Analysis of Defense Mechanisms against Bacterial Infection by Oral Streptococcus in Normal Flora

Zbl. Bakt. 285, 74-81 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Analysis of Defense Mechanisms against Bacterial Infection by Oral Streptococcus in Normal Flora ISAO FUJIMORI, KAZUHITO KIKUSHIMA, KEN-ICHI HISAMATSU, IZURU NOZAWA, REI GOTO, and YOSHIHIKO MURAKAMI Department of Otolaryngology, Yamanashi Medical University, Yamanashi, Japan Received July 18, 1995 . Revision received February 5, 1996 . Accepted April 15, 1996

Summary The incidence of oral a-streptococci with inhibitory activity against pathogens as a defense mechanism in the oral cavity was investigated in healthy individuals. Inhibitory strains were isolated from tonsil, tongue, cheek, saliva and dental plaque, and the detection rate of these strains isolated from tonsil was the highest. These results suggested that tonsillar flora is most important as a defense mechanism of the oral cavity. With respect to the effects of antibiotics against inhibitory a-streptococci, minimal inhibitory concentration of 90% of cells (MIC 90 ) of PCG, ABPC, CCL, CFIX and EM against inhibitory a-streptococci, and relative detection rates of inhibitory a-streptococci before and after antimicrobial therapy were investigated. MIC 90s of all antibiotics against these strains were low and sensitive to antibiotics tested. However, in vivo, detection rates of these strains before and after therapy did not differ significantly. Therefore, inhibitory strains were not affected by antibiotics as their MIC 90 were low during short term medication. Introduction Although group A streptococci are the major causative agents of bacterial tonsillitis, concern regarding these pathogens has decreased since the development of antibiotics. However, group A streptococcal infection remains dangerous, since an increase in the incidence of the invasive disease designated "toxic shock syndrome" (4, 9) and a resurgence of rheumatic fever in the USA (6, 17) have been reported in recent years. Furthermore, infections by Staphylococcus aureus were frequent in the upper respiratory tract and in particular, we have taken notice of infections caused by methicillinresistant Staphylococcus aureus. It has been suggested that clinical isolation of an oral streptococcus group with inhibitory activity against pathogens may be one important factor in the development of a defense mechanism against infection in the upper respiratory tract (1, 2,5,12). The purpose of this study was to investigate the clinical significance of inhibitory a-streptococci in healthy individuals and discuss the possible correlation with bacterial infection.

Defense mechanisms against oral streptococcus

75

Materials and Methods From January 1994 to March 1995, throat swabs were obtained from 245 outpatients with no infection of the upper respiratory tract of Yamanashi Medical University. These 245 healthy individuals were divided into four age groups: 80 pre-school children (3 or 4 years old), 68 school children from lower elementary classes (from 7 to 9 years old), 59 university students (from 20 to 24 years old), and 38 older patients (more than 60 years old). All individuals tested and their families gave their informed consent prior to participation in this study. Throat swabs were obtained with transwabs ENT (Medical Wire Equipment Co. ltd. England) pressed firmly into the tonsillar crypts. Furthermore, in 60 subjects (15 pre-school children, 15 school children, 15 university students, and 15 older patients), swabs were also obtained from saliva, tongue, cheek and gingiva to investigate the distribution of inhibitory a-streptococci in the oral cavity. None of the subjects had been taking any antibiotics when the specimens were obtained. All swabs were brought immediately to the microbiology laboratory, then placed on the surfaces of 5% sheep blood agar plates (Tripticase Soy, BBl), and cultured aerobically at 37°C for 24 h. Ten macroscopically different colonies of oral a-streptococci were isolated from each primary blood agar plate and streak-cultured for 24 h at 37°C. A clinical isolate of Streptococcus pyogenes (6-22, T type 12) as an indicator strain was suspended in heart infusion broth (Difco) at a concentration of 10 8 colony-forming units (CFU)/mL. Thereafter, the indicator strain was vertically inoculated straight onto the astreptococci grown on the blood agar plate. Twenty-four hours after inoculation, the growth condition of S. pyogenes was examined. An assay was considered to indicate moderate inhibitory activity (+) if interference was apparent as an area only on the overlay surface where the growth of the indicator strain and hemolysis had been inhibited. If interference was detected in an area around the overlay surface, we considered the a-streptococcus examined to have strong inhibitory activity (++) against the indicator strain. To determine the antimicrobial activities of oral astreptococci with inhibitory activity against group A streptococci, we investigated their inhibitory activities against other indicator strains such as group A streptococcus (RF-b, mucoid type T-6), group B, C, G streptococcus, E. faecalis, S. pneumoniae, S. aureus (MSSA) and H. inf/uenzae by the method described above. To determine the influence of antibiotic treatment against inhibitory a-streptococci, we calculated MIC 90 values for benzylpenicillin benzathine (DBECPCG), ampicillin (ABPC), erythromycin (EM), cefaclor (CCl) and cefixime (CFIX) for the various isolates. Furthermore, we compared the detection rate of a-streptococci before and after antimicrobial therapy consisting of DBECPCG (3000 unitslkg body weight daily), ABPC (25 mglkg body weight daily), EM (30mg/kg body weight daily), CCl (30mglkg body weight daily) and CFIX (5 mg/kg body weight daily) each administered orally for 7 days, or intravenous ABPC (50 mglkg body weight daily), cefazolin (CEZ, 50 mglkg body weight daily) or clindamycin (20 mglkg body weight daily) for four days in 10 volunteers for each treatment. a-Streptococci with strong inhibitory activity and moderate inhibitory activity against group A streptococci (6-22) were selected at random for identification. These strains were examined biochemically by the API Strep. System® (Bio Merieux, S.A., France). Comparison of detection rates was performed by the X2 test, Wilcoxon's signed rank test and the Mann-Whitney test. Differences at p < 0.05 were considered to be significant.

Results We examined the oral a-streptococci with inhibitory activity against S. pyogenes isolated from among healthy individuals. Fig. 1 shows the inhibitory strains according to

76

I. Fujimori et al.

••

.g

~ 50

o

Pre-school children

School children

University students

Elderly subjects

n=80

n=68

n=59

n=38

• : inhibition mte (%)= Number of inhibitory a-streptococci Number of a-streptococci isolated n=number of individuals tested

• =strong inhibitory mte (%) ••

I11III =modemte inhibitory mte (%)

p
Fig. 1. Comparison of isolation rate of a-streptococci with inhibitory activity against group A streptococci among the different age groups. each age group. Approximately 49% of the strains detected as oral a-streptococci isolated from school children demonstrated inhibitory activity, while 68% and 94% of the strains from university students and older individuals, respectively, exhibited such activity. Thus, the average isolation rates of inhibitory a-streptococci increased significantly with age (p < 0.01), although the detection rate of inhibitory a-streptococci in pre-school children was 75% higher than that in school children (p < 0.01). However, the detection rate of only those a-streptococci with strong inhibitory activity was relatively low and showed a similar tendency to total a-streptococci with inhibitory activity. The qualitative and quantitative composition of the a-streptococci on the species level for the 5 sources is shown below. The 3 species are shown in the order of bacterial volume: Tonsil: S. mitis (4.4 X 108), S. sanguis (7.4 X 107), S. salivarius (5.3 X 107); Saliva: S. salivarius (6.2 X 108), S. mitis (2.8 X 108), S. sanguis (4.6 X 106); Gingiva: S. sanguis (3.6 X 107), S. milleri (1.9 X 106), S. mitis (1.2 X 106); Cheek: S. mitis (6.3 X 10 7), S. salivarius (4.1 X 10 7), S. sanguis (1.2 X 107); Tongue: S. salivarius (7.6 X 107), S. mitis (3.2 X 106), S. sanguis (8.6 X 105). We investigated the distributions in the oral cavity of a-streptococci with inhibitory activity against S. pyogenes and S. aureus (Table 1). The detection rate of a-streptococci with inhibitory activity against S. pyogenes in those strains which had been isolated from the tonsils was high-

Defense mechanisms against oral streptococcus

77

Table 1. Distribution of a-streptococci with inhibitory activity against group A streptococci and S. aureus in the oral cavity Indicator strain

Inhibitory activity

S. pyogenes

+;;;;;

Source Tonsil

Tongue

Cheek

Saliva

Dental plaque

49.0%

38.5%

37.9%

41.7%

40.0%

~

*

S. aureus (MSSA)

*

++

3.4

1.4

2.4

2.7

2.3

+;;;;;

41.3

28.1

32.1

32.0

31.6

6.2

7.8

7.5

~

++

16.0

*

4.3

~

**

**

**

**

"p
78

I. Fujimori et al.

Table 2. Antimicrobial activities of oral a-streptococci with strong inhibitory activity against group A streptococci Indicator Strains

Number of strains with inhibitory activity

Inhibitory Rate (%)*

Group A streptococcus (RF-b) Group B streptococcus Group C streptococcus Group G streptococcus

57(6) 23 (3) 54(3) 44(3) 54(6) 32(0) 31 (3) 55 (4)

100.0 40.4 94.7 77.2 94.7 56.1 54.4 96.5

Streptococcus pneumoniae Enterococcus faecalis Staphylococcus aureus Haemophilus in(luenzae

( ) strains with strong inhibitory activity. * Number of strains with inhibitory activity Number of strains tested (n =57)

Table 3. Effect of oral antibiotic treatment on inhibitory a-streptococci Groups

Isolation period

Indicator strains (group A streptococcus) non-mucoid

mucoid

Benzylpenicillin benzathine

before after

53.4% 57.9

47.6% 50.6

Ampicillin

before after

52.9 59.8

53.3 55.5

Erythromycin

before after

51.5 61.2

Cefaclor

before after

56.3 62.5

Cefixime

before after

53.7 } 63.3

}*

41.5 46.3 41.7 50.9

*

43.7 46.7

before: a-streptococci isolated before antibiotic treatment. after: a-streptococci isolated after antibiotic treatment. * p
However, detection rates of a-streptococci with inhibitory activity against mucoid strains were similar before and after treatment with antimicrobial agents. On the other hand, following intravenous administration of antibiotics, detection rates of astreptococci with inhibitory activity against non-mucoid strains were not significantly different from those before treatment (Table 4). a-Streptococci with inhibitory activity

Defense mechanisms against oral streptococcus

79

Table 4. Effect of intravenous antibiotic treatment on inhibitory a-streptococci Groups

Isolation period

Indicator strains (group A streptococcus) non-mucoid

mucoid

Ampicillin

before after

49.7% 57.2

53.3% 55.5

Cefazolin

before after

53.5 61.2

51.5 46.3

Clindamycin

before after

56.3 62.5

49.7 50.9

against mucoid strains were also detected at frequencies similar to those against nonmucoid strains after intravenous antibiotic treatment. By biochemical identification of inhibitory strains against group A streptococci, approximately 60% of the strains tested with moderate inhibitory activity were shown to consist of S. sanguis and 20%, of S. mitis. The remaining 20% of strains with moderate inhibitory activity belonged to other species. In contrast, approximately 70% of the tested strains with strong inhibitory activity against group A streptococci consisted of S. salivarius. The remaining 30% of strains with strong inhibitory activity belonged to other species. Approximately 65% of the tested strains without inhibitory activity against group A streptococci belonged to S. mitis and remaining 35% of to strains, other species. All strains with strong inhibitory activity also against other pathogens were identified as S. salivarius. Discussion Normal oral flora consisting of many microbes coexisting in fine balance in the oral cavity is an important element of the non-specific host defense mechanism. Upper respiratory tract infections may be caused by disturbance of the normal flora. Sherwood (16) first reported that some bacteria produced and released bacteriocins into the extracellular environment. Thereafter, numerous studies (1, 2, 5, 12) on a-streptococci with inhibitory activity against group A ~-streptococci have been reported. Several investigations suggested that the inhibition was related to the production of hydrogen peroxide (2), reduction of pH (14) and depletion of nutrients (13). Furthermore, researchers have attempted to purify bacteriocins produced by a-streptococci which constitute part of the normal pharyngeal flora such as S. sanguis (3, 15), S. mitis (3), S. salivarius (13) and S. mutans (7) and these compounds have been named viridin A, viridin B, enocin and mutacin, respectively. However, neither the distribution of (lstreptococci with inhibitory activity in the oral cavity nor a comparison of inhibitory a-streptococci isolated from subjects of different age groups have been reported in detail. Although several methods of investigating bacterial interference have been described, the method which we have devised and described here was simpler than those

80

1. Fujimori et al.

reported previously, and made it possible not only to investigate the inhibitory activities against several indicator strains simultaneously but also to distinguish between direct inhibitory activity and expansive inhibitory activity. Hardie (8) reported that from the number of the oral streptococci in the oral cavity, tonsillar flora was composed mainly of S. mitis, S. salivarius and S. sanguis. The flora of the lingual surface consisted mainly of S. salivarius, that of the cheek mainly of S. mitis and that of dental plaque mainly of S. mutans. The results of our study suggest that there is a degree of variation with respect to species in the degree of inhibitory activity within the oral cavity. Furthermore, although S. sanguis and S. mitis, which accounted for the majority of inhibitory strains isolated from the throat, playa major role in defense, attention should also be paid to S. salivarius. Moreover, in this study, the detection rate of inhibitory a-streptococci in tonsillar flora tended to be higher than in other samples, thus emphasizing that tonsillar flora plays the most important role against bacterial infection, particularly against S. aureus, in the oral cavity. In this study, older healthy individuals showed a higher detection rate of astreptococci with inhibitory activity than younger subjects supporting the presumption that a low level of inhibitory a-streptococci facilitates colonization of the tonsils by ~-streptococci and the higher frequency of streptococcal infection of the tonsils in school children than in adults. In particular, the high detection rate of streptococci with inhibitory activity in the healthy elderly group suggested the participation of a host defense mechanism against S. pyogenes in this age group. Although it was suggested that inhibitory a-streptococci were highly sensitive to antibiotics in vitro, detection rates of inhibitory a-streptococci before and after antimicrobial therapy were not found to be significantly different and inhibitory a-streptococci were not greatly affected by antimicrobial therapy in vivo. However, prolonged administration of antimicrobial drugs may be avoided as much as possible so as not to disturb the normal oral flora. Futhermore, Roos et al. (10, 11) investigated the incidence of a-streptococci with inhibitory activity combined with clinical treatment and reported that treatment of patients with ~-streptococcal tonsillitis/pharyngitis with antibiotics followed by recolonization with a-streptococci seems to hinder further recurrence. Accordingly, we should consider selective treatment, i. e. not only surgical intervention but also antimicrobial therapy, for treatment of streptococcal infection.

References 1. Crowe, C. c., W. E. Sanders, and S. Longley: Bacterial Interference 2. Role of the normal throat flora in prevention of colonization by group A streptococci. J. Infect. Dis. 128 (1973) 527-532 2. Dajani, A. S., M. C. Tom, and D.]. Law: Viridans, bacteriocins of alpha-haemolytic streptococci. isolation, characterization, and partial purification. Antimicrob. Agents Chemother. 9 (1976) 81-88 3. Dajani, A. S., C. N. Veres, and D. J. Law: Substances that interfere with action of Viridin B, a streptococcus mitis bacteriocin. Infect. Immun. 20 (1978) 20-24 4. Edward, K. c., D. G. Jeremy, I. L. Larry, J. Kim, M. Levi, M. H. Kim, and P. M. Schlievent: Streptococcal toxic syndrome due to noninvasive pharyngitis. Clin. Infect. Dis. 14 (1992) 1074-1077 5. Grahn, E., S. E. Holm, C. Ekedahl, and K. Roos: Interference of a-hemolytic streptococci isolated from tonsillar surface on ~-hemolytic streptococci (Streptococcus pyogenes). Zbl. Bakt. Hyg. 1. Abt. Orig. A. 254 (1983) 459-468

Defense mechanisms against oral streptococcus

81

6. Griffiths, S. P. and W. M. Gersony: Acute rheumatic fever in New York City (1969 to 1988): a comparative study of two decades. J. Pediatr. 116 (1990) 882-887

7. Hamada, S. and T. Oshima: Inhibition spectrum of a bacteriocin like substance (mutacin) produced by some strains of Streptococcus mutans. J. Dent. Res. 54 (1975) 140145 8. Hardie, J. and P. D. March: Streptococci and the human oral flora. (Nelson, G. E. and W. Redman, eds.), pp.157-205, Academic Press., New York (1978) 9. Jackson, M. A., V. F. Burry, and L. C. Olson: Multisystem Group A ~-hemolytic streptococcal disease in children. Rev. Infect. Dis. 13 (1991) 783-788

10. Roos, K., E. Grahn, S. E. Holm, H.Johansson, and L. Lind: Interfering a-streptococci

as a protection against recurrent streptococcal tonsillitis in children. J. Pediatr. Otorhinolaryngo!. 25 (1993) 141-148 11. Roos, K., S. E. Holm, and L. Lind: Alpha-streptococci as supplementary treatment of recurrent streptococcal tonsillitis: A randomized placebo-controlled study. Scand. J. Infect. Dis. 25 (1993) 31-35 12. Sanders, C. c., G. E. Nelson, and W. E. Sanders Jr.: Bacterial interference. Epidemiological determinants of the antagonistic activity of the normal throat flora against group A streptococci. Infect. Immun. 16 (1977) 599-603 13. Sanders, C. and W. Sanders: Enocin: Antibiotic produced by Streptococcus salivarius that may contribute to protection against infection due to group A streptococci. J. Infect. Dis. 146 (1982) 683-690 14. Sanders, E.: Bacterial interference. Its occurrence among the respiratory tract flora and characterization of inhibition of group A streptococci. J. Infect. Dis. 120 (1969) 698707 15. Schlegel, R. and H. D. Slade: Properties of a streptococcus (Group H) bacteriocin and its separation from the competence factor of transformations. J. Bacterio!' 115 (1973) 655-661 16. Sherwood, N. P., B. E. Russel, and A. R. Jay: Studies on streptococci. New antibiotic substances produced by beta hemolytic streptococcus. J. Infect. Dis. 84 (1949) 88-91 17. Veasy, L. G., S. E. Wiedmeier, G. S. Orsmond, H. D. Ruttenberg, M. M. Boucek, S. J. Roth, V. F. Tait, J. A. Thompson, E. L. Kaplan, and H. R. Hill: Resurgence of rheumatic fever in the intermountain area of the United States. New Eng!. J. Med. 316 (1987) 421-427 Dr. Isao Fu;imori, Department of Otolaryngology, Yamanashi Medical University, 1110 Shimo-kato Tamaho-cho, Yamanashi 409-38, Japan

6 Zbl. Bakt. 285/1