Ionic interaction of oral streptococcal bacteria studied by partition in an aqueous polymer two-phase system

Ionic interaction of oral streptococcal bacteria studied by partition in an aqueous polymer two-phase system

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IONIC INTERACTION OF ORAL STREPTOCOCCAL BACTERIA STUDIED BY PARTITION IN AN AQUEOUS POLYMER TWO-PHASE SYSTEM GUDRUN WESTERGREN Department of Cariology, Faculty of Odontology. University of Gothenburg, Sweden

Summary-The net surface charge of various oral streptococci were assessed by aqueous twophase partitioning in a dextran-polyethylene glycol system. Great variability was found among individual strains within all species tested. Type 1 strains of Streptococcus sanguis serotypes which have been found to be more adherent, exposed a lower negative net surface charge than Type 2 strains.

INTRODUCTION It has become increasingly evident that the initial bacterial adhesion is of decisive importance for the hostmicrobial interaction. This principle originates from studies of the oral microflora (Gibbons and van Houte, 1975; Gibbons, 1977) but is now accepted to be essential for the virulence of a variety of other microorganisms. Various oral surfaces are selectively colonized by oral streptococci. Thus, Streptococcus salivarius is preferentially found on the dorsum of the tongue (Krasse, 1954) whereas Streptococcus mutans and Streptococcus sanguis show high alfinity for the tooth surface (Carlsson, 1967; van Houte, Gibbons and Banghart, 1970). The mechanisms involved in the adherence of different bacteria to various biological surfaces are not yet fully understood but numerous studies point to a combination of non-specific and highly specific binding mechanisms. Physico-chemical surface properties, such as charge and hydrophobicity have been demonstrated to be of importance for the host-microbial relationship in the interaction with granulocytes (Stendahl, Magnusson and Tagesson, 1977), HeLa cells (Edebo et al., 1980) and small intestinal epithelium (Perers et al., 1977). It has also been suggested that the first attachment of a microorganism to the tooth surface is influenced by charge. Thus, Olsson, Glantz and Krasse (1976) showed that strains of the same species but with different electrophoretic mobility adhered with different strength or became desorbed at different rates. In-u&o studies of the adsorption of oral streptococci to saliva-coated hydroxyapatite (HA) revealed that strains of Strep. sanguis Type 1 adhered significantly better than did the Type 2 strains (Appelbaum et al., 1979). Their results seemed to provide a body of information convenient for studying the connection between physico-chemical surface properties and ability to adhere. I therefore was interested in comparing the surface charge of two serological groups of Strep. sanguis, as well as a variety of other oral streptococci employing a two-polymer phase system. Such systems have been used to study the net surface charge of Escherichia coli and Sulmonella typhimurium, in relation to their liability to interact with phagocytic cells and to attach to mucosal surfaces (Stendahl et

al., 1977; StjernstrGm et al., 1977) and of Staphylococcus saprophyticus and Struphylococcus epidermis in relation to the adherence to urogenital cells (Colle&n er al., 1979). The distribution of the bacterial cells between the two phases, which consist of a dextranrich bottom phase and a polyethylene glycol (PEG)rich top phase, depends on the surface properties of the bacteria. By substituting part of the PEG with a positively-charged polymer, bacterial cells with pronounced negative surface charge are transposed towards the top phase. MATERIALS AND Bacterial

METHODS

strains

Designation and origin of the bacteria1 are presented in Table 1.

strains

used

Cultivation and radiouctiue labelling The strains were grown overnight (19 h) at 37 C in 9 ml of streptococcus broth (Jordan, Fitzgerald and Bowler, 1960) with the addition of l@20~1 [methyl-3H]-thymidine (18-25 Ci/mmol), The Radiochemical Centre, Amersham, England. The bacteria were washed twice in 0.01 M phosphate buffer, pH 7.3 and finally resuspended in buffer to the same optical density (ODosn = 0.5). The radioactivity of the bacterial suspensions was measured in a betaliquid scintillation counter (Rackbeta, LKB-Wallac, Sweden). Two-phase purtitioning A two-phase system (Albertsson, 1971), essentially the same as described by Stendahl et al. (1973), was prepared from stock solutions of 20 per cent (w/w) poly(ethylene glyco1)6000 (PEG, Carbowax 6000, Union Carbide, New York, N.Y., U.S.A.), 20 per cent (w/w) dextran TSOO (Pharmacia Fine Chemicals, Uppsala, Sweden), 0.1 M tris(hydroxymethyl)amino-methane buffer (tris), pH 7.0, and distilled water. A total system contained 4.4 per cent (w/w) PEG and 6.2 per cent (w/w) dextran in 0.03 M tris buffer. It was allowed to equilibrate at 4°C overnight in a separation funnel. Then the bottom (B) phase rich in dextran, and the top (T) phase, rich in PEG, were collected and stored separately at 4°C. To prepare phase systems for the partitioning studies, 2ml of B and

1035

1036

Gudrun

Westergren

Table 1. Bacterial strains Origin

Designation Strep. mutans

Strep. sanguis

Strep. salioarius

Strep. mitior

* Serotypes Ravn (1974). t Serotypes

Zinner et al., 1968 Zinner et al., 1968 Carlsson, 1967 Edwardsson, 1968 Guggenheim, 1968 Gibbons et al., 1966 Rosan, 1976 Rosan, 1976 Appelbaum et al., 1979 Appelbaum et a[., 1979 Appelbaum et a[., 1979 Westergren, 1978 Westergren, 1978 Clinical isolate Clinical isolate ATCC ATCC Rosan, 1973 Central Public Health Laboratories, Colindale, London, England Ranhand, 1974 ATCC ATCC Carlsson, 1968 Carlsson, 1968 Carlsson, 1968 ATCC ATCC ATCC ATCC

AHT (a)* BHT (b)* KPSKZ = JC2 (c)* B13 (d)* OMZ 176 (d, g)* LM7 (e)* G9B (Type 1)t 66 x 49 (Type l)t 47A2 (Type l)t 38 (Type l)t 5913 (Type l)t G26-S (Type l)t G26-R (Type 1)t G30 (Type 1)t G53 (Type 1)t 29667 (Type 1)t 29668 (Type 1)t M-5 (Type 2)t Challis (Type 2)t

WE4 (Type 2)t 10556 (Type 2)t 10558 (Type 2)t MPSI = JC8 HTSI = JC6 MVSI = JC13 8618 903 15909 15912 according

according

to Bratthall

(1970) and Perch,

Kjems

and

to Rosan (1976).

2 ml of T phase were pipetted into graded test tubes. For tests with positively charged PEG, 2.5 per cent of the PEG had been exchanged with bis-trimetdylamino [(CH,),N+]-PEG (TMA-PEG) already in the preparation of the stock solutions. The TMA-PEG was kindly provided by Dr 0 Stendahl, University of LinkBping, Sweden. To the graded tubes with the different phase systems was added 0.1 ml of a suspension of bacteria. The tubes were inverted (20 times) for mixing and kept for 2 h at 4°C for separation of the phases. After reading the volumes of the total phase system and the B phase, 0.5 ml samples were withdrawn from the T and B phases and, after mixing on a Whirlimixer (Fisons Scientific Apparatus, Loughborough, Leicestershire, England), also from the rest containing the material adhering to the interface. Quantification of the bacteria was made in the beta-liquid scintillation counter and the distribution of the bacteria was calculated from the number of bacteria in the samples and the volumes of the phases.

RESULTS The partition of various oral streptococci in the two different two-phase systems is shown in Tables 2 and 3. The figures refer to percentages of bacteria in the T and B phases and are mean values of two separate experiments in duplicate with two different cultures. The rest of the bacteria (making a total of 100 per cent) adhered to the interphase between T and B. In the basal system with uncharged PEG, the bacteria accumulated mainly at the interphase and in the dextran-rich B phase. Only a few test strains showed slight affinity for the PEG-rich T phase. Experiments with the two-phase system containing positively charged TMA-PEG revealed that strains with high negative surface charge as well as strains with low negative charge could be found within all species tested. Several of the bacterial strains showed a tendency to move to the T phase and some strains, such as Strep. mutans strain BHT, Streptococcus mitior strain ATCC 15909, Strep. salivarius strain HTSI

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Partition of oral streptococci

Table 2. Distribution of oral streptococci in a dextran-PEG system Bacteria (percentage) in T and B phases Basal, uncharged PEG Strain

Positively charged TMA-PEG

T

B

T

B

2(l) 2(l) 2(O) 2(O) 2(f) 2(O)

74(2) 25(8) 5(4) 21(10) 29(2) 5(O)

2(l) 38(3) 20(2) 2(l) 2(l) 17(3)

140) 4(3) 3(2) 70) 6(4) 2(3)

2(2) 7(3) 2(l)

9(3) lo(5) 66(7)

17(10) 74Ul) 31(6)

4(l) 5(O) 3(f)

l(0) 2(O) 2(O) 2(O)

14(2) 20(7) 28(15) 20(7)

1l(O) 5(2) 39(6) 2(O)

2(O) 5(l) 3(2) 6(l)

Strep. mutans

AHT (a) BHT (b) KPSKZ (c) B13 (d) OMZ 176 (d, g) LM7 (e) Strep mitior ATCC 903 ATCC 15909 ATCC 15912 Strep saliuarius

MPSl 8618 HTSl MVSl

Figures in brackets represent the range (2 experiments performed).

Table 3. Distribution

of Strep. sanguis strains in a dextran-PEG

system

Bacteria (percentage) in T and B phases

Basal uncharged PEG Strain

T

B

31) l(O)

6W)

Positively charged TMA-PEG T

B

Type1

G9B

38 66 x 49 B-4 47-A2 5913 G26-S G26-R G30 G53 ATCC 29667 ATCC 29668 Type 2 M-5 Challis WE4 ATCC 10556 ATCC 10558

4(2) 412) l(0) f(0) 2(O) 2(l) 2(L) 4(f) l(0) 2(3)

l(O) 3(2) 3(2) 4(l) 2(l)

15(6) 27(9) 29(8) 17(O) 21(7) 30(2) 17(l) 9(9) 20(4) 11(8) 9(8)

31(5) 4(l) 19(3) 1l(5) 3(O) 7(4) 6(5) 3(O) 14(5) 17(10) 13(l) 2(2)

5(5) 5(3) 7(O) 19(3) 2(O) 3(O) 6(2) 2(2) 3(l) 4(O) 2(O) 4(3)

30(f) 29(7) 31(3) 29(9) 400)

20(3) 49(11) 68(l) 7(3) 32(5)

4(f) 5(3) 5(2) lo(7) 3(4)

Figures in brackets represent the range (2 experiments performed).

.I--. Gudrun

1038 PEG TMA-PEG

PEG TMA-PEG

l

. i -c_ .t”.

4

Strep. saflguls

Type 1

Type 2

Fig. 1. Partition of Strep.sunguisstrains of serotypes 1 and 2 in a dextran-PEG system with either uncharged PEG or positively charged TMA-PEG. Dotted lines indicate the

means of the percentage of bacteria collecting T-phase.

in the

(Table 2) and Strrp. sang& strains WE4 and Challis (Table 3) seemed to expose very high negative surface charges, as indicated by the high percentage of cells collecting in the T phase. When the two sero-groups of Strep. sanguis were compared, the Type 1 strains were in a considerably lower proportion in the T phase of the TMA-PEG system than the Type 2 strains (10.8 & 8.6 per cent versus 35.2 i: 23.9 per cent) (Fig. 1). The difference was significant (0.001 < p < 0.01) by Student’s t-test. DISCUSSION

A number of oral streptococci have previously been investigated with regard to surface charge (Olsson ef ul., 1976) by determining the electrophoretic mobility of the bacterial cells in an electric field, using a particle-micro electrophoresis apparatus. Some of these strains were included in my present study and the results from the two-phase partitioning tests confirmed most of the data obtained by Olsson et al. Thus, the partition of Strep. sanguis strains ATCC 10556 and ATCC 10558 in the TMA-PEG system indicated that the strain ATCC 10558 was more negatively-charged than strain ATCC 10556, as shown by the higher affinity of strain ATCC 10558 for the T phase (Table 3). This is consistent with the results by Olsson ut al. who demonstrated a higher zeta-potential for strain ATCC 10558 than for strain ATCC 10556. Furthermore, Strep. mutans strain BHT, which they found to have the highest zeta-potential of all strains tested, was also highly negatively charged in my study (Table 2), although not as highly charged as some strains of other species. All Strep. sanguis strains I used responded clearly to either anti-G9B serum (Type 1) or anti-M-5 serum (Type 2). as determined by the Ouchterlony technique (Ouchterlony, 1958) or by immunofluorescence (Bratthall, 1972). Strains which showed obvious crossreactions were not included. Appelbaum et a/. (1979) demonstrated that strains of Strep. sunguis Type 1

Westergren

adhered better to saliva-coated HA than did the Type 2 strains, as indicated by the Langmuir adsorption isotherm. Type 1 Strep. sanguis strains were less negatively charged than the Type 2 strains (Table 3 and Fig. 1) indicating that a high negative net surface charge does not favour the adherence of Strep. sanguis to salivacoated HA. A supporting finding is that strain G26-S was transposed to the TMA-PEG phase to approximately the same extent as was the more adherent mutant strain G26-R. These two morphological variants have previously been compared with regard to their adherence ability and the results revealed better adherence of the R-form (Westergren, 1978). Strep. sanguis has high affinity for the tooth surface in viuo and appears to be the first streptococcal species to colonize newly erupted teeth (Carlsson et al., 1970). Strains of this organism have been isolated from the heart tissues of subjects with subacute endocarditis (Weinstein and Rubin, 1973) and Henriksen and Henrichsen (1975) reported on the isolation of twitching, polarly fimbriated strains of Strep. sanguis from the human pharynx. Two such twitching strains (ATCC 29667 and ATCC 29668) I included have been referred to as a newly described serological group W (Henriksen and Eriksen, 1977) but as they distinctly responded to anti-G9B serum (Type 1) and showed no cross-reaction with anti-M-5 serum (Type 2), I consider them to be Type 1 strains. Their partition in the two-phase systems agreed well with the other Type 1 strains. Growth conditions and environmental factors, on the whole, are known to effect surface properties of the bacteria (Bleiweis et al., 1976; Ellwood, 1976; Shockman et al., 1976). To ensure good reproducibility, the test strains were always cultured in the same medium for exacly 19 h, when they were expected to have reached stationary phase of growth. A TMA concentration of 2.5 per cent was found to be convenient for distinguishing between bacterial strains with net surface charge within the same range. Attempts were also made to use 5 per cent TMA, but this high concentration increased the positive charge of the PEG phase to such an extent that a large proportion of the bacterial strains tended to collect to the T phase at nearly 100 per cent. Two-phase systems preferentially have been used to study physico-chemical surface properties of Gramnegative bacteria (Stendahl et al., 1973; Magnusson et al., 1978). However, my study shows that the twophase system is also a suitable method for the assessment of surface charge of oral microorganisms which might well be used to study the influence of various substances and environmental factors on the net surface charge of oral streptococcal cells. Acknowledgements-I am indebted to Dr 0 Stendahl, University of Link&&. Sweden. for heloful discussions during the preparatibnof this paper. The-study was supported by the Swedish Medical Research Council (project No. 4548. Principal investigator Bo Krasse). REFERENCES

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Partition

of 01*al streptococci

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