versão impressa ISSN 0001-3714
Rev. Microbiol. v.30 n.4 São Paulo out./dez. 1999
Elerson Gaetti-Jardim Júnior; Mario Julio Avila-Campos*
Departamento de Microbiologia, Instituto de Ciências Biomédicas-ICB, Universidade de São Paulo-USP, São Paulo, SP, Brasil
Submitted: June 07, 1999; Returned to authors for corrections: September 02, 1999; Approved: December 02, 1999
Fusobacterium nucleatum is indigenous of the human oral cavity and has been involved in different infectious processes. The production of bacteriocin-like substances may be important in regulation of bacterial microbiota in oral cavity. The ability to produce bacteriocin-like substances by 80 oral F. nucleatum isolates obtained from periodontal patients, healthy individuals and Cebus apella monkeys, was examinated. 17.5% of all tested isolates showed auto-antagonism and 78.8% iso- or hetero-antagonism. No isolate from monkey was capable to produce auto-inhibition. In this study, the antagonistic substances production was variable in all tested isolates. Most of the F. nucleatum showed antagonistic activity against tested reference strains. These data suggest a possible participation of these substances on the oral microbial ecology in humans and animals. However, the role of bacteriocins in regulating dental plaque microbiota in vivo is discussed.
Key words: Fusobacterium nucleatum, bacteriocin-like substance, oral bacteria.
Anaerobic bacteria comprise a large percentage of the oral and gut indigenous microbiota (9). Some anaerobic bacteria possess several potentially pathogenic factors, particularly Gram-negative rods which appear to be present in several anaerobic infections, such as periodontal diseases (23).
Fusobaterium nucleatum is indigenous of the human oral cavity and has been involved in several infectious processes such as, sinusitis, osteomyelitis, brain or liver abscesses (3, 9, 14,17). Also, this organism constitutes a considerable part of the subgingival microbiota of gingivitis in children and adults and of periodontitis in juveniles and adults (18).
The microbial composition of the established dental plaque may be controlled by nutrient requirements or production of antagonistic substances (6). However, the nature of the inhibitory substances is still a matter of discussion. Microbial antagonistic substances were first studied by Gratia and Fredericq (11), who showed the iso-inhibitory activity produced by Enterobacteriaceae. These colicin-like substances were called bacteriocins (13). The knowledge of bacteriocin production has been extended to Gram-positive and Gram-negative aerobes, facultative and strict anaerobes.
From an ecological viewpoint, there are few studies concerning production of antagonistic substances, such as bacteriocins, in Gram-negative anaerobes indigenous to the human microbiota, particularly, in oral F. nucleatum from human and animal origin. However, the production of metabolic products such as bacteriocin-like substances may be important in ecological processes to the colonization of periodontal tissues by F. nucleatum or other members from human indigenous microbiota (5).
In this study, the production of bacteriocin-like substances by F. nucleatum isolated from humans and Cebus apella monkeys is reported. The monkeys were used because of anatomic similarities with the human oral cavity.
MATERIALS AND METHODS
A total of 80 oral F. nucleatum isolates were tested for their bacteriocinogenic activity. Fourty-nine isolates were obtained from 30 patients with adult periodontitis (age range 18 - 40 years), with clinical and radiographic evidence of periodontal disease, including pockets of depth equal or exceeding 5 mm, 21 isolates from 20 healthy individuals (age range 20 - 30 years) from the Clinic of Periodontology, University of São Paulo, SP, Brazil, and 10 isolates were from 10 Cebus apella monkeys without evidence of periodontal disease (Núcleo de Procriação do Macaco Prego, São Paulo, SP, Brazil). Subgingival bacterial samples were taken using sterilized absorbent paper points (Dentsply Ind. & Co. Ltda., Rio de Janeiro, RJ, Brazil), which were introduced into the periodontal pocket or gingival sulcus for 60 s and then transferred to tubes containing 2.0 ml of Ringer-PRAS solution, pH 7.2, under CO2 flux (22). The bacterial isolation and identification to the species level was performed by using Omata & Disraely selective agar as described previously (1, 10) and by using conventional biochemical tests (12, 24).
The isolates were tested for their inhibitory activity against themselves and against F. nucleatum ATCC 10953, F. nucleatum ATCC 25586, Bacteroides fragilis ATCC 23745, Eubacterium lentum ATCC 25559 and Peptostreptococcus anaerobius ATCC 27337. The bacteriocinogenicity was determined by using a double layer method (4).
The bacterial inoculum was prepared in brain-heart infusion broth (BHI, Difco) supplemented with 0.5% extract yeast (12). 10 ml of 24 h cultures were inoculated in 5 equidistant spots onto BHI agar (BHI-A), using a standard platinum loop. Plates were incubated at 37oC for 72 h under anaerobic conditions (10% CO2/ 90% N2). After incubation, the cells were killed by exposure to chloroform for 30 min (8). Residual chloroform was allowed to evaporate and BHI-A plates were overlayed with 3.5 ml of BHI-soft agar (0.7%) inoculated with 0.1 ml of a 48 h culture of each indicator. After 48 h of incubation under anaerobic conditions, the plates were evaluated for the presence of bacteriocin-like substances. The inhibitory halo was measured. Experiments were done in duplicate.
Test for the presence of bacteriophages
A piece (3.0 mm in diameter) of agar at the inhibitory halo was removed aseptically (26). This block of agar was homogeneized, placed onto BHI-A and overlayed with 3.0 ml of BHI-soft agar inoculated with 0.1 ml of the indicator culture. After 48 h of incubation under anaerobic conditions, the plates were evaluated for the presence or absence of lytic zones. Tests were done in duplicate.
Table 1 shows the inhibitory activity of 80 oral F. nucleatum isolates recovered from periodontal patients, healthy individuals and monkeys, tested against themselves. Auto-antagonism was observed in nine isolates from periodontal patients, and in five isolates from healthy individuals. However, no auto-antagonism was observed among the monkey isolates. Iso-antagonism was not observed only in three isolates from periodontal patients, seven isolates from healthy individuals and six monkey isolates. The non-producing hetero-antagonism isolates were six from patients, nine from healthy individuals, and one from monkey.
All isolates showed antagonistic activity against all tested reference strains (Table 2). None of the F. nucelatum isolates from periodontal patients or monkeys produced antagonistic substances against F. nucleatum ATCC 10953. On the other hand, none of the isolates obtained from healthy individuals was capable of inhibiting B. fragilis ATCC 23745. None of the F. nucleatum isolates obtained from healthy individuals or monkeys produced antagonistic substances against P. anaerobius ATCC 27337. However, only 3 (6.1 %) of periodontal isolates inhibited this microorganism (Table 2). All the diameters of the inhibitory zones were between 10 and 13 mm (data not shown). In tests for the presence of phages, no lytic zone with the same indicator organisms was observed, indicating that the inhibition halos were not due to the presence of bacteriophages.
The production of antagonistic substances may play an important role in the microbial colonization of the human or animal oral cavity, leading to ecological alterations in the indigenous microbiota. Because bacteriocin-like substances are produced by a high number of oral microorganisms, a constant and complex intra- or inter-specific regulation can be expected (8).
Among F. nucleatum isolated from humans (periodontal patients and healthy individuals) and from Cebus apella monkeys, in terms of auto-, iso- and hetero-antagonism, 14 (17.5%) presented auto-antagonism and 63 (78.8%) iso- or hetero-antagonism. Interestingly, the inhibitory activity produced by isolates from periodontal patients was those intense than that produced by isolates from healthy individuals or monkeys (data not shown). Significant differences among hetero-antagonism levels from periodontal and from healthy isolates were observed (Table 1), suggesting an association with the predominance of this organism in periodontal sites.
Auto-antagonism is more prevalent in Gram-positive than in Gram-negative organisms (21, 28). In some Gram-negative bacteria, such as Pseudomonas solanacearum and B. fragilis, this phenomenon has not been observed (7, 8, 16). Our results show auto-inhibition in 18.4 % and 25 % of the periodontal and healthy isolates, respectively. No auto-antagonism was observed in animal isolates. Azevedo et al. (2) showed that the frequency of bacteriocin-producing strains among oral streptococci, including auto-, iso- and hetero-antagonism, is variable.
Antimicrobial proteins are produced by several pathogenic and non-pathogenic bacteria. However, little is known about antagonistic substances in F. nucleatum. Auto-antagonism among the tested F. nucleatum was observed in a small number of isolates, where as, iso- and hetero-antagonism were observed in a large number of them, suggesting that the bacteriocin-like activity in F. nucleatum is variable. This can have a great ecological significance, particularly in colonization or regulation of the autochtonous microbiota of the host (15).
Tagg et al. (25) showed that bacterial immunity to their own auto-antagonistic products is due to the synthesis of a specific protein which acts as an immunity factor, and the formation of a complex between bacteriocin and this factor would protect the bacteriocin-producing strain.
The diameters of the inhibition zones varied from 10 to 13 mm with a higher frequency of 13 mm and with sharp delimitation (data not shown). These data are similar to those reported by Farias et al. (8). Bacteriophages were not observed since the inhibitory action was not transmissible as shown by the tested method (26), excluding the possibility of phages causing the bacteriocin-like effect have been suggested by several authors (14, 20).
Bacteriocin synthesis appear to be an unstable characteristic since some microorganisms lose and recover the capacity to produce it. However, Oliveira and Drozdowicz (19) suggested that the capability to produce bacteriocin is expressed by a small proportion of the bacterial population. On the other hand, it also is possible that one isolate may produce more than one antagonistic substance with different physico-chemical and biological properties. However, some components of the growth medium have been implicated in the induction of bacteriocins (25). However, Weerkamp et al. (27) suggested that some components of the nutrient media protect some organisms against bacteriocin action. The chemical nature, genetic determinants and environmental factors that affect the phenotypic expression of the bacteriocin-like production are not totally determined. Since the production can be masked by presence of similar or different substances, studies about characterization and purification of these products should help to a better knowledgement of its action on oral microbiota of human and animal origin.
The authors would like to thank to Dr. Brent Lasker for his critical review of this manuscript, and Andemir da Silva and João Paulo Ribeiro for their technical assistance. This study was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Grant 91/0483-0, São Paulo, SP, Brazil.
Atividade semelhante a bacteriocina de Fusobacterium nucleatum orais isolados de primatas humanos e não-humanos
Fusobacterium nucleatum é indígena da cavidade oral humana e tem sido envolvido em diferentes processos infecciosos. A produção de substâncias semelhantes a bacteriocinas pode ser importante na regulação da microbiota bacteriana da cavidade oral. A capacidade de produzir substâncias tipo bacteriocina de 80 isolados de F. nucleatum orais, obtidos de pacientes com doença periodontal, indivíduos sadios e macaco Cebus apella, foi avaliada. 17,5% de todos os isolados mostrou auto-antagonismo e 78,8% iso- ou hetero-antagonismo. Nenhum isolado de macaco foi capaz de produzir auto-inibição. Neste estudo, a produção de substâncias antagonístas foi variável em todos os isolados testados. A maioria dos F. nucleatum mostrou atividade antagonísta para as cepas de referência testadas. Esses dados sugerem a possível participação dessas substâncias sobre a ecologia microbiana em humanos e animais. Entretanto, o papel das bacteriocinas na regulação da microbiota da placa dental in vivo é discutida.
Palavras chave: Fusobacterium nucleatum, substância tipo bacteriocina, placa dental.
1. Avila-Campos, M. J.; Raymundo, N. L. S.; Farias, L. M.; Silva, M. A. R.; Carvalho, M. A. R.; Damasceno, C. A. V.; Pereira, L. H.; Cisalpino, E. O. Isolation and identification of strains of Bacteroides fragilis group from the digestive tract of Callithrix penicillata marmosets. Lab. Animals, 24: 68-70, 1990.
2. Azevedo, R. V. P.; Zelante, F.; Ito, I. Y. Detecção de cepas de Streptococcus mutans produtoras de substâncias semelhantes a bacteriocina (mutacina). Rev. Facul. Odontol. de Ribeirão Preto, 22: 67-74, 1985.
3. Bartlett, J. G. Anaerobic infections of the lung and pleural space. Clin. Infect. Dis., 16: 248-255, 1993.
4. Booth, S. J.; Johnson, J. L.; Wilkins, T. D. Bacteriocin production by strains of Bacteroides isolated from human feces and the role of these strains in the bacterial ecology of the colon. Antimicrob. Agents Chemother., 11: 718-724, 1977.
5. Bolstad, A. I.; Jensen, H. B.; Bakken, V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin. Microbiol. Rev., 9: 55-71, 1996.
6. Carlsson, j. Growth of Streptococcus mutans and Streptococcus sanguis in mixed culture. Arch. Oral Biol., 16: 963-965, 1971.
7. Cuppels, D. A.; Hanson, R. S.; Kelman, A. Isolation and characterization of a bacteriocin produced by Pseudomonas solanacearum. J. Gen. Microbiol., 109: 295-303, 1978.
8. Farias, L. M.; Carvalho, M. A. R.; Damasceno, C. A. V.; Cisalpino, E. O.; Vieira, E. C. Bacteriocin-like activity of Bacteroides fragilis group isolated from marmosets. Res. Microbiol., 143: 151-159, 1992.
9. Finegold, S. M. Anaerobic Bacteria in Human Disease. Fourth Edition. Academic Press, New York, USA, 1977.
10. Gaetti-Jardim, Jr. E.; Zelante, F.; Avila-Campos, M. J. Oral species of Fusobacterium nucleatum from human and environmental samples. J. Dent., 24: 345-348, 1996.
11. Gratia, A.; Fredericq, P. Diversité des souches antibiotiques de Bacterium coli et étendue variable de leur champ d'action. C. R. Soc. Biol., 140: 1032-1033, 1946.
12. Holdeman, L. V.; Cato, E.; Moore, W. E. C. Anaerobe Laboratory Manual. Fourth Edition, Blacksburg: Virginia Polytechnic Institute and State University, USA, 1977.
13. Jacob, F.; Lwoff, A.; Siminovitch, E.; Wollman, E. Définition de quelques termes relatif à la lysogénic. Annals Inst. Pasteur, 84: 222-224, 1953.
14. Jetten, A. M,; Vogels, G. D.; Windt, F. Production and purification of a Staphylococcus epidermidis bacteriocin. J. Bacteriol., 112: 235-242, 1972.
15. Jousemies-Somer, H.; Savolainen, S.; Mäkitie, A.; Ylikoski, J. Bacteriologic findings in peritonsilar abscess in young adults. Clin. Infect. Dis., 16: 292-298, 1993
16. Mossie, K. G.; Jones, T. D.; Robb, F. T.; Woods, D. R. Characterization and mode of action of a bacteriocin produced by a Bacteroides fragilis strain. Antimicrob Agents Chemother., 17: 838-841, 1979.
17. Moore, W. E. C; Moore, L. V. H. The bacteria of periodontal diseases. Periodontology 2000, 5: 66-77, 1994.
18. Moore, L. V. H.; Moore, W. E. C.; Riley, C.; Brooks, C. N.; Buroneister, J. A.; Smibert, R. M. Periodontal microflora of HIV positive subjects with gingivitis or adult periodontitis. J. Periodontol., 64: 48-56, 1993.
19. Oliveira, R. G. B.; Drozdowicz, A. Bacteriocin in the genus Azospirillum. Rev Microbiol, 12: 42-47, 1981.
20. Reeves, P. Defective bacteriophages, In: Reeves, P. The bacteriocins. Second Edition. Springer-Verlag, Heidelberg, Berlin, pp. 17-18, 1972.
21. Ryan, F. J.; Fried, P.; Mukai, F. A colicin produced by cells that are sensitive to it. Biochem. Biophys. Acta, 18: 131, 1955.
22. Slots, J. Selective medium for Actinobacillus actinomycetemcomitans. J. Clin. Microbiol., 15: 606-609, 1982.
23. Slots, J.; Genco, R. F. Black-pigmented Bacteroides and Capnocytophaga species and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival and tissue destruction. J. Dent. Res., 63: 412-421, 1984.
24. Summanen, P. H.; Baron, E. J.; Citron, D. M.; Strong, C.; Wexler, H. M.; Finegold, S. M. Wadsworth Anaerobic Bacteriology Manual. Fifth Edition. Singapore: Star Publishing Company, 1993.
25. Tagg, J. R.; Dajani, A. S.; Wannamaker, L. W. Bacteriocin of Gram-positive bacteria. Bacteriol. Rev., 40: 722-756, 1976.
26. Turner, J. W.; Jordan, H. V. Bacteriocin-like activity within the genus Actinomyces. J. Dent. Res., 60: 1000-1007, 1981.
27. Weerkamp, A.; Vogels, G. D.; Skotinicki, M. Antagonistic substances produced by streptococci from human dental plaque and their significance in plaque ecology. Caries Res., 11: 245-256, 1977.
28. Yamada, M.; Takazole, I.; Okuda, K. Bacteriocinogenicity of oral Bacteroides species. Bull Tokyo Dent College, 28: 55-61, 1978.