanine/NaHCO₃ (pH 9·0) and L-alanine/glucose/NaHCO₃ (pH 6·8). D-alanine was unable to replace L-alanine (Ando 1971). D-alanine was a more effective inhibitor of spore germination in proteolytic Cl. botulinum. Although addition of D-alanine at a concentration ratio to L-alanine of 1 :  1 had no effect on germination triggered by L-alanine/L-lactate/NaHCO₃ (pH 6·7), it completely inhibited germination when added at a ratio of 30 : 1 (Ando 1973b). D-alanine and D-cysteine both inhibited germination of spores of proteolytic Cl. botulinum type A and B (strains 62A, B-aphis and Ba410) induced by L-alanine/thioglycollate/NaHCO₃ and by L-cysteine/thioglycollate/NaHCO₃. Neither D-alanine nor D-cysteine acted as germinants in the presence of thioglycollate/NaHCO₃ (Rowley and Feeherry 1970; Montville et al. 1985). The present study has confirmed previous findings (Ando 1971, 1974) that D-lactate is able to replace L-lactate in germination of spores of non-proteolytic Cl. botulinum.

This study of the effect of an extensive range of factors by means of an automated turbidometric system has improved understanding of spore germination in non-proteolytic Cl. botulinum; it provides information relevant to the control of this pathogen in foods, and provides a basis for detection of viable spores in foods. Spore germination in non-proteolytic Cl. botulinum types B, E and F shares many common features with germination in other clostridia. Three pairs of germinants (L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate) were particularly effective in triggering germination. Changes in pH and temperature failed to distinguish more than one type of germination system in non-proteolytic Cl. botulinum types B, E and F. Accordingly, a working hypothesis is that in the food environment, there is a single type of spore germination system. The trigger is an amino acid together with L-lactate. The most effective amino acids are L-alanine, L-cysteine and L-serine. Other compounds are able to substitute for the amino acid and L-lactate, but reduce the rate and extent of germination. Non-essential factors (NaHCO₃, sodium thioglycollate and heat shock) increase the rate and extent of germination, probably by activating the germination receptor, making the receptor more responsive to the amino acid and L-lactate. This type of germination system appears distinct from those described for L-alanine, AGFK mixture and inosine in Bacillus species.

ACKNOWLEDGEMENTS

This research was funded by the competitive strategic grant of the BBSRC.

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