Journal of Applied Bacteriology 1987,62,269-213 24 1 8/ 10/86

Factors affecting the germination of spores of Bacillus anthracis

R . W . TITBALL & R . J . MANCHEE Chemical Defence Establishment, Porton Down, Salisbury SP4 OJQ, U K

Received 6 October 1986 and accepted 20 November 1986

TITBALL, R.W. & MANCHEE, R.J. 1987. Factors affecting the germination of spores of Bacillus anthracis. Journal of Applied Bacteriology 62,269-273.

Spores of Bacillus anthracis germinated poorly at high cell densities unless the alanine racemase inhibitor 0-carbamyl-D-senne was added to the germination medium. Spores derived from a variety of strains of B. anthracis germinated opti- mally at 22°C. No correlation was found between rate of spore germination and virulence or between susceptibility of animal species to anthrax and spore germi- nation rate using sera from those animals as the germination medium.

Bacillus anthracis has been recognized as the aetiological agent of anthrax for over 100 years. In the 1800s the disease caused widespread losses of livestock in Europe (Whitford 1979) and even today significant numbers of animals in Third World countries die as a result of the disease. The pathogenesis of anthrax is still only partially understood. Usually the disease occurs as a cutaneous infection (Borts 1972) or more rarely as pulmonary (Laforce et al. 1969) or intestinal forms (Borts 1972) according to the route of entry of the infective spores. Germi- nation of the spore is an essential pre-requisite for growth of the organism and establishment of the disease. The bacterium then replicates rapidly, entering the bloodstream and becoming disseminated to various organs (Keppie et al. 1955). Ingestion of the bacterium by host phagocytes is limited because of the production of a poly-glutamic acid capsule (Sterne 1937). Death eventually occurs as a result of the action of a tri-partite toxin produced by the bacterium (Smith & Keppie 1954).

We set out to examine some germination characteristics of the anthrax spore. It is known that L-alanine, tyrosine and adenosine can induce spore germination in uitro (Hills 1949, 1950), and we report here the effect of other environmental factors which may be relevant to germination in uivo.

Materials and Methods

PRODUCTION OF S P O R E S

A selection of virulent and attenuated strains of B. anthracis was used (Table 1). The bacteria were normally grown on the surface of new sporulation agar in Roux bottles (Williams et al. 1979). After incubation at 37°C for 72 h the spores were harvested by washing them from

Table 1. Virulence of the strains of Bacillus anthracis used

Toxin Capsule Strain Virulence production possession

Vellum + + + M36 + + + ATCC 944 + + + Ames + + + 107 + + + NH6 + + + Penicillin

resistant + + + Sterne -

BI - LVS - ST-I - d-Vollum - 6-Ames - &Sterne -

- + + + +

- - - + +

- - - -

2 70 R. W . Titball and R. J . Manchee the surface growth with 25 ml of sterile distilled water (SDW) per bottle and heating the suspen- sion at 60'C for 60 min to kill vegetative cells and heat shock the spores. Strains B1; ST-I and ATCC 944 sporulated very poorly on this medium and these strains were grown on nutri- ent agar containing 0.01% MnSO, which improved spore yields. The spores were har- vested by washing the suspension five times with SDW. once with 0.03 mol/l HCl and four times with SDW. with centrifugation at 4000 g for 20 min at 4'C. The purified spore suspen- sions were diluted to contain 2 x lo8 viable spores 'ml before use unless otherwise stated.

Results

E F F E C T O F M E D I A O N SPORE G E R M I N A T I O N

The germination medium was an important factor influencing the germination of spores of the virulent Vollum strain of B. anthracis (Table 2). A small proportion became heat-

Table 2. Germination of Bacillus anthracis Vollum strain spores in a selection of liquid

media

Percentage Germinating medium germination

G E R M I N A T I O N M E D I A

L-Alanine or D-alanine (Sigma) were dissolved in 20 mmolil phosphate buffer at p H 7.1 before use. 0-Carbamyl-D-serine (OCDS) was kindly donated by the International Minerals and Chemical Corporation (Terre Haute, USA). Nutrient broth (Oxoid) was prepared as directed by the manufacturer.

A S S E S S M E N T OF S P O R E G E R M I S A T I O N B Y

O P T I C A L D E N S I T Y C H A N G E

Spores were normally mixed with an equal volume of germination medium and incubated in a waterbath. The optical density of spore sus- pensions was determined at 450 nm, a wave- length which was found to be the most sensitive for detecting the increase of light transmission through a suspension of spores as they germi- nate.

ASSESSMENT O F SPORE G E R M I N A T I O N BY C H A N G E IN H E A T S E N S I T I V I T Y

Spore suspension (0.1 ml) was mixed with ger- mination medium (0.9 ml) and then, at pre- determined intervals, 0.1 ml volumes of this mixture were dropped into 9.9 ml volumes of SDW containing 0.1% Tween 80 at 60T, and held at this temperature for 60 min to kill any spores rendered heat-sensitive by the initial stages of germination. Samples of the suspen- sion (0.25 ml) were plated on nutrient agar and incubated at 37'C for 24 h before the colonies were counted. In some experiments, colonies were also counted after incubation for 48 h.

Sterile distilled water I Phosphate buffer

(10 mmolil, pH 7.1) 9 Phosphate buffer + L-alanine 22 Nutrient broth 18 Rabbit serum I Phosphate buffer + L-alanine

+ OCDS 65 Nutrient broth + OCDS 96 Rabbit serum + OCDS 95 Phosphate buffer + D-alanine 14 Phosphate buffer + OCDS 11 Phosphate buffer + o-alanine

+ OCDS 13

Spores (1.3 x 10s/ml final concentration) were incubated with germinating solution for 90 min at 21'C and the proportion of spores germinated was determined by measuring their loss of heat resistance.

OCDS, 0-Carbamyl-D-serine.

sensitive when mixed with distilled water (7%) or phosphate buffer (9%). In an L-alanine solu- tion 22% of the spores germinated within 90 min and limited germination was also observed in nutrient broth or rabbit serum. The addition to L-alanine solution of OCDS resulted in a marked increase in the proportion of spores that germinated within 90 min. D-Alanine or OCDS alone did not promote spore germi- nation. Spores from the attenuated Sterne strain of B. anthracis were also found to germinate poorly in L-alanine solution but germination was enhanced by the addition of OCDS.

We examined the effect of the concentrations of OCDS and L-alanine plus OCDS on the rate of spore germination. An increase in OCDS concentration up to 6 mmol/l produced a corre- sponding increase in the rate of germination, but higher concentrations (up to 12.5 mmol/l) had virtually no further effect.

Bacillus anthracis germination 27 1 0.3

E g 0.2 *

Time (min)

Fig. 1. The effect of ~-alanine concentration and 0- carbamyh-serine (OCDS) on the germination of Bacillus anthracis spores. Spores of B. anthracis (Vellum strain, 1.2 x W / m l final concentration) were incubated at 24°C with phosphate buffer (10 mmol/l, pH 7.1) containing OCDS (6 mmol/l) and ~-alanine: 0, 150 rnmol/l; 0, 15 mrnol/l; ., 1.5 mmol/l; 0, 0.05 mmol/l; A, no ~-alanine; A, no L-alanine or OCDS.

The effect of L-alanine concentration on the rate of germination was determined by incu- bating spores with various concentrations of L- alanine in phosphate buffer plus OCDS (6 mmol/l) (Fig. 1). After 24 h incubation, over 99% of the spore population incubated with all but the lowest concentration of L-alanine (0.05 mmol/l) had germinated.

T H E EFFECT OF S P O R E D E N S I T Y A N D

T E M P E R A T U R E O N G E R M I N A T I O N

Spore density had a profound effect on germi- nation. At a final concentration of 1.4 x 10' spores/ml, only 27% of spores incubated with 75 mmol/l L-alanine germinated within 90 min. At spore densities of 1.4 x lo5 or 1.4 x lo3 spores/ml, 61% or 63% respectively of the spores germinated in 90 min.

In a medium containing 75 mmol/l L-alanine plus 6 mmol/l OCDS maximum germination was observed at 22°C. At 37°C the rate of ger- mination was reduced considerably (Fig. 2). At 9°C germination proceeded at a low level.

100

00

6

5 s?

c 60 c

D 40

20

0 I I I I I I 1 13 17 21 25 29 33 37

Temperature ("C)

Fig. 2. The effect of temperature on the germination of Bacillus anthracis spores. Spores (Vollum strain, 1 x 108/ml final concentration) were incubated with L-alanine (75 mmol/l) and 0-carbamyl-o-serine (6 mmol/l) in phosphate buffer (10 mmol/l, pH 7.1) and the change in optical density of the suspension determined. 0, 60 rnin incubation; A, 120 min incu- bation.

R E L A T I O N S H I P OF G E R M I N A T I O N RATE TO

V I R U L E N C E OF B. anthracis

A selection of strains of B. anthracis o f known virulence was grown on nutrient agar + O~OlO/o MnSO, until they sporulated. The spores were harvested and washed as described above. Sus- pensions of these spores (0.1 ml, 8 x lo5 spores/ ml) were incubated with 0.9 ml of nutrient broth and the numbers of heat-resistant spores determined at intervals up to 120 min. The results indicated that over 90% of the spores of any strain germinated within 60 min, and over 98% of the spores germinated within 120 min. This experiment was repeated using sheep or mouse serum as the germinant. In sheep serum, over 90% of the spores of any strain germinated within 15 min. In mouse serum at least 70% of the spores germinated within the same time. In both cases little difference in germination rate between strains was observed because germi- nation was so rapid.

The rate of germination of Vollum strain spores in a variety of animal sera was measured in two ways. Firstly, spores, at a final concentra- tion of 8 x 104/ml, were incubated in serum at 37°C and the number of heat-resistant spores remaining at various times was determined. Large variations in the germination rates were

272 R. W . Titball and R . J . Manchee Table 3. Germination rates of spores and alanine

levels in sera

Initial Animal germination Serum Serum species rate alanine tyrosine serum (spores min ml) (mmol,']) (mmolil)

Foetal bovine 1.28 x lo4 0.35 0.03 Human 1.25 x lo4 0.19 0.04 Goat 1.15 x lo4 0.08 0.03

Rat 0.96 x lo4 0.20 0.08 Sheep 0.78 x lo4 0.01 0.03 Dog 0.37 x lo4 0.16 0.02 Mouse 0.26 x lo4 0.18 0.04 Rabbit 0* 0.14 0.06 Horse O* 0.11 0.07 Guinea pig 0 0.13 0.02

Pig 1.1 x 104 0.27 o w

* Germination detected after 15 min.

observed (Table 3). Germination was most rapid in human or foetal bovine serum and slowest in guinea pig serum. Similar results were obtained using higher spore concentra- tions (1 x 1O8/ml) and measuring optical density to indicate the rate of germination in serum + OCDS (6 mmol/l). The levels of the amino acids tyrosine and alanine in the sera tested were determined using a Technicon amino acid analyser.

Discussion

The amino acid L-alanine has been reported to be an effective germination inducer for spores derived from many Bacillus species (Wolf & Thornley 1957; Thornley & Wolf 1961; Gould & Dring 1972). Our results show that only limited germination of B. anthracis spores occurred in L-alanine solution, nutrient broth or serum. A similar result has been reported for the related bacterium B. cereus and in this case L- alanine-induced germination could be stimu- lated by OCDS, an analogue of D-alanine which binds to the enzyme alanine racemase (Gould 1966). The resultant enzyme inactivation pre- vents conversion of L-alanine to the generation inhibitor D-alanine by spore racemase. When OCDS was added to our media complete germi- nation of B. anthracis spores occurred, indicat- ing that these spores probably possess alanine racemase activity. Our observation that OCDS stimulated germination in nutrient broth or serum indicates that L-alanine is involved in germination in these media.

At low concentrations of spores, germination

can proceed in the absence of OCDS supporting the suggestion of Hills (1949) that a greater pro- portion of spores germinate as the density is decreased. Similar results have been reported for B. cereus by Vas & Proszt (1957). It is apparent that high densities of spores contain sufficient alanine racemase to cause rapid formation of D-

alanine, which inhibits germination at an early stage.

Alanine racemase is known to be essential for the formation of D-alanine, and its subsequent incorporation into cell wall peptidoglycan of other Bacillus species (Berberich et al. 1968; Buxton & Ward 1980). Our results suggest that this enzyme may also represent a mechanism controlling the germination of high concentra- tions of spores. This may be important for ensuring that, even if spore germination does occur in environments such as soil (Van Ness 1971), a reservoir of spores is still maintained.

Surprisingly, spores of B. anthracis, a potent pathogen of homeothermic animals, germinated optimally at 22°C whereas spores of the closely related soil bacterium and low grade patho- gen, B. cereus, germinated best a t 30"-32"C (OConnor & Halvorson 1961; Thornley & Wolf 1961). Spores of B. megaterium or B. licheniformis have also been reported to germi- nate optimally at higher temperatures (Thornley & Wolf 1961). The implications of the low optimal germination temperature for spores of B. anthracis are not known, but it may be important for the establishment of cutaneous infection.

The detailed kinetics of germination of spores of B. anthracis following entry into a host are unknown. The possibility that speed of germi- nation might influence the course of infection has been considered. Our results, however, indi- cate that differences in the rates of spore germi- nation between strains are small and d o not correlate with virulence of the parent strain. The number of spores which are required to estab- lish disease in a susceptible host is generally less than 10 (Lincoln et al. 1967; Whitford 1979) and our results suggest that at this concentration germination will proceed very quickly with any of the strains investigated.

Hachisuka (1969) suggested that the ability of a bacterium to germinate and grow in uiuo could explain the natural innate resistance of some animal species to anthrax. These conclu- sions were based on results indicating that

Bacillus anthracis germination 213

spores germinated rapidly in serum from the mouse-an animal which is very susceptible to anthrax, but germination was slow in serum from the rat, which is known to be difficult to infect with B. anthracis.

Our results do not support this proposal. For example, the pig, rat, dog and man are all con- sidered to be relatively resistant to infection with B. anthracis (Lincoln e t al. 1967; Whitford 1979) but we found that germination of the spores was not slowest in these sera as would be predicted from Hachisuka’s hypothesis. Lincoln et al. (1967) also failed to correlate spore germi- nation within phagocytes with susceptibility of an animal to anthrax.

Germination of B. anthracis spores is known to be triggered by L-alanine, and tyrosine may also he an additional requirement (Hills 1949, 1950). Although L-alanine appears to be the major germination-inducing chemical in serum, our results suggest that there are other factors which also influence the rate of spore germi- nation.

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