Bacterial interference in vitro : Comparison between a quantitative kinetic and a cocultivation blood agar test method

APMIS 102: 810416. IYY4 Coprrixhr 0 A P M I S 1994 . . - Printed in Denmark . All righrs rew-vid

Bacterial interference in vitro Comparison between a quantitative kinetic and a cocultivation blood agar test

method

ANDERS JOHANSSON.' AXEL BERGENHOLTZ' and STIG E. HOLM2

Departments of 'Periodontology and 'Clinical Bacteriology, University of UmeA, UmeA, Sweden

Johansson, A,, Bergenholtz, A. & Holm, S. E. Bacterial interference in vitro: Comparison between a quantitative kinetic and a cocultivation blood agar test method. APMIS 102: 810-816, 1994.

The aim of the present study was to compare two methods for estimation of bacterial growth interference between various bacteria using a Bioscreen robot analyzer, allowing kinetic documentation, and a cocultivation test on blood agar plates. Six laboratory strains with different virulence and growth requirements were used: Actinobacillus uctinomycetemcomituns, Porphyromonas gingivalis, Fu- sobacterium nucleatum, Streptococcus mitis, Staphylococcus aureus, and Staphylococcus epidermidis. The interference activity was correlated with a reference system of Streptococcus sanguis (strain a 89) and Streptococcus pyogenes (group A streptococci, GAS serotypes T 9 and T 22). The methods used and results obtained were as follows: 1 . Estimation of synergistic and antagonistic bacterial inter- ferences using a Bioscreen robot analyzer. Suspensions of viable bacteria were added to microtiter plates with different concentrations of UV light-killed bacteria in liquid media. The Bioscreen analyzer monitored bacterial growth every 10 min for 24 h giving kinetic data during the growth period. Synergisms as well as antagonisms were demonstrated between the tested bacterial strains which have not earlier been reported in the literature. However, the antagonistic effect observed between the six strains was less than that induced by the S. sanguis strain on the two strains of S. pyogenes. 2 . Cocultivation of bacterial strains on blood agar surface with precultivated or simultaneously stamped interfering bacteria indicated no detectable interference between the six tested bacterial strains, while the S. sanguis strain inhibited the growth of S. pyogenes strains as well as the hemolysis around the colonies. The Bioscreen method was found more sensitive for testing bacterial interference compared to the commonly used blood agar test.

Key words: Oral bacteria; bacterial interference; Bioscreen method.

Anders Johansson, Department of Periodontology, University of UmeA, S-901 87 UmeA, Sweden.

Microbial interference has been known for more than 60 years (6, 7). The genesis of this phenom- enon is multifactorial (2, 23, 29) but often correlated with production of bacteriocins or bacteriocin-like substances, which regulate the balance between microorganisms in the normal flora (20, 26). Antagonistic interactions afforded by the normal flora versus pathogens, which may act as protection against diseases, have been

~~

Received February 24, 1994. Accepted September 9, 1994.

verified between bacterial species in the human throat (9, 16), on the skin (26), and on the teeth (5 ) . Bacterial interference in the gingival pocket is not fully clarified mainly due to difficulties in evaluation of the individual activity of a certain microorganism among the diversity of bacterial species in the crevice and methodological prob- lems.

The aim of the present study was to evaluate methods to estimate growth interaction between various bacteria by cocultivation on blood agar plates or by using a robot analyzer system (Bio- screen), allowing kinetic documentation of bac-

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BACTERIAL INTERFERENCE I N VITRO

terial growth. The bacterial species tested were selected based on their various virulence and growth requirements.

MATERIALS AND METHODS

Bacterial strains

killed, were as follows:

43718

The laboratory strains of bacteria, viable and UV-

Actinobacillus actinomycetemcomitans ( A a ) A TCC

Porphyromonas gingivalis (Pg) A TCC 33277 Fusobacterium nucleaturn (Fn) (clinical isolate

Streptococcus mitis ( S m ) (Clinical isolae a 5 (9)) Staphylococcus aureus (Sa) A TCC 6538 Staphylococcus epidermidis ( S e ) A TCC 12228 Streptococcus sanguis (Ss a89a) (clinical isolate nr.

1 in (16)) Streptococcus pyogenes serotypes T9 and T22 (Sp

T9, Sp T22) All bacterial strains were cultivated on blood agar

plates except for P. gingivalis, which was cultivated on plates supplemented with hemin and vitamin K (BGA plates) (12).

For liquid cultivation E-MEM (Flow, UK), without phenol red and antibiotics, with 25%) TY medium ( l l ) , 10% fetal calf sera (Flow, UK), and 20 mM HEPES (Sigma, USA), was used for all bacterial strains. This composition of growth culture medium (CM) was shown to stimulate growth of all the bacterial strains used in the present study.

Quantification of bacteria in suspensions was estimated by reading optical density (OD) at 500 nm on a spectrophotometer (Vitatron CMP, Vital Scientific, Netherlands).

BN1 la-d (3))

Viable log phase bacteria The bacterial strains were cultivated aerobically

(Sa, Se, Sm) or anaerobically (Aa, Pg, Fn) on agar plates at 37°C for 2 4 4 8 h. Inoculates were then transferred to 4 ml CM in 10 ml glass tubes and incubated for 20-24 h at 37°C until the culture reached the stationary phase. From the bacterial suspensions 20-160 p1 (depending on the species) was transferred to new tubes containing 4 ml CM and incubated for 4-8 h at 37°C until the log phase (ODSoonm 0.5-1.0) was reached.

Bacterial interference on blood agar Cocultivation test (9). The edge of a sterile glass

slide was dipped in a Petri dish containing log phase bacteria in CM. Excess fluid was removed by blotting on a sterile filter paper. The edge of the glass slide was stamped on a blood agar plate and the interfering bacteria likewise stamped at right angles to the former strain. Each bacterial strain was stamped on

six different blood agar plates. The growth inter- ferences at the zones of interaction were recorded and documented photographically.

1. Under aerobic conditions a) aerobic bacteria were simultaneously cross-

stamped and cultivated for 24 h at 37°C. b) anaerobic bacteria were precultivated anaer-

obically for 48 h at 37 "C, cross-stamped with the same bacteria, and cultivated aerobically for another 48 h.

c) aerobic bacteria were precultivated aerobically for 24 h at 37"C, transferred to anaerobic conditions, cross-stamped with anaerobic bacteria, and cultivated anaerobically for 48 h at 37°C.

2. Under anaerobic conditions a) anaerobic bacteria were simultaneously cross-

stamped and cultivated for 48 h at 37°C. b) aerobic bacteria were precultivated aerobically

for 24 h at 37"C, cross-stamped with the same bacteria, and cultivated anaerobically for another 24 h period.

c) anaerobic bacteria were precultivated for 48 h at 37 "C, transferred to aerobic conditions, cross-stamped with aerobic bacteria, and cultivated aerobically for another 24 h at 37°C.

Growth interaction was classified according to cri- teria described earlier (9). Thus:

N o interaction. The test bacteria grow to the very edge of the interfering bacteria maintaining the same colony size.

Slight inferaction. The test bacteria do not grow all the way to the edge of the interfering bacteria and/or the colonies at the zone of interference are signifi- cantly smaller.

Strong interaction. A zone of growth inhibition is demonstrated at the interaction zone.

Bacterial interference using Bioscreen A microprocessor-controlled photometer (21, 28),

Bioscreen robot analyzer (Labsystems, Finland), was used for monitoring microbial growth in microtiter plates (Labsystems, Finland). Bacterial growth in each of the 100 wells was presenteg as turbidometric curves (growth curves). Changes in optical density of various pure or mixed bacterial cultures were measured every 10 min for 24 h. The interference between strains was determined by adding viable bacteria of the different strains to various concentrations of UV- killed bacteria in liquid medium.

UV irradiation of bacterial strains (1). CM (4 ml) containing bacteria in log phase was transferred to a 60 mm sterile Petri dish (Flow, UK). The bacterial suspensions from each of the six strains were left under a UV lamp (Germicidal, 30 W, Germany) at a distance of 50 mm for 30 min. The depth of medium should not exceed 2 mm for effective killing. Killing efficacy was tested ( 100% killing) by recultivation of treated bacteria on blood agar plates. The suspen-

81 1

JOHANSSON et a1

sions were always used immediately after UV irradiation.

Kinetic documentation of growth interference (Bio- screen). UV light-killed bacteria (200 pl) at a concen- tration of ODSoonm 3X10-3-10-’ in CM were pip- etted into 100-well microtiter plates. Then 200 pl of viable log phase bacteria (S. aureus, S. epidermidis, S. rnitis ODSoonm A . actinornyceterncornitans, P. gingivalis, F. nucleaturn ODSoonm lop2) in CM was added.

The bacterial suspensions were allowed to grow for 24 h at 37°C in the Bioscreen analyzer. Observations were made every 10 min at OD450nm. The interactions were documented by analysis of the 24 h growth curves. Each experiment was repeated six to seven times.

For estimation of bacterial growth by the Bio- screen analyzer under anaerobic conditions the microtiter plates were sealed with vacuum grease (Fluka, LabKemi, Sweden) in the anaerobic box be- fore being transferred to the Bioscreen incubator.

Bioscreen data handling. Raw data from Bioscreen were converted to ASCII. Three different parameters were processed:

1) “The Growth rate” estimated as the maximal mean value of relative growth (generation time) measuring the increase in optical density (OD) during a 100 rnin period (Fig. lb). This illustrates bacterial interference during the maximal growth period (bacteria in log phase).

2 ) “The Growth density” estimated as the increase in OD from time 0 until the time when the mean value of relative growth during a 50 min period fell to under 15% of maximal relative growth. The break- point for the increasing OD was set to half the 50 min period (Fig. lb). This shows interference of bacteria permitted to grow to stationary phase.

3) “The Growth index” indicates the mean value of bacterial growth. The growth in the control cuvettes was set to index 100 for each strain and compared to samples where UV-killed bacteria had been added.

Significant synergism or antagonism between bacteria is when the 95% confidence limit ( 2 t X standard error) of the outgrowth in the control cuvettes for one bacterial species (Table 1) is separated from the 95% confidence limit for outgrowth of the same species in cuvettes where interacting bacteria (Table 2 ) are present.

The series of measurements for the different cuvettes were normalized according to the formula:

d,= d, - min (d ,.. . ,dT) + 0.3

where d,=the recorded value at time t and

0.3=the initial density.

The relative Growth rate was computed as

rt=(dt-dt-l)/dt-l

Cow. U V a 89a G R , GD O D ~ ~ ~ 1d2 7

1 1 0 ’ 5 0 T

C Conc. UV-a89a 100

Fig. I. Growth of S. pyogenes T9 in the presence of interfering S. Sanguis (a89a): a) simultaneously stamped on blood agar surface and incubated for 24 h at 37°C. b) Bioscreen growth curves as support for estimation of Growth rate and Growth density. Growth of bacteria without interfering strain (no UV-killed bacteria) is set at index 100 (mean of n=6). c) growth curves converted into parameters (for definition of Growth rate and Growth density see text). Mean of n=6, standard error (see Table 2) .

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BACTERIAL INTERFERENCE IN VITRO

TABLE 1. Growth of the various viable bacterial strains in the absence of interfering bacteria. Mean 222standard error. Optimum growth of each species in the absence of interfering bacteria was set at index 100. A. actinomycetemcomitans (Aa), F. nucleatum (Fn), I? gingivalis (Pg), S. aureus (Sa), S. epidermidis (Se), S. mitis (Sm), S.

pyogenes T9 ( T9), and S. pyogenes T22 (T22) Aa Fn PP Sa Se Sm T9-~ t T22-~t

Growth rate 100219 10025 10025 100211 10027 100?2 100212 100215 Growth density 10024 10023 100?2 100212 100+3 10021 10026 100?10 No. ofobservations n=16 n=18 n=18 n=18 n=18 n=18 n = l l n = l l

t c rs max s=t-9

10 t = l I . .... T

The point of maximum relative growth tmax was de- fined by computing a 10-period moving average for each time t = l 1, ..., T.

The Growth density was measured at time td, when a 5-period moving average fell to under 15% of the relative rate at P a x .

tC2 z rs s=t-2

5 , where t=tmax, ..., T

RESULTS

Interference on blood agar No visible signs of interference were observed

between the six bacterial strains, either with simultaneous or precultivated interfering bacteria in any combination using the blood agar plate method. The S. sanguis strain (a 89a) caused a slight inhibition of the growth of S.p T9 and S.p T22 at the zone of interaction where the hemolysis around the growth line of the S.p T9 and the S.p T22 was also inhibited (Fig. la).

Interference using Bioscreen Growth index of the different viable bacterial

strains in the presence of various interfering species (UV-killed) in different concentrations is presented in Table 1.

Significant antagonism or synergism caused by interfering bacteria present in growth medium of the viable bacteria was found in the following combinations:

Viable A . actinomycetemcomitans with interfering A . actinomycetemcomitans, S. aureus, S. epidermidis or S. mitis in growth medium

showed significant antagonism in Growth density.

Viable F. nucelatum showed significant antagonism in Growth density to all of the six tested interfering UV-killed bacterial species, while in Growth rate only UV-killed bacteria of same species (F. nucleatum) showed significant antagonism.

Viable P. gingivalis showed significant antagonism in both Growth rate and Growth density to all of the six tested interfering UV-killed bacterial species.

Viable S. aureus showed significant antagonism in both Growth rate and Growth density to interfering UV-killed S. mitis, while interfering A . actinomycetemcomitans or F. nucelatum revealed significant antagonism only in Growth rate.

Viable S. epidermidis showed significant antagonism in both Growth rate and Growth density to interfering UV-killed A . actinomycetemcomitans, F. nucleatum, S. epidermidis or S. mitis, while S. aureus revealed significant antagonism only in Growth density.

Viable S. mitis showed significant antagonism in Growth density to all six tested interfering UV-killed bacterial species, while in Growth rate only four of these ( A . actinomycetemcomitans, F. nucleatum, P. gingivalis or S. mitis) showed significant antagonism.

Significant synergism could be shown with viable S. mitis when low concentrations (ODSo0 3X 10-3-10-2) of interfering bacteria were present (S. aureus or S. epidermidis in Growth rate and P. gingivalis or S. epidermidis in Growth density).

Viable S. pyogenes (T9 and T22) revealed significant antagonism in both Growth rate and Growth density when UV-killed S. sanguis (a- 89a) was present in growth medium. This antagonism was extremely strong compared t o that between any of the other six bacterial strains tested.

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JOHANSSON er al,

TABLE 2. Growth index of each of eight viable bacterial strains (bold type) with various interfering species (italicized) in different concentrations. Mean22Xstandard error based on n=6 for viable A. actinomycetemcomitans (Aa), E nucleatum (Fn), F! gingivalis (Pg), S. pyogenes T9 (T9) , and S. pyogenes T22 (T22) , and on

n=7 for viable S. aureus (Sa), S. epidermidis (Se), and S. mitis (Sm) (interfering a=a89a streptococci) Growth rate Growth density

Concentrations ( ODSoonm) of interfering bacteria 3 ~ 1 0 - ~ ~ X I O - ~ lo-' 3 ~ 1 0 - ~ ~ X I O - ~ 10-l

Aa-Aa Fn pg su Se Srn

Fn- An Fn pg sa Se Srn

Fn pg Sa Se Srn

Sa-Aa Fn pg sa Se Srn

Se-Aa Fn pg sa Se Srn

Sm-Au Fn pg s a Se Srn

Pg-Aa

T9-a T22-CY.

9823 10324 9822 93215 9923 0121 0226 9 6 t 11 0226 98210 0128 0127 91 2 7 902 18 9728 9828

1002 10 9626 9626 9423 9626 9728 9224 91 5 5 9925

104 t7 10329 10823 1 0 3 t 6 10028 10126 9726

10422

10426 1106+31 101 t 2 11026

9923 9423 9724 9126 9523 8826 9923 9422 9723 9523 9823 9722 9828 94210 9828 85213 9826 90211 97210 9426

10229 97216 10326 100212 9027 -1 88211

8927 -1 9 4 t 1 3 1m1 9425 184+101

9427 184-+41 78217 1-1 9125 8227 90212 79220 9328 81220 8728 81220 82210

109210 94212 10026 9328 1075 11 114220 99215 9 4 t 1 2 92222 90213

10627 98213 9826 -1

9623 -1 107210 98216

-1 10524

9923 m

100+8 9325 99511 76214

118230 108224 105e42

7823 7823 7826 7825 8522 91 2 5 83222

772 22 81 2 14 88229 97 2 20

164-+261

m 170+111 m i i E 6 l 1m1 70220

87246 792 13 78224

IiE-id

!3E7il M I 171+131

164+171 174+171 Bi.37 176'-51 -1

115228 872 17

101210 9824

9 5 t 6 9827

104212 9728 992 1

101 2 4 9923 93218

10427 10422 94215 942 12 9624 9524 9623 9623 9825 9623

10325 10126 101 2 4 9 5 t 1 2

10629 9357 9522

10025 9823 9824

10326 9527

10226 9 8 t 4

10322 10325 10022 10523

1106.tlI

9 7 t 6 94210 9726 93210 9826 9823 9826 9429 9624 9657 9622 9524 9627 9824 181-+121 9325 m

10127 9627 92214 [m]

10122 186+141 9623 -1

-1 184+61 9523 188+81

9524 184t71 194+31 186+-21

9728 -1

98210 9428 10126 92211 103210 107% 12 9727 86222

101 2 12 101 2 10 89510 9329 9926 9725 9824 9625 9927 10527 9528 9627 93227 10027 9824 9625

10026 -1 10023 -1 10023 102210 rn rn 9926 9927 m

107210 10329 97216 l E - i 4 m I W I

T I significant antagonism -j significant synergism

DISCUSSION

Commonly used methods in determination of bacterial interference are interference on agar plates, interference in broth, agar overlay plates, and interference in vivo (9, 19).

In the present study significant antagonisms

as well as synergisms between the tested bacteria were detected with the Bioscreen analyzer, while no visible bacterial interference was observed on the blood agar plates. the Bioscreen method has the advantage that it is possible to document quantitative kinetic changes during the whole growth period; these can be converted

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BACTERIAL INTERFERENCE IN VITRO

owlh density

h rate

Conc UV-Aa 100

Fig. 2. Growth index parameters of P. gingivalis in the presence of various concentrations of UV-killed A. actinornyceterncornitans.

to Growth rate and Growth density parameter data, which can be statistically analyzed. The breakpoints for the two parameters depend on the growth of each species and mirror interference at a specific time for each experiment (Fig. lb). The results also show that the changes occur at between 4 and 12 h of cultivation, which might explain why no visible changes were detected on the blood agar plates. On the other hand, cocultivation of bacteria on the blood agar surface showed the ability of an S. sanguis strain to inhibit the hemolysis around the S. pyogenes colonies. Furthermore the ex- tent of interference between the two antagonistic strains of streptococci obtained with the agar surface method could be quantified with the Bioscreen method. The interference tests in the present study were performed between some bacterial strains that are commonly found in the mouth flora and have pathogenic potential (17, 18, 24). Interference between these bacteria has not been reported earlier in the literature even though A. actinomycetemcomitans, S. aureus, S. epidermidis, and S. rnitis are well-known producers of bacteriocins or bacteriocin-like substances active against other species (4, 10, 13, 26, 30). The strongest antagonism obtained between the tested bacterial strains was the ability of A. uctinomycetemcornitans to inhibit growth of P. gingivafis (Fig. 2), which was about half that obtained between viable S. pyogenes strains and the S. sanguis strain (Fig. 1). In a previous study presence of UV-killed P. gingivafis in culture medium of growing A. actinomycetemcomitans was shown to neutralize the cytotoxicity, while with UV-killed S. aureus present the Au cytotoxicity increased (14).

Bacterial interference is a multifactorial phe- nomenon comprising competition of nutrition,

pH changes, production of fatty acids and hydrogen peroxide, and release of enzymes and toxins (15, 23, 25, 29). The six different bacterial species tested in the present study were selected based on their various virulence and growth requirements. The present study estimates the amount of various UV-killed bacterial species that can be added to culture medium without decreasing growth of the viable strains. This in- formation will be of importance in further interaction studies estimating various effects of bacterial interference, but cannot contribute to clarifying the pathogenesis of periodontal diseases.

The clinical significance of the present results must be judged with caution as the interference studies were performed in vitro and using only two bacteria at a time. The number of bacterial species analyzed was low in relation to the high number in normal mouth flora.

Recent reports seem nevertheless to indicate that bacteria in the normal throat flora can pro- tect against infection by p-hemolytic streptococci in man (9, 16).

There are a number of reports confirming that the normal bacterial flora on the body plays a protective role against invasion of pathogens (9, 16, 26, 29), though it still remains unknown whether there is such a capacity in the gingival pocket preventing periodontal diseases.

As the periodontal flora is complex and more than 325 distinct species are found in the human mouth (22), periodontal diseases may not be de- fined simply by the presence of periodontal pathogens but more likely as a function of interactions between bacteria as well as between host and bacteria (8, 20, 27).

We thank Carin Olofsson, Department of Clinical Bacteriology, University of Umei, for technical ad- vice.

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Bacterial interference in vitro : Comparison between a quantitative kinetic and a cocultivation blood agar test method