Growth from Spores of Nonproteolytic Clostridium botulinum in Heat-Treated Vegetable Juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Historical and Contemporary NaCl Concentrations Affect the Duration and Distribution of Lag Times from Individual Spores of Nonproteolytic Clostridium botulinum

In this study we determined the effect of NaCl concentration during sporulation (0 or 3.0% [wt/vol] added NaCl) and subsequent growth (0 or 2.0% [wt/vol] added NaCl) on the distributions of times associated with various stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B. The effects of NaCl on the probability of germination and the probability of subsequent growth were also determined. Spore populations exhibited considerable heterogeneity at all stages of lag phase for each condition tested. Germination time did not correlate strongly with the times for later stages in the lag phase, such as outgrowth and doubling time. Addition of NaCl to either the sporulation or growth media increased the mean times for, and variability of, all the measured stages of the lag phase (germination, emergence, time to one mature cell, and time to first doubling). There was a synergistic interaction between the inhibitory effects of NaCl in the sporulation medium and the inhibitory effects of NaCl in the subsequent growth medium on the total lag time and each of its stages. Addition of NaCl to either the sporulation medium or the growth medium reduced both the probability of germination and the probability of a germinated spore developing into a mature cell, but the interaction was not synergistic. Spores formed in medium with added NaCl were not better adapted to subsequent growth in suboptimal osmotic conditions than spores formed in medium with no added NaCl were. Knowledge of the distribution of lag times for individual spores and quantification of the biovariability within lag time distributions may provide insight into the underlying mechanisms and can be used to improve predictions of growth in food and to refine risk assessments.     

Role of GerKB in Germination and Outgrowth of Clostridium perfringens Spores

Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.Spores of Bacillus and Clostridium species can break dormancy upon sensing a variety of compounds (termed germinants), including amino acids, nutrient mixtures, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), and cationic surfactants such as dodecylamine (20). Nutrient germinants are sensed by their cognate receptors, located in the spore''s inner membrane (6), which are composed of proteins belonging to the GerA family (10, 11). In Bacillus subtilis, three tricistronic operons (gerA, gerB, and gerK) expressed uniquely during sporulation in the developing forespore each encode the three major germinant receptors, with different receptors responding to a different spectrum of nutrient germinants (5, 9, 20). Null mutations in any cistron in a gerA family operon inactivate the function of the respective receptor (9, 11). In contrast, Clostridium perfringens, a gram-positive, spore-forming, anaerobic pathogenic bacterium, has no tricistronic gerA-like operon but only a monocistronic gerAA that is far from a gerK locus. This locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream of and in the opposite orientation to gerKA-gerKC (Fig. ​(Fig.1A)1A) (16). GerAA has an auxiliary role in the germination of C. perfringens spores at low germinant concentrations, while GerKA and/or GerKC are required for l-asparagine germination and have partial roles in germination with KCl and a mixture of KCl and l-asparagine (AK) (16). In contrast to the situation with B. subtilis, where germinant receptors play no role in Ca-DPA germination (12, 13), GerKA and/or GerKC is required for Ca-DPA germination (16). The partial requirement for GerKA and/or GerKC in C. perfringens spore germination by KCl and AK suggests that the upstream gene product, GerKB, might also have some role in KCl and AK germination of C. perfringens spores. Therefore, in this study we have investigated the role of GerKB in the germination and outgrowth of C. perfringens spores.Open in a separate windowFIG. 1.Arrangement and expression of gerKB in C. perfringens SM101. (A) The arrangement of the gerK locus in C. perfringens SM101 is shown, and the locations of the primers used to amplify the upstream regions of the gerKB gene and the putative promoters of gerKB and gerKA are indicated. The gerKB promoter was predicted to be within the intergenic regions between gerKB and the gerK operon. (B) GUS specific activities from the gerKB-gusA fusion in strain SM101(pDP84) grown in TGY vegetative (filled squares) and DS sporulation (open squares) media were determined as described in the text. Data represent averages from three independent experiments with the error bars representing standard deviations, and time zero denotes the time of inoculation of cells into either TGY or DS medium.To determine if gerKB is expressed during sporulation, 485 bp upstream of the gerKB coding sequence, including DNA between gerKB and gerKA, was PCR amplified with primer pair CPP389/CPP391, which had SalI and PstI cleavage sites, respectively (see Table S2 in the supplemental material). The PCR fragment was cloned between SalI and PstI cleavage sites in plasmid pMRS127 (17) to create a gerKB-gusA fusion in plasmid pDP84 (see Table S1 in the supplemental material). This plasmid was introduced into C. perfringens SM101 by electroporation (3), and Em^r transformants were selected. The SM101 transformant carrying plasmid pDP84 was grown in TGY vegetative growth medium (3% Trypticase soy, 2% glucose, 1% yeast extract, 0.1% l-cysteine) (7) and in Duncan-Strong (DS) (4) sporulation medium and assayed for β-glucuronidase (GUS) activity as described previously (23). Vegetative cultures of strain SM101 carrying plasmid pMRS127 (empty vector) or pDP84 (gerKB-gusA) exhibited no significant GUS activity, and strain SM101 grown in DS medium also exhibited no significant GUS activity (Fig. ​(Fig.1B1B and data not shown). However, GUS activity was observed in sporulating cultures of SM101(pDP84) (Fig. ​(Fig.1B),1B), indicating that a sporulation-specific promoter is located upstream of gerKB. The expression of the gerKB-gusA fusion began ∼3 h after induction of sporulation and reached a maximum after ∼6 h of sporulation (Fig. ​(Fig.1B).1B). The decrease in GUS activity observed after ∼6 h is consistent with the GerKB-GusA protein being packaged into the dormant spore where it cannot be easily assayed and thus with gerKB being expressed in the forespore compartment of the sporulating cell (8). These results confirm that, as with the gerKA-gerKC operon (16), gerKB is also expressed only during sporulation.To investigate the role of GerKB in C. perfringens spore germination, we constructed a gerKB mutant strain (DPS108) as described previously (14-16). A 2,203-bp DNA fragment carrying 2,080 bp upstream of and 123 bp from the N-terminal coding region of gerKB was PCR amplified using primers CPP369 and CPP367, which had XhoI and BamHI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). A 1,329-bp fragment carrying 134 bp from the C-terminal and 1,195 bp downstream of the coding region of gerKB was PCR amplified using primers CPP371 and CPP370, which had BamHI and KpnI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). These PCR fragments were cloned into plasmid pCR-XL-TOPO, giving plasmids pDP67 and pDP69, respectively (see Table S1 in the supplemental material). An ∼2.2-kb BamHI-XhoI fragment from pDP67 was cloned into pDP1 (pCR-XL-TOPO carrying an internal fragment of gerAA), giving plasmid pDP68, and an ∼1.4-kb KpnI-BamHI fragment from pDP69 was cloned in pDP68, giving pDP73 (see Table S1 in the supplemental material). The latter plasmid was digested with BamHI, the ends were filled, and an ∼1.3-kb NaeI-SmaI fragment carrying catP from pJIR418 (1) was inserted, giving plasmid pDP74. Finally, an ∼4.8-kb KpnI-XhoI fragment from pDP74 (see Table S1 in the supplemental material) was cloned between the KpnI and SalI sites of pMRS104, giving pDP75, which cannot replicate in C. perfringens. Plasmid pDP75 was introduced into C. perfringens SM101 by electroporation (3), and the gerKB deletion strain DPS108 was isolated as described previously (18). The presence of the gerKB deletion in strain DPS108 was confirmed by PCR and Southern blot analyses (data not shown). Strain DPS108 gave ∼70% sporulating cells in DS sporulation medium, similar to results with the wild-type strain, SM101 (data not shown).Having obtained evidence for successful construction of the gerKB mutant, we compared the germinations of heat-activated (80°C; 10 min) gerKB and wild-type spores as previously described (16). Both the gerKB and wild-type spores germinated identically and nearly completely in 60 min at 40°C in brain heart infusion (BHI) broth as determined by the fall in optical density at 600 nm (OD₆₀₀) of germinating cultures and phase-contrast microscopy (data not shown). This result suggests that GerKB plays no essential role in spore germination in rich medium. The role of GerKB in C. perfringens spore germination was also assessed with individual germinants identified previously (16). Heat-activated wild-type and gerKB spores germinated similarly with high (100 mM) concentrations of KCl, l-asparagine, and AK, all in 25 mM sodium phosphate (pH 7.0), and in 50 mM Ca-DPA adjusted to pH 8.0 with Tris base (Fig. 2A to D). These results were also confirmed by phase-contrast microscopy (data not shown). However, with lower (10 to 20 mM) concentrations of KCl, l-asparagine, and AK, gerKB spore germination was very slightly (Fig. ​(Fig.2A)2A) to significantly (Fig. 2B and C) slower than that of wild-type spores. These results suggest that while GerKB is not essential for germination with high concentrations of KCl, l-asparagine, or AK, it plays a significant role in germination with low l-asparagine and AK concentrations and, further, that GerKB is not required for Ca-DPA germination. This latter finding is similar to the situation with B. subtilis spores where germinant receptors play no role in Ca-DPA germination (19, 20). However, in C. perfringens spores, GerKA and/or GerKC do play a significant role in Ca-DPA germination (16).Open in a separate windowFIG. 2.Germination of spores of C. perfringens strains with various germinants. Heat-activated spores of strains SM101 (wild type) (filled symbols) and DPS108 (gerKB) (open symbols) were incubated at an OD₆₀₀ of 1 at 40°C with high (squares) and low (triangles) germinant concentrations of 100 and 10 mM KCl (A), 100 and 20 mM l-asparagine (B), 100 and 10 mM AK (C), and 50 mM Ca-DPA (D) as described in the text, and at various times the OD₆₀₀ was measured. No significant germination was observed when heat-activated spores of SM101 and DPS108 were incubated for 60 min at 40°C in 25 mM sodium phosphate buffer (pH 7.0) (data not shown). The data shown are averages from duplicate determinations with two different spore preparations, and error bars represent standard deviations.Bacterial spores can also germinate with dodecylamine, a cationic surfactant (19). In B. subtilis spores, dodecylamine induces germination most likely by opening channels composed, at least in part, of SpoVA proteins (22), allowing release of the spores'' Ca-DPA (19). Spores of B. subtilis lacking all three functional germinant receptors release DPA, as do wild-type spores, upon incubation with dodecylamine (19), while C. perfringens spores lacking GerKA-GerKC incubated with dodecylamine release DPA slower than wild-type spores (16). However, when C. perfringens gerKB spores at an OD₆₀₀ of 1.5 were incubated with 1 mM dodecylamine in Tris-HCl (pH 7.4) at 60°C (2, 16), gerKB spores released their DPA slightly faster than wild-type spores (Fig. ​(Fig.3)3) when DPA release was measured as described previously (16). These results suggest that GerKB has no role in dodecylamine germination.Open in a separate windowFIG. 3.Germination of spores of C. perfringens strains with dodecylamine. Spores of strains SM101 (wild type) (filled squares) and DPS108 (gerKB) (open squares) were germinated with dodecylamine, and germination was monitored by measuring DPA release as described in the text. There was no significant DPA release in 60 min by spores incubated similarly but without dodecylamine (data not shown). Error bars represent standard deviations.Previous work (16) found that C. perfringens spores lacking GerKA-GerKC had lower viability than wild-type spores on rich medium plates, and it was thus of interest to determine gerKB spore viability, which was measured as previously described (14, 16). Strikingly, the colony-forming ability of gerKB spores was ∼7-fold lower (P < 0.01) than that of wild-type spores after 24 h on BHI plates (Table ​(Table1),1), and no additional colonies appeared when plates were incubated for up to 3 days (data not shown). The colony-forming ability of spores lacking GerKA and GerKC determined in parallel was ∼12-fold lower than that of wild-type spores (Table ​(Table1).1). Phase-contrast microscopy of C. perfringens spores incubated in BHI broth for 24 h under aerobic conditions to prevent vegetative cell growth indicated that >90% of wild-type spores not only had germinated but had also released the nascent vegetative cell, while >85% of gerKA gerKC and gerKB spores remained as only phase-dark germinated spores with no evidence of nascent cell release (data not shown), as found previously with gerKA gerKC spores (16). The fact that >85% of gerKB spores germinated in BHI medium in 24 h but most of these germinated spores did not progress further in development strongly suggests that GerKB is needed for normal spore outgrowth (and see below) as well as for normal spore germination.

TABLE 1.

Colony formation by spores of C. perfringens strains^a

Survival and Outgrowth of Clostridium botulinum Type E Spores in Smoked Fish

Chub injected in the loin muscle with 10(6)Clostridium botulinum type E spores were smoked to an internal temperature of 180 F (82.2 C) for 30 min, sealed in plastic bags, and incubated at room temperature (20 to 25 C) for 7 days. Viable type E spores were found in practically all such fish. Toxin formation by the survivors in the smoked fish was dependent on the brine concentration of the smoked fish. A brine concentration of 3% or higher, as measured in the loin muscle, inhibited toxin formation. Six different type E strains gave similar results. Only a few hundred of the million spores in the inoculum survived the smoking. Moisture in the atmosphere during smoking did not reduce the incidence of fish with type E survivors.     

Hypochlorite Injury of Clostridium botulinum Spores Alters Germination Responses

[This corrects the article on p. 1361 in vol. 45.].     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.     

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg⁻¹. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.     

Ultraviolet Sensitivity of Bacillus subtilis Spores upon Germination and Outgrowth

A strain of Bacillus subtilis, UVSSP-42-1, which produces ultraviolet (UV)-sensitive spores and vegetative cells, was found to possess germinated spores 25 times more UV resistant than the resting spores. This relative resistance achieved upon germination was associated with the transition of the heat-resistant refractile spores to the heat-sensitive phase-dark forms. Several generations of outgrowth were required before the cells attained the level of UV sensitivity characteristic of the vegetative cell. The UV sensitivity of germinated spores was compared with other strains with various combinations of mutations affecting deoxyribonucleic acid repair capabilities. The presence of hcr and ssp mutations which are known to abolish the removal of photoproducts from deoxyribonucleic acid did not alter significantly the sensitivity of the germinated forms. However, the addition of the recA mutation and, to some extent, the pol mutation increased the UV sensitivity of the germinated spores. These results indicate that deoxyribonucleic acid repair mechanisms dependent on the recA gene are active in the germinated spores. The chemical nature of the damage repaired by the recA gene product is not known. This study indicates that the life cycle of sporulating bacilli consists of at least three photobiologically distinct forms: spore, germinated spore, and vegetative cell.     

Macromolecular Syntheses During Germination and Outgrowth of Bacillus subtilis Spores

Alanine and glucose used jointly are known to be necessary and sufficient for spore germination in Bacillus subtilis 168. By testing them separately, we have verified that alanine provokes optimal phase-darkening of the spores but inhibits macromolecular syntheses, while glucose is specifically needed for initiating those syntheses. By using them in succession we obtained evidence suggesting that: (i) sporal modifications which lead to phase-darkening must occur before macromolecular synthesis can start; (ii) the amino acid pool, on which the early protein synthesis is solely dependent, expands during incubation in alanine which allows degradative but prevents synthetic activities; and (iii) progression of degradations in alanine not promptly followed by syntheses in glucose produce a metabolic imbalance in the germinating spore. A sharp transition in the origin of building blocks was shown by using a tryptophan-defective mutant. At first the synthesis of proteins depended on pre-existing amino acids from turnover of sporal material since it occurred in the absence of any exogenous amino acid and its rate remained unaltered by supplying either all amino acids except tryptophan or tryptophan alone. Eventually, protein synthesis became dependent strictly on exogenous tryptophan and strongly on the supply of several other amino acids, not required later during vegetative growth. Clearly, by the start of outgrowth, all building blocks must be provided either by endogenous de novo synthesis or by exogenous supply.     

Ultrastructural Analysis During Germination and Outgrowth of Bacillus subtilis Spores

Electron microscopy of thin sections of dormant and germinating spores of Bacillus subtilis 168 revealed a progressive change in the structure of the cortex, outer spore coat, and inner spore coat. The initial changes were observed in the cortex region, which showed a loose fibrous network within 10 min of germination, and in the outer spore coat, which began to be sloughed off. The permeability of the complex outer spore layers was modified within 10 min, since, at this time, the internal structures of the spore coat were readily stainable. A nicking degradation action of the laminated inner spore coat began at 20 min, and this progressed for the next 20 min leading to the loosening of the inner spore coat. By 30 min, the outer spore coat showed signs of disintegration, and at 40 min, both the outer and inner spore coats were degraded extensively. At 30 to 40 min, a period just preceding net deoxyribonucleic acid synthesis, mesosomes became very prominent in the inner spore core and the cell wall began to thicken around the spore core. At 50 min, an emerging cell was observed, and by 60 min, there was clear evidence for elongation of the emerging cell and the presence of two nuclear bodies. At 90 min, elongation had been followed by the first cell division. There was evidence for spore coat fragments at the opposite poles of the dividing cell.     

Requirements for Germination of Clostridium sordellii Spores In Vitro

Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, d-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of l-phenylalanine and l-alanine but not in that of bicarbonate or l-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.Clostridium sordellii is an anaerobic, Gram-positive, spore-forming bacterium that is commonly found in soil and in the intestines of animals (4). Many C. sordellii strains are nonpathogenic; however, virulent strains cause lethal infections in several animal species, such as hemorrhagic enteritis in foals, sheep, and cattle (5, 10, 16, 28), omphalitis in foals (43), and wound infection in humans (4, 35).C. sordellii also can cause life-threatening necrotizing infections after gynecological procedures (4). In addition, fatal cases of C. sordellii endometritis following medical abortion with a mifepristone-misoprostol combination have been reported recently (13, 19, 56). The increased use of mifepristone-misoprostol for medical abortion may result in larger numbers of C. sordellii infections (38, 40).Although C. sordellii rarely has been identified in the genital tract, a correlation between gynecological procedures and C. sordellii-mediated toxic shock syndrome is apparent (19). Pregnancy, childbirth, or abortion may predispose some women to acquire C. sordellii in the vaginal tract (19). Under these conditions, C. sordellii infections result in an almost 100% mortality rate.Since there is no national system for tracking and reporting complications associated with gynecological procedures, the identification of the true rates of reproductive tract infections in women is not readily available (8). Therefore, the number of known C. sordellii-associated infections, although low, may be underreported (19, 29). Furthermore, unsafe abortion practices in developing countries cause large mortality rates due to complicating infections (24, 34). In many cases, however, the causative agent of the abortion-associated sepsis have not been characterized (24). Thus, the worldwide morbidity and mortality associated with C. sordellii infections is not currently known.C. sordellii produces several virulence factors. The two major toxins are the lethal toxin (TcsL) and the hemorrhagic toxin (37, 46). The lethal toxin produced by C. sordellii is causally involved in enteritis of domestic animals and in systemic toxicity following infections of humans (46). Furthermore, TcsL is associated with rapid mortality in C. sordellii endometritis rodent models (26). Interestingly, TcsL cytopathic effects are increased at low pH, a characteristic found in the vaginal tract (48). The hemorrhagic toxin is not well characterized, but it has been reported to cause dermal and intestinal necrosis in guinea pigs (6, 52).C. sordellii, like other Bacilli and Clostridia species, has the ability to form metabolically dormant spores that are extremely resistant to environmental stresses, such as heat, radiation, and toxic chemicals (42, 55). Upon encountering a suitable environment, spores germinate into vegetative cells, the form that is responsible for toxin production and disease onset (39, 54).In most cases, the germination process initially is triggered by the detection of low-molecular-weight germinants by a sensitive biosensor (39, 54). This sensor consists of a proteinaceous germination (Ger) receptor encoded, in general, by a tricistronic operon. Spore germination requirements have been studied most extensively for Bacilli and can be initiated by a variety of factors, including amino acids, sugars, and nucleosides (20, 30).Spore germination in the Clostridia generally requires combinations of multiple germinants. The germination of spores of proteolytic Clostridium botulinum types A and B was triggered by a defined three-component mixture comprised of l-alanine (or l-cysteine), l-lactate (or sodium thioglycolate), and sodium bicarbonate (3). In contrast, the optimum germination of spores of nonproteolytic C. botulinum types B, E, and F required binary combinations of l-alanine-l-lactate, l-cysteine-l-lactate, and l-serine-l-lactate (45).Clostridium difficile is a human pathogen that can cause fulminant colitis (11). Interestingly, C. difficile does not encode any known Ger receptors (53). However, it is likely that germination receptors exist, because C. difficile spores must germinate in order to complete their life cycle. While C. difficile germination receptors remain elusive, the spores of C. difficile germinate in rich medium supplemented with bile salts (62). More recently, taurocholate (a bile salt) and glycine (an amino acid) were shown to act as cogerminants for C. difficile spore germination (57, 61).Clostridium bifermentans is a close relative of C. sordellii (14). The minimum requirement for C. bifermentans spore germination was the presence of l-alanine, l-phenylalanine, and l-lactate (59). In addition, an unknown factor present in yeast extract was suggested to enhance germination (59). However, the Ger receptors involved in C. bifermentans spore germination are not known.Even though many Bacilli and Clostridia species use similar metabolites as germinants, the mechanisms of germinant recognition remain to be elucidated. Unfortunately, the multimeric interactions of Ger receptor complexes and the hydrophobic nature of the Ger receptor subunits have hindered our understanding of the mechanism of germinant recognition.To understand the molecular determinants of germinant recognition, we recently applied kinetic methods to study bacterial spore germination (1, 2, 18). Spore germination can be analyzed quantitatively by fitting optical density (OD) decreases to the Michaelis-Menten equation (2). The kinetic parameters obtained allow the determination of the apparent binding affinity (K_m) of spores for the different cogerminants and the maximum rate of spore germination (V_max). In these instances, K_m refers to the concentration of substrate required to reach half of the maximal germination rate. These parameters can, in turn, be used to determine the mechanism of germination and potential interactions between germination receptors. Furthermore, by comparing apparent K_m values to germinant concentrations in vivo, models for spore-germinant complex distribution can be proposed, and rate-limiting steps for the germination process can be derived. Thus, kinetic analysis can yield information on spore activation even if the identities of the germination receptors are not known.Using this procedure, we were able to determine the mechanism for Bacillus anthracis germination with inosine and l-alanine. In turn, this information was used to design nucleoside analogs that inhibit B. anthracis spore germination in vitro and protect macrophages from anthrax cytotoxicity (2).Since C. sordellii germination receptors have not been identified, we used chemical probes and kinetic methods to investigate the conditions necessary for spore germination. We found that C. sordellii spores germinate better at slightly acidic pH. Furthermore, germination rates varied slightly from 25 to 40°C. We also found that C. sordellii spores have an absolute requirement for a small amino acid, a basic amino acid, an aromatic amino acid, and bicarbonate (NaHCO₃) for efficient germination. Kinetic analysis showed allosteric interaction for the putative l-phenylalanine and l-alanine germination receptors. In contrast, l-arginine or bicarbonate recognition followed typical Michaelis-Menten kinetics. The implication of germinant recognition and host environment is discussed.     

Germination of spores from Clostridium botulinum B-aphis and Ba410

The germination of spores from 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-1 in the absence of NaCl and was not affected by 2% NaCl. A salt-tolerant derivative, strain Ba410, germinated at rates of 0.16% min-1 in CTB and 0.04% min-1 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-Alanine- and 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.     

Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.     

Effect of Sodium Chloride and pH on the Outgrowth of Spores of Type E Clostridium botulinum at Optimal and Suboptimal Temperatures

The sodium chloride inhibition of spore outgrowth of four strains of type E Clostridium bolulinum was determined in a Trypticase-peptone-glucose (TPG) medium. At 16, 21, and 30 C, spores of three strains required 5.0% and one strain 4.5% salt for complete inhibition during 1 year of incubation. At 8 and 10 C, spores of the four strains required 4.5% salt for definite inhibition. Salt concentrations slightly lower than those providing inhibition tended to extend spore outgrowth time at low temperatures. The minimal pH permitting outgrowth of type E spore inocula was affected by the concentration of reducing compound present in the system. When either 0.02% sodium thioglycolate or 0.05% L-cysteine hydrochloride was used, outgrowth at 30 and 8 C occurred at much lower pH levels than when 0.2% thioglycolate was added. At 30 C, spores of one strain showed outgrowth in TPG medium as low as pH 5.21 with an inoculum of 2 million spores per replicate tube. At a 10-fold higher inoculum, the same strain showed outgrowth at pH 5.03 in one of five replicate tubes. At 8 C, spore outgrowth of the four strains occurred at pH 5.9, but not at pH 5.7, in TPG medium containing L-cysteine hydrochloride.     

Mechanism of Nitrite-Induced Germination of Clostridium perfringens Spores

A study has been undertaken to understand the mechanism(s) of the nitrite-induced germination of Clostridium perfringens S40 spores. An increase in germination rates of the spores in response to increasing NaNO2 concentrations was entirely dependent on both pH and temperature of incubation. Low pH and high temperature were effective in accelerating the germination rate, the maximal germination level being reached at pH 4.0 and 60°C in the presence of 0.5 M NaNO₂. On the basis of germination rate, the activation energy (μ) for the nitrite-induced germination calculated was approximately 9.9 kcal/mol. Germination was greatly stimulated after pretreatment of spores with DTT at pH 10.5 to remove the coats. Furthermore, cortical fragments prepared from spores of the same organism were lysed not only by lysozyme but also by NaNO_2. Hexosamine-containing material was also solubilized by these reagents. However, nitrite, unlike lysozyme, released a considerable amount of free hexosamine as well. These results suggest that nitrite-induced germination may involve an interaction of sodium nitrite as nitrous acid with some component of the cortex. A possible mechanism of nitrite-induced germination is discussed.     

The Effect of Oxygen on the Germination and Outgrowth of Clostridium butyricum Spores and Changes in the Oxidation-Reduction Potential of Cultures

S ummary . The E_h fall observed during incubation of Clostridium butyricum spores occurred during germination and emergence, not during the log phase; it is attributed to the H₂ tension resulting from metabolism. When the O₂ tension in the medium was increased, the E_h fell only after a few spores outgrew and replicated; germination of remaining spores then followed. It is suggested that the few cells able initially to metabolize can (a) elaborate NADH etc. which reduce the O₂ tension to a level non-inhibitory for the remaining spores, and (b) produce the H₂ tension recorded by the Pt electrode.