Growth of Streptococcus mutans in a chemostat

GROWTH

OF STREPTOCOCCUS

MUTANS

IN A CHEMOSTAT

D. C. ELLWWD, J. R. HUNTER and V. M. C. LONGYEAR Microbiological Research Establishment, Porton, Near Salisbury, Wiltshire, England Summary-A strain of Streptococcus mutans isolated from a carious lesion of a patient and known to cause caries in hamsters and monkeys were grown in a complex medium in a chemostat. There were changes in the ability of the organism to stick to surfaces, glucose utilization, and acid production of the organism when grown at different dilution rates.

INTRODUCTION There is now a great deal of evidence that certain strains of streptococci isolated from human carious lesions cause caries in gnotobiotic rats (Orland et al., 1955; Fitzgerald, Jordan and Stanley 1960), hamsters (Krasse. 1966) and in monkeys (Bowen. 1969). These streptococci, mainly Streptococws mutans, can be isolated often in high yield from dental plaque of patients (Car&son, 1967) and experimental animals (Krasse and Edwardsson, 1966). The growth characteristics of Strep. mutans have been studied by Tanzer, Wood and Krichevsky (1969) who showed that the organisms grow linearly, as a plaque-type deposit on a glass rod, when incubated in a sucrose-containing medium, whereas in media containing other sugars no deposit is formed, and the organisms grow exponentially. Washed Strep. mutuns, harvested from the stationary phase in batch cultures, catabolize glucose to lactic acid in 80 per cent yield (Tanzer, Krichevsky and Keys, 1969). These organisms can produce sufficient acid to cause demineralization of the tooth surface in the presence of glucose and this suggests a mechanism by which tooth decay might be caused. The study of Strep. mutans in a chemostat with growth-limiting substrates may be useful in understanding the behaviour of organisms which undergo quite marked changes in nutritional levels in vivo, as food is ingested into the oral cavity at intervals. This paper describes the growth of Strep. mutans in a chemostat, in a complex medium using glucose as the growth limiting substrate. MATERIALSAND

METHODS

Organism Strep. mutans strain Ingbritt re-isolated from a carious lesion in a monkey was obtained from Dr. W. H. Bowen, and maintained in tryptone-yeast-sucrose broth and on mitis-salivarius agar (Difco formula). Growth conditions The culture was grown in a Porton chemostat (Herbert, Phipps and Tempest, 1965) of 500ml working capacity. The temperature was maintained at 37”C,

and the pH was automatically controlled at 6.5 k 0.1. Foaming proved not to be a problem and was easily suppressed by automatic hourly additions of 0.1 ml of Silicone RD (Hopkins and Williams. Chadwell Heath, Essex, England). The culture was stirred by an externally driven impeller, and anaerobic conditions were maintained by passing a metered flow of 5 per cent CO1 : 95 per cent NZ into the reactor at 500 ml/min of which 350 ml entered the reactor down the hollow impeller shaft; 150 ml were used to sweep aerosol from the medium input port. thus preventing growth-back into the medium line. The gas mixture was sterilized by passing through sterilecotton wool filters. Medium was pumped into the reactor at the appropriate dilution rate (proportion of culture volume replaced/hr) by a Miniature Flow Inducer (Watson-Marlow. Marlow, Buckinghamshire, England) and the culture allowed to reach equilibrium for at least ten mean generation times, at each dilution rate before being assayed. The culture medium was similar to that devised by Cybulska and Pakula (1963) modified by using yeast extract powder rather than yeast cells. Two-hundred g of Oxoid L37 peptone dissolved in I litre of deionized waterwasdialyzedthree timesagainst2litreofdeionized water for 24 hr at 4°C. yielding 4 litre of diffusate. Twohundred and fifty g Oxoid L21 yeast extract powder dissolved in 800ml deionized water was treated in a similar way. Nine point five 1. of each diffusate were combined, 900g Casamino Acids (Difco Detroit, Michigan, U.S.A.) and 60 g K,HP04 were dissolved in the mixture and the whole sterilized by autoclaving (121“C. 30 min). Two-hundred g glucose dissolved in 1 litre of deionized water was sterilized by autoclaving in a separate vessel and added aseptically to the bulk. Determination ofthe gerieration time in batch conditions The reactor was loaded with 500 ml of medium and brought to 37°C. After inoculation with 20ml of a heavy suspension of organisms from the bottom of an 18 hr seed culture. growth was monitored for optical density (O.D.) and microscopically over 12 hr while being stirred as a batch culture (i.e. medium was not being pumped in). At the end of this time, the medium 659

6hO

D. C. Ellwood.

J. K. Hunter

and V. M. C. Longyear

pump was switched on at the lowest How rate and the culture allowed to equilibrate under continuous conditions.

3or

The O.D. of the culture was measured in a Spectronic ~Ospcctrophotometer (Bausch & Lomb. Rochcstar. NY. U.S.A.). at 540 nm after I :20 dilution in physiological saline. Bacterial dry weights were determined by taking duplicate 5 ml samples of killed culture (IO per cent formalin by vol. for I hr), centrifuging, wlishing the cells once in distilled water, and drying the washed deposit of cells at 105’C for 18 hr. Culture samples were examined daily by phase-contrast microscopq and were plated out onto mitis-salivarius and ordinary nutrient agar plates. incubated in 5 per cent CO, : 95 per cent NJ and examined after 2 days at 37 c‘.

The amount of glucose remaining in the culture supernatant after growth of the organisms was determined by using the Glucostat reagents obtained from Worthington Biochcmicals. Freehold, NJ. U.S.A. The glucose utilized was then calculated from the initial and final concentrations observed.

By the method of Barker and Summerson

(1941).

Samples of 0 I ml of 1 in 70 dilution of culture supernatants were analysed directly on a Technicon TSM amino-acid analyser. using norleucine as an internal standard. The amounts utilized were calculated from the amounts present in the medium before and after bacterial growth.

Samples of I ml of culture supernatant were treated with IO per cent sodium acetate w,iv (I ml) and 95 per cent w/v ethanol (2.5 ml). and kept at -4 C for 17 hr. The samples after this treatment were centrifuged (3000 g. 30 min) and the ppt washed with NaAciethanol ( I :4) solution three times by centrifugation. The ppt was suspended in I ml water and 0.1 ml samples were assayed fortotal hexose by the phenol method of Dubois (‘I II/. I 1956). RESULTS

The organism was first grown as a batch culture, and the results given in Fig. I show that in the exponential phase the mean generation time (MGT) of this strain of Sr~,cp. ITI~~~LIIIS in the modified Cybulska and Pakula medium was 1% hr. However, as growth continued. agglomerates of 30-40 organisms began to form. and growth became progressively linear. By the end of the growth phase. most of the organisms were present in these agglomerates. In the chemostat experiments. the dilution rate was varied through a ten-fold range from D = 0.05 to D = 0.5 hr. Total flow rates. including alkali addition. were

Fig.

I. The Initial stirred batch culture of Str.r/~. mur~m showing an exponential growth characteristic.

25 ml to 250 ml/hr, while the temperature and pH were kept constant. The yields of organisms are shown in Table I. The initial experiments were carried out at a low dilution rate and the dilution rate was then incrcascd stepwise until cvidcnce of washout M;IS observed. This was found to occur at a dilution rate of 0,5/hr which would be equivalent to a mean generation time of I.4 hr calculated from the usual equation MGT = (log,2/D) = (0.69310.5) = 1.38 hr This is somewhat shorter than the initial MGT found in batch culture. Microscopic examinations of the culture showed that at low dilution rates the bacteria were distributed evenly in lanceolate pairs and short chains (4-6 cells). However. as the dilution rate was increased the organisms formed longer chains of more spherical bacteria which tended to stick together to form large agglomerates of many organisms (over 200). On switching otf Ihc stirrer. the culture quicki! linmcd ;I precipitate of thcso agglomerates. This tendency was maximal at the highest dilution rate. at which point most of the Table I. Growth of Strrp. UIU~I,S in a chemostat at 37 C pH 6.5 in the modified medium of Cybulskd and Pakula (1963). with glucose as the growth limiting substrate Dilution rate (per hr)

O.D. (540 nm)

Dry wt (mg/ml)

PO5 0.10 0.22 0.33 0.50

5.7 6.0 6.2 6.4 3.0

I.58 1.72 I ,78 I.36

Growth of Strrptococcus mutum in a chemostat

661

Table 2. The effect of growth rate on the utilization of glucose and formation of lactic acid by Strep. fnutu/rs when grown in the modified media of Cybulska and Pakula (1963) at 37°C and pH 65 in a chemostat

Dilution rate (per hr)

Residual glucose @g/ml)

0050 0,104 0,201 0.221 0.332 0.564

20 20 20 20 20 2400

Glucose utilization (%)

Lactic acid (mg/ml)

Conversion* of glucose to lactic (%)

0.85 2.1 3.6 4.3 9,38 7.8

8.5 21 36 43 93.8 102

99% 99.x 99.8 99.8 99.8 76

* Calculated by Lactic acid mg/ml/Glucose utilized mg/ml x 100. Mean of three determinations variation 1 5 per cent. organisms were in the agglomerate form with comparatively few isolated pairs and short chains. Plates 1 and 2 are scanning electron micrographs illustrating these two conditions. On return to the low dilution rate. the agglomerates dispersed at a rate which precluded simple wash-out. It was also noticed at low dilution rates that the walls of the reactor and stirrer baffles. and the projections of the telemetering controls into the culture, remained free of any adhering organisms, but concomitant with the development of the spherical agglomerates at high dilution rates the organisms seemed to become more “sticky”. and quickly coated all surfaces in the reactor. The amounts of residual glucose and lactic acid present in the culture supernatants are given in Table

2. The glucose utilization and the amount of lactic acid formed from glucose (expressed as a percentage of the glucose utilized) may be calculated. Since over 995 per cent of the initial concentration of glucose in the medium was utilized, the culture was glucose (carbon) limited. When the dilution rate was adjusted to O.S/hr, the glucose utilization fell to 76 per cent, which indicates that the culture was no longer glucose limited. However, at this dilution rate the culture was starting to wash out. The conversion of glucose to lactic acid also varied with the dilution rate as shown in Table 2. At low dilution rates only 10 per cent of the glucose was converted to lactate. whereas at fast dilution rates all the glucose utilized was converted to lactic acid.

Table 3. Amino acid utilization by Strep. /~tutans grown at 37-C, pH 6.5 in a modified Cybulska and Pakula medium in a chemostat

Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine

p moles/ml present in original medium 20.4 19.2 24.4 53.6 39.2 IS.6 23.2 21.8 6.4 14% 30.6 3.0 9.4 7% 24.2 9.2 0.16

Mean of three determinations

variation

PO5

“;, Utilized at dilution rate 0.3 0.1 0.2

6 26 9 4 13 7 3 6 0

3 24 21 14 21 23 13 6 0

1 I

1 II

0 20 0 9 3 4

9 22 0 8 15 6

+ IO per cent.

20 24 11 28 16 23 27 17 14

8 26 5 45 21 29 16 10

15 27 12 32 17 27 32 23 34 24 29 6 45 19 25 19 17

@5 22 17 23 31 18 24 30 18 16 22 30 23 35 25 24 19 13

(162

D. C‘. Ellwood.

J. R. Hunter

The utilization of amino acids by this organism N hen the dilution rate was changed was also followed and the results are shown in Table 3. At the fastest growth rate (D = 05:hr) all the amino acids in the medium were used to some extent. In contrast. at the lowest dilution rate (D = @OS.!hr) onlv three amino acids. threonine. proline and phcnylalaninc. wcrc used to any extent.

The organism nas nominalI) @ucose-limited throughout these experiments. but m a complex medium of this kind it is diflicult to establish precisely the limitation being applied. particularly at high dilution rates. Clearly though. from the amount of glucose converted into lactic acid. a profound change took place in the glucose metabohsm of this organism as the dilution rate was increased. At low rates. the major part of glucose was utilized to provide the carbon 'lb cell production and only IO per cent was converted to I:ictatc: at high dilution rates. all the glucose utili/cd has converted to lactic acid and the carbon rcyuircd by the organisms for energy and growth must have been derived from another source, presumably the amino acids in the medium. It is of interest to compare the amino-acid utilization I-csults. with those amino acids found in play~~e lx C’ritchley (1969). The predominatmg amino acids ill plaque are glutamic acid and alanme. and these wcrc‘ two of the amino acids mainly used by this organism when growing at D = @S/hr. The two sulphur-containIng amino acids, methioninc and cysteinc. were absent l’rom plaque. suggesting that the organism might bc sulphur-limited. In our medium. both wcrc prehcnt Icysteine at a very low concentration) and wcrc mainI> used only at the faster growth rates. No cxtracellular or intracellular polysaccharide was produced bq this organism when growing under these glucose-limited conditions. No slime layer or stick!; material could bc dctectcd in washings of the organisms. nor could any be seen on the bacterial surfaces by scanning electron microscopy. However. adherence of the faster-growing bacteria to the internal hurfaccs of the reactor showed that thcrc \\;ts ;I change in their surface properties and that their agglomeration was not a mechanical ct’fcct due to intorlinking of long chains of the streptococci. Gibbons and Socransky( 1462)showed that. when cariogcnic streptococci arc incubated in the presence of’ excess sugar. the! lay down intracellular polysaccharide which is metabolized

to acid

when

the

sugar

in the

medium

becomes depleted. Electron microscopy of plaque streptococci has shown the presence of intracellular polysaccharide (Critchley. 1969). Holmc (1957) showed that Esci~ri&~~r c~li B when grown under glucose limitation IlilS ;I 10% inlracellular glycvgcn content. whereas when grown under nitrogen limitation. glycogen could account for over 20 per cent of the hacterial drl wt. As glycogen-containing bacteria arc

and V M. C. Longyear

found in plaque. it is tempting to suggest that some of the organisms in plaque are nitrogen-limited in their growth. The relationship between total conversion of the available glucose to acid and the change in the surfxc properties of the bacteria to become sticky when grown at faster rates. may be important in dental caries. in that it is known that breakdown of the tooth surf&x is related to acid production by oral bacteria and their adhesion to the tooth surface. ~c~~,rt)~~/ct/y~,rt~~,~lr,s We art: most grateful to Dr. W. H. Bowcn of the Royal College of Surgeons of England ExperImental F.htabhshmcnt at Downe, Kent. for much helpful ad\icc and criticism. Our thanks are due to Mr. G. R. G. Mood) and Mr. G. R. A. Kirk, Miss T. Overton. Miss H. Prince and Miss A Goddard for excellent experimental asslstancc which made this work possible. We are also indchtcd to the Department of Health and Social Security for tinawxl support and for permission to publish this paper. REFERENCES

Barker S. B. and Summerson W. H. 1941. The calorimetric deter-mjnation oflactic acid in biological material. J. biol. <‘licr,l. 138, 535 554. Bowen W. H. 1969. The induction of rampant dental caries in monkeys (~~ucucu ins). Curies Rrs. 3, 227-237. (‘arlsson J. 1967. Presence of various types of non-haemolytic streptococci in dental plaque and in other sites of the oral cavity in man. &font. Rrc. 18, 57-76. (‘ritchley P. 1969. The breakdown of carbohydrate and protein matrix of dental plaque. Caries Rcs. 3, 249-265. (lbulska J. and Pakula R. 1963. Streptococcal polyglucosidaze. I j\ medium suitable for polyglucosidase producIlOll E\j’. M?d. Microhiol. (Pohd) 15, I X7-198. Dubols M.. Gilles K. A.. Hamilton J. K., Rebers P. A. and Smith F. 1956. Calorimetric method for determination of sugars and related substances. .4t1ui~t. Chem. 28, 350-356. Fitrgerald R. J.. Jordan H. V. and Stanley H. R. 1960. Experimental caries and ginglval pathologic changes in the gnorohiotic rat. .I. dent. Rr.5. 39, 923 93X. Glhhclns R. J. and Socranskq S. S. 1962. Intracellular polysaccharide storage bq organisms in dental plaques. Archs or~ciiBiol. 7, 73-~80. Herbet-t D.. Phipps P. J. and Tempest D. W. 1965. The chemostat: destgn and instrumentation. Lab. Ptact. 14, 11SO-

I lhl. culture studies on glycogen synHolmc T 1957. Continuous thc\ls in E.whoichict Ai B. Acta chn scund. 11, 763. Kr,~s\c B. lY66. Human streptococci and experimental c:trIcs in hamsters. 4rchs OI.U[Biol. II, 429-436. Kras\c B. and Fdwardason S. 1966. The proportional distrlhution ofcaries-inducing streptococci in various parts of the oral cabit) of hamsters. Avclts oru[ Biof. II, 1137

I I-l?. Orland F. J.. Blayney J. R.. Harrison R. W.. Reyniers J. A., Trcxlcr P. C.. Ervin R. F.. Gordon H. A. and Wagner M. IY55. Experimental caries in germ-free rats inoculated \I ith enterococci. J. Arrr. Dent. Ass. 50, 259-272. Tan~r J. M.. Krichevsky M. I. and Keyes P. H. 1969. The mctaholic fate of glucose metabolized by a washed \tationarl phase caries-conducive streptococcus. Cuvics Kc,\. 3, I67 177. Tantcr J M.. Wood W. 1. and Krichevsky M. I. 1969. Linear gl-owth kinetics of plaque-forming streptococci in the prcsencc of sucrose‘ J. <,c’rl.Uic~rohi0l. MI. 115 I ii

Growth

of Strummccus

mutam in a

chemostat

Rbumb-Une souche de Streptococcus mutans, isolCe &partir d’une l&ion carieuse humaine et provoquant des caries chez le hamster et le singe, a CtCincubbe dans un milieu complexe. En effectuant des cultures a des dilutions varites, on observe des modifications dans la propriM d’adhbrer & des surfaces, dans l’utilisation du glucose et dans la production acide par le microorganisme.

Zusammenfassung-Ein aus einer kariiisen L&ion eines Patienten isolierter Stamm Streptococcus mutans, der beim Hamster und Affen Karies verursacht, wurde im Chemostaten mit einem Komplexmedium kultiviert. Hinsichtlich der Eigenschaft des Organismus, an Ober&hen anzuhaften, Glukose zu verwerten und SBure zu produzieren, gab es beim Wachstum in verschiedenen Verdtinnungsgraden Unterschiede.

PLATE

I

OVERLEAF

663

D. C‘. Ellwood,

J. R. Hunter

and V. M. C. Longyear

Figs. 7 and .3. The scanning electron microscopic appearance of Strep. IIWUIIS when grown at different bacteria. at a dilution rates. 3. Dispersed bacteria. at a dilution rate of OG5,‘hr. ( x 7240). 3. Agglomerated rate of 0.5;hr. ( x 5500).

Growth of Streptococcus mittam in a chemostat

A.O.B. f.p. 664