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Title:
THE EFFECT OF MANDELATE AND LACTATE ON SPORE GERMINATION
Document Type and Number:
WIPO Patent Application WO/2019/020346
Kind Code:
A1
Abstract:
A method of chemically inducing germination of bacteria of the group Bacillus cereus sensu lato, comprising the step of administering to a bacterial population a germinant comprising:inosine; one or more amino acids; andmandelate and/or lactate.

Inventors:
BISHOP, Alistair (School of Biological and Marine Sciences, University of PlymouthDrake Circus, Plymouth Devon PL4 8AA, PL4 8AA, GB)
Application Number:
EP2018/068448
Publication Date:
January 31, 2019
Filing Date:
July 06, 2018
Export Citation:
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Assignee:
UNIVERSITY OF PLYMOUTH (Drake Circus, Plymouth Devon PL4 8AA, PL4 8AA, GB)
International Classes:
A01N25/00; A01N63/00; A62D3/02; C12N3/00; C12R1/085
Domestic Patent References:
WO2014193746A12014-12-04
WO2017117089A12017-07-06
Foreign References:
US6656919B12003-12-02
Other References:
ANDO Y: "THE GERMINATION REQUIREMENTS OF SPORES OF CLOSTRIDIUM-BOTULINUM TYPE E", JAPANESE JOURNAL OF MICROBIOLOGY, vol. 15, no. 6, 1971, pages 515 - 525, XP009507320, ISSN: 0021-5139, DOI: 10.1111/j.1348-0421.1971.tb00613.x
T.O. OMOTADE ET AL: "The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies", JOURNAL OF APPLIED MICROBIOLOGY., vol. 117, no. 6, 1 December 2014 (2014-12-01), GB, pages 1614 - 1633, XP055498143, ISSN: 1364-5072, DOI: 10.1111/jam.12644
OZGUR CELEBI ET AL: "The Use of Germinants to Potentiate the Sensitivity of Bacillus anthracis Spores to Peracetic Acid", FRONTIERS IN MICROBIOLOGY,, vol. 7, 29 January 2016 (2016-01-29), XP055498157, ISSN: 1664-302X, DOI: 10.3389/fmicb.2016.00018
H. LUU ET AL: "Cooperativity and Interference of Germination Pathways in Bacillus anthracis Spores", JOURNAL OF BACTERIOLOGY, vol. 193, no. 16, 17 June 2011 (2011-06-17), US, pages 4192 - 4198, XP055498164, ISSN: 0021-9193, DOI: 10.1128/JB.05126-11
ALISTAIR H BISHOP: "Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato", 29 September 2017 (2017-09-29), XP055497974, Retrieved from the Internet [retrieved on 20180807], DOI: 10.1128/AEM
Attorney, Agent or Firm:
GREENWOOD, Matthew et al. (BRYERS LLP, 7 Gay Street, Bath Bath and North East Somerset BA1 2PH, BA1 2PH, GB)
Download PDF:
Claims:
CLAIMS

I . A method of chemically inducing germination of bacteria of the group Bacillus cereus sensu lato, comprising the step of administering to a bacterial population a germinant comprising:

inosine;

one or more amino acids; and

mandelate and/or lactate.

A chemical germinant for inducing germination of bacteria of the group Bacillus cereus lato, the germinant comprising:

inosine;

one or more amino acids; and

mandelate and/or lactate.

3. A method or germinant as claimed in claim I or claim 2, in which the germinant includes only mandelate.

4. A method or germinant as claimed in any preceding claim, in which mandelate is used at a concentration in the range 0.5 mM to 1 00m M.

5. A method or germinant as claimed in claim I or claim 2, in which the germinant includes only lactate. 6. A method or germinant as claimed in any preceding claim, in which lactate is used at a concentration in the range 5 mM to 1 00 mM.

7. A method or germinant as claimed in any preceding claim, in which both mandelate and lactate are present in the germinant.

8. A method or germinant as claimed in any preceding claim, in which the amino acid is one or more selected from: L-alanine, histidine, methionine, valine, serine, phenylalanine.

9. A method or germinant as claimed in any preceding claim, in which amino acid is used at a concentration in the range I mM to 1 00 mM.

1 0. A method or germinant as claimed in any preceding claim, in which the germinant is administered at a pH in the range 5-9.

1 1 . A method or germinant as claimed in any preceding claim, in which the pH is approximately 7.

1 2. A method or germinant as claimed in any preceding claim, in which inosine is used at a concentration in the range 0. 1 mM to 1 0 mM. 1 3. A method of counteracting bacteria of the group Bacillus cereus sensu lato, comprising the step of administering a germinant comprising:

inosine;

one or more amino acids; and

mandelate and/or lactate.

14. A method as claimed in claim 1 3, in which the method provides one or more of: decontamination; hazard management; prevention of food contamination; bioterrorism counteraction; biodefence; disinfection; sterilisation; healthcare facility disinfection; and food- handling facility disinfection.

1 5. A method of decontaminating a malicious release of Bacillus anthracis comprising chemically inducing germination by administering a germinant comprising:

inosine;

one or more amino acids; and

- mandelate and/or lactate.

Description:
THE EFFECT OF MANDELATE AND LACTATE ON SPORE GERMINATION

The present invention relates to the potentiating effect of mandelate and lactate on chemically- induced germination of members of Bacillus cereus sensu lato.

Introduction

Bacterial endospores are a highly resistant form into which some bacterial species are able to differentiate ( I ), typically in response to nutrient limitation. These spores are a very long-lived, latent form that can revert to a state capable of growth and multiplication when conditions become favourable. A limited number of bacterial genera are capable of forming spores. Among these are organisms that are of public health interest, for example: Bacillus anthracis is the most important bacterial threat agent and is the causative agent of anthrax; Bacillus cereus is a common agent of food-borne disease and Clostridium difficile can cause severe diarrhoeal illness, even leading to death. Spores are far more difficult to inactivate than other bacteria and also the replicating forms of the same species: this is true of methods using heat, antimicrobial chemicals, desiccation, pressure and radiation.

Due to the high chemical resistance of spores, they are difficult to inactivate by disinfectants and only a few such chemicals are truly sporicidal. For wide-spread decontamination of hospital wards the concept of 'germinate to decontaminate' has been raised (3). This has also been applied as a suggested as means to remove spores of 6. anthracis after a malicious release (4, 5). A further advantage, given the relative ineffectiveness of chemical sporicides in soil, is to exploit the poor persistence of 6. anthracis in soil once germinated (6).

A number of triggers are capable of causing these spores to begin the process of returning to the vegetative form. These include heat shock, high pressure and also a number of chemicals that are somewhat specific to particular species (2). The limited number of chemicals that are capable of triggering germination have been well established for several decades. For members of the Bacillus cereus sensu lato group, which includes 6. anthracis and 6. cereus, L-alanine and inosine are amongst the most powerfully active agents. The mostly widely used experimental model, Bacillus subtilis will, for example, germinate when exposed to a mixture L-asparagine, D-glucose, D-fructose and potassium ions (7). A prior heat shock is required to activate these spores and make them receptive to the chemical

I germinants. The less well-studied Clostridium difficile also requires a heat shock but the chemical germinants appear to be less well defined: glycine and bile salts have been shown to act as co- germinants (8). For some clinical isolates, however, amino acids were insufficient and rich nutrient media were required for germination to occur; bile salts, however, were not required (9).

Members of Bacillus cereus sensu lato ( 10) are interesting in that they do not require a prior heat shock to become induced to germinate by the appropriate chemicals. For Bacillus anthracis and Bacillus thuringiensis a powerful combination of chemical nutrients is L-alanine and inosine ( I I ). The stereo specificity for the amino acid is crucial, with D-alanine acting as an inhibitor ( 1 2). Germinant receptors (GRs) have been identified on the inner membrane of the spore for the specific chemical germinants active on the Bacillus species ( 1 3, 14, 1 5). Each having specificity for one or more compounds, as has been hypothesized for 6. anthracis, for example ( 1 6). This complexity is increased by positive and negative interactions between some of the chemical germinants ( I I ). Other 'non-nutrient' chemicals such as calcium dipicolinate and dodecylamine can trigger germination in both Bacillus and Clostridium species. In the former group this has been shown to be independent of binding to any GRs ( 1 7) but such germinant pathways may be involved for Clostridium species. In the absence of other nutrients, the extent of germination in 6. cereus sensu lato when triggered by chemical germinants halts with the loss of calcium dipicolinate, phase brightness and enhanced resistance to heat and anti-microbial compounds. These features typify the sporulated state. This is an ideal termination stage from an applied point of view because the germinated spores are now much more susceptible to decontamination measures but are not able to replicate and, potentially, worsen the contamination problem.

The limited number of specific chemical germinants, active on bacterial spores has remained unchanged for decades. Woese et al. ( 19) examined the effect on a number of amino acids, focusing also on analogs of L-alanine. More recently, a number of chemicals have been screened for activity as inhibitors of germination (20, 2 1 ). In connection with the present invention a number of chemicals were screened for activity on spores of 6. cereus sensu lato. The activity of two chemicals, mandelate and lactate, on spore germination forms the basis of the present invention and is described herein.

Summary

Two novel chemicals were found to have activity in the germination of these spore species: mandelate and lactate. The former being more active on a molar basis than the latter. Neither were capable of triggering germination on their own: both required inosine and one of a small number of amino acids to be present also. Under these circumstances both mandelate and lactate had a significant potentiating effect on spore germination beyond that of inosine and the amino acid on their own in terms of the rate and extent of germination elicited. This was demonstrated to be clearly dose-dependent for mandelate with a minimum threshold for activity found to be at 0.5 mM, reaching saturation at about 1 00 mM.

According to an aspect of the present invention there is provided a method of chemically inducing germination of bacteria of the group Bacillus cereus sensu lato, comprising the step of administering to a bacterial population a germinant comprising: inosine; one or more amino acids; and mandelate and/or lactate.

A further aspect provides a chemical germinant for inducing germination of bacteria of the group Bacillus cereus sensu lato, the germinant comprising: inosine; one or more amino acids; and mandelate and/or lactate.

In some embodiments the germinant includes only mandelate (i.e. no lactate).

Mandelate (with or without lactate) may be used at a concentration in the range 0.5 mM to l OOm M.

In some embodiment the germinant includes only lactate (i.e. no mandelate).

Lactate (with or without mandelate) may be used at a concentration in the range 5 mM to 1 00 mM.

In some embodiments both mandelate and lactate are present in the germinant.

The amino acid may be one or more selected from: L-alanine, histidine, methionine, valine, serine, phenylalanine.

The amino acid component may be used at a concentration in the range I mM to 1 00 mM.

The germinant may be administered at a pH in the range 5-9; for example approximately 7.

Inosine may be used at a concentration in the range 0. 1 mM to 1 0 mM. Two example of applications of the present invention are related to what is called a 'germinate to decontaminate' strategy. By adding germination-active chemicals spores can be 'tricked' into germination and so losing their enhanced resistance to inactivation. This then allows them to be destroyed by conditions that kill other bacteria. Food-grade forms of both chemicals could be used to help eradicate spores of 6. cereus in the food industry. Similarly, the newly-identified chemicals could be added to cocktails of germinants which would be sprayed over areas that have been contaminated with spores of 6. anthracis. For both applications the addition of mandelate and/or lactate would decrease the amount of the (more expensive) conventional germinant chemicals required and would increase the rate and extent of germination. Furthermore, in the latter example, mandelate would be expected to be less easily degraded by environmental bacteria than the germinants currently proposed.

Neither mandelate nor lactate were active on any Clostridium species. Endospores of the genus Bacillus can be triggered to germinate by a very limited number of chemicals. Mandelate was shown to have a powerful additive effect on the level and rate of germination produced in non-heat shocked spores of 6. anthracis Sterne and 6. thuringiensis using nutrient chemical germinants. It had no germinant effect on its own but was active in a dose-dependent manner at concentrations above 0.5 mM. The rate of germination produced in 6. anthracis spores by 1 00 mM L-alanine with 1 0 mM inosine was equalled by 25% of these germinants when supplemented with 1 0 mM mandelate. The activity was highest around neutral pH values but the addition of mandelate maintained much higher levels of germination between pH values of 5 - 9 when used with L-alanine and inosine compared to these germinants alone. Lactate also had a potentiating effect on germination in the presence of L- alanine and inosine, this was further increased by mandelate. Ammonium ions also enhanced L- alanine and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant; a mutant of 6. cereus lacking the germinant receptor used by phenylalanine did not respond differently from the wild type strain. Binding to spores was implicated for mandelate to exert its additive effect on germination. There was no effect when mandelate was used in conjunction with non- nutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes or C. difficile.

The number of chemicals that can induce germination in the species related to 6. cereus has been defined for many years and they conform to specific groups of chemical. Mandelate has a different structure from these germination-active compounds and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of 6. cereus in food-borne disease and 6. anthracis as a threat agent. The destruction of spores of 6. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of L-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of 'germinate to decontaminate' strategy may, thus, become more practicable.

A further aspect of the present invention provides a method of counteracting bacteria of the group Bacillus cereus sensu lato, comprising the step of administering a germinant comprising: inosine; one or more amino acids; and mandelate and/or lactate.

The method may provide or relate to one or more of: decontamination; hazard management; prevention of food contamination; bioterrorism counteraction; biodefence; disinfection; sterilisation; healthcare facility disinfection; and food-handling facility disinfection.

The present invention also provides a method of decontaminating a malicious release of Bacillus anthracis comprising chemically inducing germination by administering a germinant comprising: inosine; one or more amino acids; and mandelate and/or lactate.

Different aspects and embodiments of the invention may be used separately or together.

Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

The present invention is more particularly shown and described, by way of example, in the accompanying drawings.

The example embodiments are described in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternative forms and should not be construed as limited to the examples set forth herein.

Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail below as examples. There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included. Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealised or overly formal sense unless expressly so defined herein. RESULTS

Potentiating effect of mandelate on spores responsive to alanine and inosine.

The maximal level of germination of 6. anthracis Sterne spores was found at concentrations of about 100 mM L- alanine and 10 mM inosine. Using 15% of these concentrations produced the lowest level of germination shown in Fig. I . This equated to 50% of the spore population becoming phase dark. The addition of 0. 1 mM mandelate had no effect but progressively increasing this concentration to I , 5, and 10 mM mandelate dramatically potentiated the germination response (Fig. I ). This resulted in the proportion of phase dark spores being 70, 80 and 90% of the population, respectively. The minimal concentration of mandelate required under these conditions was 0.5 mM. Mandelate on its own at any concentration had no germinating effect. The increase in germination was proportional to the amount of mandelate added; it had no potentiating effect on the germination induced by L-alanine or inosine separately at any concentration of any of the chemicals.

Fig. I Relative stimulation of germination in 6. anthracis Sterne spores, monitored by Tb-DPA fluorescence. Both (R)-(-)and (S)-(+) enantiomers of mandelic acid and mixtures thereof produced identical results. There was no toxic effect on the germinated spores at pH 7.2, even up to mandelate concentrations of 100 mM.

Key:

Fig. 1 Relative stimulation of germination in B. anthracis Sterne spores, monitored by terbium-dipicolinic acid fluorescence. L-alanine, 100 mM; inosine, 10 mM ; L-alanine, 15 mM; inosine, 1.5 mM; mandelate, 100 mM - ; L-alanine, 15 mM; inosine, 1.5 mM; mandelate, 10 mM X ; L-alanine, 15 mM; inosine, 1.5 mM mandelate, 5 mM '· ; L-alanine, 15 mM; inosine, 1.5 mM; mandelate, 1 mM X ; L-alanine, 15 mM, inosine, 1.5 mM, mandelate, 0.5 mM 4 ; L-alanine, 15 mM; inosine, 1.5 mM mandelate, 0.1 mM - ; L-alanine, 15 mM; inosine, 1.5 mM + . Effect of heat shock. Activating spores by heat shock negated the increase in germination produced by mandelate. This was true of 6. anthracis and, as illustrated in Fig. 2, of Btcry-.

Fig. 2 Response of heat shocked and non-heat shocked spores of Btcry- to L-alanine and inosine, with and without mandelate.

Key:

Fig. 2. Response of heat shocked and non-heat shocked spores of Btcry to L-alanine and inosine, with and without mandelate. L-alanine (10 mM) and inosine (1 mM), no heat shock♦ (10%) ; L-alanine (10 mM) and inosine (1 mM) plus mandelate (5 mM), no heat shock ; L-alanine (10 mM) and inosine (1 mM) plus mandelate (10 mM), no heat shock A ; L-alanine (10 mM) and inosine (1 mM), after heat shock X ; L- alanine (10 mM) and inosine (1 mM) plus mandelate (5 mM) after heat shock ; L-alanine (10 mM) and inosine (1 mM) plus mandelate (10 mM), after heat shock X .

From microscopic examination, the final levels of phase dark spores were, for non-heat shocked spores: L-alanine and inosine alone, 10%; germinants with 5 mM mandelate, 40% and germinants with 10 mM mandelate, 70%. After heat shock, all of the spore preparations were over 95% phase dark.

Interactions with lactate and ammonium ions. Mandelate was not the only compound found to potentiate the triggering effect of L-alanine and inosine. Lactate, while ineffective on its own, was also able to increase the germinating effect of the germinants (Fig. 3). Its effect was not as marked as mandelate, however, because, for a given concentration of L-alanine and inosine, the addition of 10 mM mandelate produced a greater level of germination than the addition of 25 mM lactate. When mandelate and lactate were added together to the germinants they produced an additive effect, resulting in the greatest rate and extent of germination (Fig. 3). Surprisingly, D-and L- forms of lactate had identical effects and combinations of the two were additive.

The same stimulation of germination induced by L-alanine and inosine with mandelate, ammonium ions and lactate was observed in Btcry- although it was less pronounced than in 6. anthracis (Fig. 3). As with 6. anthracis, lactate had no effect alone with the germinants but had an additive effect in the presence of mandelate.

Ammonium ions were found to promote the stimulation by mandelate of alanine/ inosine- induced germination. It is noteworthy that they did not increase spore germination in the absence of mandelate (Fig. 3). The inclusion of all three stimulants had a small but reproducible promotion of germination beyond the combination of mandelate and ammonium ions or mandelate and lactate. FIG 3 Germination of spores of 6. anthracis Sterne in the presence of L-alanine ( 1 0 mM) and inosine ( I mM) with and without supplementation by mandelate, lactate and ammonium ions. Key:

Fig. 3 Germination of spores of B. anthracis Sterne in the presence of L-alanine (10 mM) and inosine (1 mM) with and without supplementation by mandelate, lactate and ammonium ions. With NhUCI (25mM) fl ; L- alanine (10 mM), inosine (1 mM) alone A ; with lactate (25mM)♦ ; with mandelate (5 mM) X ; with mandelate (5 mM) and NhUCI (25mM) X ; with mandelate (5 mM) and lactate (25 mM) ; with mandelate (5 mM), lactate (25 mM) and NhUCI (25mM) + .

Kinetics of the mandelate and lactate effects. A double reciprocal plot of the rate of germination of Btcry- spores over 1 0 min in varying concentrations of mandelate and of L- alanine and inosine was constructed (data not shown). The lines do not intersect at a single point, indicating that mandelate does not have to be at its putative receptor at the same time as L-alanine and inosine are at theirs (20). There is an almost doubling of affinity of the spores for mandelate over a four-fold range in L-alanine and inosine concentration, indicating a degree of co-operativity between the germinants and the adjuvant. The apparent germination V max increases with increasing concentration of L-alanine plus inosine and indicates that mandelate binds at a different site. The apparent V max and K m values are shown in Table I .

Table I . Apparent V max and K m values for 20 mM mandelate with different concentrations of L- alanine and inosine for spores of Btcry-. 95% confidence intervals are shown in parentheses. The interactions of lactate with L-alanine and inosine were much more complex. Linear relationship between the rate of germination and the concentration of lactate for given concentrations of L-alanine and inosine were not observed. No deductions about the interactions between these germinant chemicals were, therefore, possible.

The maximum rate of decrease in optical density produced in 6. anthracis Sterne spores with L- alanine ( 100 mM) and inosine ( 1 0 mM) was 0.0054 OD units/ min. Half of this rate, termed C50, was produced by 1 5% of this concentration of both germinants was supplemented with 0.8 mM mandelate. Equally, if only 2.5% of both germinants was used, the C50 value was restored by the addition of 1 00 mM mandelate. The maximal rate of germination was produced by 1 5% of the optimal germinant concentration by the addition of 1 0 mM mandelate.

Receptor binding seems to be involved in mandelate. Two approaches were taken to demonstrate that mandelate binds to spores in order to stimulate L-alanine/ inosine induced germination. Pre-incubation of spores of either 6. anthracis Sterne or 6. thuringiensis cry- in mandelate (2 mM) followed by centrifugation and resuspension in buffer produced the same rate and extent of germination in L-alanine/ inosine as spores incubated throughout in 2 mM mandelate with these germinants. Similarly, spores that had been pre-incubated as above but where the mandelate was then diluted to a concentration of 0.02 mM (a non-active concentration, Fig. I ) with a solution of L-alanine and inosine again produced an identical germination response to those where the concentration of mandelate was 2 mM throughout the assay (data not shown).

Interaction with other amino acids. Phenylalanine has a powerful effect on germination in 6. anthracis in combination with inosine, acting through gerS ( 1 6). Given the structural similarity between this amino acid and mandelate it was considered possible that the same germination receptor was used.

When added separately and in combination with sub-maximal levels of L-alanine plus inosine to Btcry- it was evident that there was no competition but rather an additive effect of mandelate and phenylalanine (Fig. 4). This was true when saturating levels ( 1 00 mM) of both were used.

FIG 4 Interactions of mandelate and phenylalanine in combination with the germinants L- alanine ( 1 5 mM) and inosine ( 1 .5 mM). Key:

Fig. 4 Interactions of mandelate and phenylalanine in combination with the germinants L-alanine (15 mM) and inosine (1.5 mM). Non-heat shocked Bt cry ' spores were incubated at 25 °C with the germinants L-alanine and inosine alone ; germinants with 20 mM phenylalanine X ; germinants with 10 mM mandelate · ;

germinants with 100 mM phenylalanine ; germinants with 100 mM phenylalanine and 10 mM mandelate H ; germinants with 100 mM mandelate♦ and germinants with 100 mM phenylalanine and 100 mM mandelate The error bars represent standard deviation.

Positive and negative interactions have been identified between some of the amino acids that can contribute to spore germination ( I I , 1 6). Mandelate had an additive effect for all of the combinations with amino acids tested (Table 2). Surprisingly, the negative interaction between methionine and valine ( I I ) (Table 2) appeared to be relieved when mandelate was added. An alternative interpretation is that it simply exerted an additive effect with one or both of the amino acids present.

Germinant combination Percentage

germination

Inosine 3.1 (0.9)

Inosine + histidine 30.4 (3.6)

Inosine + histidine + mandelate 94.8 (3.4)

Inosine + methionine + valine 46.8 (3.3)

Inosine + methionine + valine + 76.4 (4.1)

mandelate

inosine + alanine 41.3 (3.2)

lnosine+ alanine + mandelate 98.2 (3.1)

Inosine + serine 35.8 (2.7)

Inosine + serine + mandelate 94.8 (3.0)

Inosine + valine 76.4 (4.4)

Inosine + valine + mandelate 94.8 (3.1)

Inosine + methionine 35.8 (2.8)

Inosine + phenylalanine 76.4 (4.6)

Inosine + phenylalanine + 94.8 (2.9)

mandelate

Table 2. Percentage germination as judged by microscopic evaluation of phase dark spores after 20 minutes at 25°C. Standard deviation is shown in parentheses after the mean values [29/ 1 1 / 1 6 and 1 /2/ 1 7].

Dependence on pH value. The optimum pH value for germination with L-alanine and inosine using non-heat shocked spores of Btcry- spores was around pH 7.0 (Fig. 5). The same was true when mandelate (25 mM) was added but higher rates of germination were evident and were also maintained over a broad range of pH values. When heat shocked spores were used, this difference disappeared, as shown in Fig. 2, and near complete germination across the pH range was observed in all case.

FIG 5 Dependence on pH value of germination of non-heat-shocked Btcry spores after incubation at 25°C for 20 min. The germinant in each case was 1 0 mM L-alanine plus I mM inosine: these germinants alone; these germinants with mandelate 25 mM).

Key:

FIG 5 Dependence on pH value of germination of non-heat-shocked Btcry spores after incubation at 25°C for 20 min. The germinant in each case was 10 mM L-alanine plus 1 mM inosine: UJ these germinants alone; S these germinants with mandelate 25 mM).

Effect of mandelate on 6. subtilis, B. atrop aeus and Clostridium, spp. Spores of all of these bacteria had to be heat-shocked for any appreciable germination to occur. With all of the strains used there was no difference in the rate or extent of germination using mandelate. There was no effect on heat-shocked spores.

DISCUSSION

Other screening programs may have been under-taken but, particularly if they have been unsuccessful, have not been reported. The discovery of mandelate as a compound active in the germination of some Bacillus species opens up a new line of investigation in the fundamental biology of spore germination. It is not a 'nutrient germinant' such as the purines and amino acids, nor is it a spore constituent like calcium dipicolinate. Unlike this and the other well- known non-nutrient germinant, dodecylamine, mandelate does seem to bind to the spore and interact with the germinants that are required for its activity to be apparent. The GR used by aromatic acids in the 6. cereus group does not, however, seem to be used by mandelate: there is a lack of competition with phenylalanine and the normal response to mandelate of the IA5 mutant of 6. cereus, which lacks this receptor, argue against its involvement. Having a different chemical structure to the other known germination- active chemicals increases the possibility that other such chemicals may exist.

The mechanism of action of mandelate is unknown. The identity and specificity of any putative GR for mandelate has not been investigated. Mandelate had a potentiating effect on spore germination with all of the amino acids tested, when inosine was present (Table 2). This, perhaps, argues against it operating through the GRs used by these amino acids ( 1 6, 1 9). There is a precise requirement for the L- isomer of amino acids. It was surprising that the R- and S-

I I stereoisomers of mandelate were equally active. Related chemicals like mandelonitrile were shown in the screening program to have no activity. Moreover, methyl anthranilate was found to have an inhibitory effect on L-alanine-induced germination of 6. subtilis (22). As found by Woese et al. ( 19) lactate alone was ineffective at triggering spore germination. When combined with L-alanine and inosine there was a potentiating effect on germination (Fig. 2) although it required a higher concentration than mandelate to achieve the same effect. Lactate has a similar molecular structure to alanine and, conceivably, operates by interaction with the GR that recognises the amino acid. It is important to note, however, that lactate has no activity in the absence L-alanine and/or inosine. Pyruvate also has a similar structure but was found to be ineffective, indicating a degree of molecular selectivity by the GR if, indeed, that is the mechanism. It is surprisingly that both stereo-isomers of mandelate and lactate had equal effect on promoting germination. Given the structural similarity it might be surmised that lactate causes its effect through interaction with the alanine GR. If this were so the acute specificity that is shown for the D- and L- forms of the amino acid is completely absent with respect to lactate. Similar to the findings here it has been shown that L-lactate, while not capable of inducing germination on its own, increased the rate and extent of germination in C. botulinum in the presence of L-alanine and also some other amino acids (23). It is of interest that this effect was, as shown here, irrespective of the stereoisomer used while there was an absolute requirement for the L- form of alanine. Lactate has, however, been shown to have an inhibitory effect on the germination of spores of C. perfringens (24).

Ammonium ions have previously been reported to have a stimulatory effect on B. cereus germination using I mM L-alanine (25). This finding was not reproduced here and even the presence of much higher concentrations of L-alanine and inosine (25 mM and 2.5 mM, respectively) did not benefit from the addition of ammonium ions (25 mM). This combination, when supplemented with mandelate, however, produced a much higher level of germination in β. anthracis spores than with L-alanine, inosine and mandelate alone (Fig. 3). Ammonium ions were found to stimulate the germinating effect of L-alanine and inosine (7) in one strain of 6. cereus but it was reported to be inhibitory in another (26). The mechanism for this is unknown and has not yet been explored further.

Mandelate has never previously been associated with bacterial spore germination. Mandelic acid is known for its antibacterial effects at acidic pH values (27, 28) and as a mild exfoliant cosmetic (29) and in the treatment of certain dermatological conditions such as inflammation. The (R)-form is a key intermediate in the production of semi-synthetic penicillins and cephalosporins (30). It also has a long history of usage by oral dosage as a derivative of methenamine (3 1 ) for persistent urinary tract infections. It is, therefore, conceivable that mandelate could be used in the food industry to increase the germination of 6. cereus spores prior to inactivation. Btcry- was used in this study to represent 6. cereus for this reason and because it has been used as a simulant for 6. anthracis (32, 33).

Another area of applied relevance for this work is in the decontamination of 6. anthracis. To achieve a 'germinate to decontaminate' regime for 6. anthracis over a wide area would require large amounts of L-alanine and inosine. Furthermore, the outdoor application of these nutrients might be hampered by their being readily metabolised by soil micro-organisms. The application of concentrated solutions of L-alanine and inosine were successful in the laboratory at promoting the 'self-decontamination' by microcosms of B. anthracis spores. The logistics and effectiveness of transferring this to the field have yet to be demonstrated. The data presented herein show that the addition of mandelate to L-alanine and inosine would greatly decrease the requirement for these chemicals to achieve the same level of germination. This could either mean less of the latter chemicals would be needed in the germinant cocktail or, the efficacy of the cocktail could be maintained as they were utilised by soil micro-organisms. Mandelate is not a conventional nutrient of micro-organisms although, of course, it is subject to degradation by certain micro-organisms (35, 36). Given the restricted presence of the mandelate racemase degradation pathway it would be assumed that mandelate would have a greater persistence in the environment than the nutrient germinants.

When mandelate and lactate are used together a synergistic effect is observed. MATERIALS AND METHODS

Spore production. The wild type strain of 6. cereus 569 (UM20. 1 ) and its gerlA5 mutant (AM 1 3 14) were obtained from Prof. Anne Moir (University of Sheffield, U.K.). Spores of these strains and 6. anthracis Sterne, B. thuringiensis subsp. kurstaki HD- I cry- ('Btcry '), B. atrophaeus NCTC 1 0073 and 6. subtilis ATCC 55405 and 1 33 were produced and washed as previously described (33). C. difficile strains 1 634 and 1 8 1 3 were a gift from Prof. Les Baillie (University of Cardiff, U.K.) and strain 1 3566 was purchased from NCTC (Salisbury, U.K.) and were grown and purified according to the methods of Edwards and McBride. Clostridium sporogenes strain 70 1 792 was purchased from NCIMB (Aberdeen, U.K.) and was grown in anaerobic jars on reinforced Clostridium medium (Oxoid, Basingstoke, U.K.) vegetative cells were scraped from the plates and used to inoculate the sporulation medium of Yang et al. (39). The harvesting and washing of spores was as described above (Edwards and McBride). All spores were stored in sterile distilled water at 4°C for up to three months. The heat shock treatments used were 70°C for 30 min. for 6. anthracis, Btcry and 6. subtilis while 80°C for 1 0 min. was used for C. sporogenes and C. difficile. All heat-shocked spores were stored on ice and used within 8 h.

Germinants. The standard germinant mix used for 6. anthracis and Btcry- was inosine ( 1 0 mM) and L-alanine ( 100 mM) in phosphate buffer, pH 7.2 (50 mM). Other pH values were obtained using acetate buffer (pH 5.0); phosphate buffer (pH 6-8) and CHES (pH 9.0) all at 50 mM final concentration. The germinants used for 6. subtilis were D-glucose ( 1 0 mM), D-fructose ( 10 mM) and potassium chloride ( 10 mM), termed 'GF ' solution, with and without supplementation with L-valine (2 mM) and L-asparagine (2 mM). Stock solution of mandelic acid (0.5M and 0. 1 M) were adjusted to pH 7.2 with sodium hydroxide solution. For C. sporogenes the germinants used were L-alanine (50 mM), L-lactate (25 mM) and sodium bicarbonate (25 mM) in 25 mM Tris, pH 7.4. Spores of C. difficile were germinated in sodium taurocholate ( 1 0 mM) and L-glycine (50 mM) in 25 mM Tris, pH 7.4 or Brain Heart Infusion broth (Oxoid, Basingstoke, U.K.) with and without sodium taurocholate ( 1 0 mM). Dodecylamine was used at concentrations between I and 1 0 mM. Calcium dipicolinate was used at a concentration of 60 mM. All chemicals were obtained from Sigma Aldrich (Gillingham, U.K.). Mandelic acid was also purchased from Fisher Scientific (Loughborough, U.K.) and Organics Merck Millipore (Watford, UK). Germination assays. At least two separate preparations of spores were used to derive the data presented. Triplicate readings were taken for all data points. Germination assays were assessed in 96 well microtitre plates and the decrease in absorbance at 595 nm measured in a plate reader (Tecan, Mannedorf, Switzerland). For members of the 6. cereus group all assays were carried out at 25°C. For other bacteria the germination temperature was 37°C. Released spore DPA was measured by measuring its fluorescence with Tb 3+ as previously described (40). The extent of germination was also monitored at the end of all experiments by the examination of over 1 00 spores by phase-contrast microscopy. D-cycloserine (5 g/ml) was initially incorporated into assays but alanine racemase was not found to be a confounding factor so its inhibitor was subsequently omitted.

Receptor binding. To demonstrate that binding of mandelate to spores is involved in its stimulatory effect of alanine-inosine-induced germination two approaches were used. First, spores were incubated in mandelate (2 mM) in 50 mM phosphate buffer, pH 7.2 for 1 0 min at 25°C and then centrifuged ( 1 3,000 x g for 5 min). The supernatant was removed and the spores were resuspended in phosphate buffer. They were then added to a solution of L-alanine (20 mM) and inosine (2 mM). The rate and extent of germination was then compared to spores that had not been pre-incubated in mandelate but were in L-alanine (20mM) and inosine (2 mM) in phosphate buffer, with and without mandelate (2 mM). Alternatively, spores were incubated for 10 min at 25°C in mandelate (2 mM) in 50 mM phosphate buffer, pH 7.2. This suspension was then diluted 1 / 100 with L-alanine (20mM) and inosine (2 mM) in phosphate buffer. Germination was then monitored in comparison with the positive and negative controls used above.

Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

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