Abstracts

Microbiologia

Antimicrobial effect of human serum on oral Fusobacterium nucleatum isolates from humans and monkeys

Atividade antimicrobiana do soro humano sobre isolados de Fusobacterium nucleatum obtidos de humanos e macacos

Elerson GAETTI-JARDIM JÚNIOR^** PhD, Department of Pathology, Faculdade de Odontologia de Araçatuba - UNESP PhD, Department of Pathology, Faculdade de Odontologia de Araçatuba - UNESP

Mario Julio AVILA-CAMPOS^*** PhD, Department of Pathology, Faculdade de Odontologia de Araçatuba - UNESP PhD, Department of Pathology, Faculdade de Odontologia de Araçatuba - UNESP

GAETTI-JARDIM JÚNIOR, E.; AVILA-CAMPOS, M. J. Antimicrobial effect of human serum on oral Fusobacterium nucleatum isolates from humans and monkeys. Rev Odontol Univ São Paulo, v. 13, n. 1, p. 1-4, jan./mar. 1999.

The susceptibility of eighty F. nucleatum isolates from periodontal patients, healthy subjects and Cebus apella monkeys to human pooled sera was tested. The resistance to serum bactericidal effects was observed in 46.9% of the isolates from periodontal patients, 28.6% of the healthy subjects and 40% of the monkeys. These results support the hypothesis that serum plays an ecological role by controlling the microbial population inside either the gingival crevice or periodontal pocket.

UNITERMS: Bacteria, anaerobic; Periodontal diseases; Microbiology.

INTRODUCTION

Fusobacterium nucleatum is a Gram-negative non-spore forming anaerobic rod, that belongs to the indigenous oral, intestinal and vaginal microflora, and it has been associated in many infectious processes such as sinusitis, osteomyelitis, brain and lung abscesses, and particularly periodontal diseases^2,11,16,18. It has also been suggested that F. nucleatum plays an important role in the formation and maturation of dental plaque¹⁷.

The pathogenic role of anaerobic bacteria has been demonstrated in experimental infections using animal models^4,5,6. These anaerobes produce toxic compounds such as hydrogen sulfide, methylmercaptans^9,22, butirate and propionate³, which adhere to lymphocytes²⁶ oral epithelial cells, erythrocytes, fibroblasts, leukocytes, collagen IV, laminin and fibronectin^10,27,28.

Many factors may influence the host-parasite relationship in infections produced by these amphibiontic bacteria that belong to the normal microflora. One of these factors is the microbial susceptibility to serum which participates in the hosts defense response against microbial invasion²⁴.

On the other hand, F. nucleatum habitates periodontal pockets and gingival crevices, where the serum components are in high levels and flow to the oral cavity, supplying nutritional and microenvironmental factors to bacterial survival. However, there are few reports on bactericidal effect of human serum against periodontopathogens.

Thus, the aim of this study was to evaluate the bactericidal effect of human pooled sera on oral F. nucleatum isolates of human and animal origin.

MATERIAL AND METHODS

A total of eighty F. nucleatum isolates were recovered from thirty periodontal patients, twenty healthy subjects and ten Cebus apella monkeys (Núcleo de Procriação do Macaco-Prego, São Paulo, SP, Brazil): forty nine, twenty one and ten isolates, respectively. These isolates were tested for their susceptibility against the bactericidal effect of human sera. The bacterial isolation was performed by using Omata & Disraely selective agar, incubated under anaerobic conditions (90% N₂, 10% CO₂), at 37^oC, for 4 days^1,12, and identification of the isolates was carried out by using biochemical tests^14,23.

Bacteria were grown briefly in PY-glucose broth, under anaerobic conditions, at 37^oC, for 48 h²⁴. Then, they were collected (3.000 x g, 8 min, 4^oC), washed with blank solution¹⁴ and resuspended in the same solution to obtain a concentration of approximately 5 x 10⁶ CFU/mL, as determined by OD_550nm of 0.7 (DME-21 spectrophotometer, Digimed, SP, Brazil).

Sera A, B, O, AB, Rh-positive and negative from healthy human adults were pooled and stored at -70^oC in aliquots of 1.0 ml and thawed just before use. One mL of bacterial suspension and 1.0 mL of human serum were mixed and incubated under anaerobic conditions for 2 h. Then, a 0.1 mL aliquot of each mixture was removed at zero time and after 2 h. The number of viable bacteria was assayed after a two-fold serial dilution and plating on blood agar plates (Brain heart infusion agar supplemented with 0.5% yeast extract enriched with 5% defibrinated sheep blood). Inoculated plates were incubated under anaerobic conditions, at 37^oC, for 3 days. F. nucleatum ATCC 10953, F. nucleatum ATCC 25586 and Bacteroides fragilis ATCC 23745 were also included in the tests.

Isolates were considered susceptible when presenting a 25% decrease of initial inoculum. Tubes containing bacteria mixed with inactived sera (56^oC x 30 min) were used as control. Statistical analysis was performed using a comparative Fischer’s test.

RESULTS

Table 1 shows that values for resistance or susceptibility reached approximately 50% in periodontal patient and monkey isolates. However, most of isolates from healthy subjects were susceptible. All of the isolates were grown in inactived sera.

There are few reports about susceptibility of F. nucleatum against the bactericidal activity of human serum, although a relationship between serum resistance and virulence has been demonstrated in Gram-negative bacteria⁸. Studies have shown that bacterial strains recovered from infectious processes are more resistant to serum than those from normal microflora⁷. Namavar et al.¹⁹ (1991) showed that non-virulent strains of B. fragilis were more susceptible to serum than the virulent ones.

During the evolution of periodontal disease an increase of gingival fluid and exsudate in crevice and in pockets is observed, which contains several serum factors. On the other hand, pathogens are exposed to serum bactericidal effects that appear to be a microbial regulation process, where resistant strains could invade periodontal tissues. However, susceptible strains would be eliminated from that environment and it could become an important host defense mechanism²⁴.

Serum factors are involved in acting as bactericidal mechanisms, such as a complementary system²⁴, which is an important factor for bacterial opsonization in apical and marginal periodontopathies^20,21. The role of the serum and its bactericidal activity in periodontal diseases remains unclear.

Monkey isolates have shown similar behaviour to those from periodontal patients. This could be explained by the close contact of the animals with maintenance people that eventually resulted in bacterial cross-contamination.

Horiba et al.¹⁵ (1992) have shown that LPS from F. nucleatum induces the complementary activation. This activation can occur by a classical or alternative pathway^13,20. On the other hand, Ter Steeg et al.²⁵ (1987) by using subgingival plaque treated with human serum, showed a selection of Bacteroides species, Peptostreptococcus micros and F. nucleatum.

Our results suggest that serum plays an ecological role by controlling the microbial population inside either the gingival crevice or periodontal pockets. However, other studies must be done to characterize the relationship between antimicrobial serum activity and pathogenesis of periodontal diseases.

Acknowledgments

We thank Mr. David Whaley who reviewed this manuscript, and Andemir da Silva and João Paulo Ribeiro for their technical assistance. This study was supported partially by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Grant 91/0483-0, São Paulo, SP, Brazil.

GAETTI-JARDIM JÚNIOR, E.; AVILA-CAMPOS, M. J. Atividade antimicrobiana do soro humano sobre isolados de Fusobacterium nucleatum obtidos de humanos e macacos. Rev Odontol Univ São Paulo, v. 13, n. 1, p. 1-4, jan./mar. 1999.

Foi avaliada a susceptibilidade de oitenta isolados de F. nucleatum obtidos de pacientes com doença periodontal, indivíduos sadios e Cebus apella (macaco-prego) frente ao soro humano. A resistência à atividade bactericida do soro foi observada em 46,9% das fusobactérias isoladas de pacientes com doença periodontal, 28,6% das obtidas de indivíduos sadios e em 40% das fusobactérias de primatas não humanos. Esses resultados suportam o conceito de que o soro possui um papel ecológico em controlar a população microbiana no interior do sulco gengival ou bolsa periodontal.

UNITERMOS: Bactérias anaeróbias; Doenças periodontais; Microbiologia.

BIBLIOGRAPHIC REFERENCES

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Recebido para publicação em 24/03/98

Reformulado em 18/11/98

Aceito para publicação 13/01/99

** PhD, Departament of Microbiology, Institute of Biomedical Sciences - USP

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