Fall 2005

Scientific Article

Spirochete evasion into murine peritoneal tissue produces resistance to antibiotics cholorhexidine
(peridex) and Tetracycline HCI
Submitted by John D. Miller, 4th year student at the University of Southern California

INTRODUCTION

Periodontal diseases arise from problems in the gum sulcus usually because of an increase or change in the type of bacteria. Spirochetes are represented throughout subgingival and supragingival plaque and are the predominant microflora in areas of periodontal disease and tissue deterioration. (1,2) They are undetectable or in low numbers at sites with good periodontal health. (3,11) Studies suggest that some spirochetes damage periodontal tissues by secreting enzymes and/or cytotoxic substances. (6)

To date, the most successful treatment for periodontal disease is to remove bacterial plaque. (12) Antibiotics are one of the standard treatments performed to remove bacteria. Some bacteria are resistant to repeated administration of antibiotics. A major goal of dentistry is to prevent recurrence of periodontal disease. To do this, oral spirochetes must be eliminated in order to keep spirochetes from reproducing to log-phase.

Several species of Treponema including T. denticola, T. socranskii and T. maltophilum invasively penetrated the subgingival tissue. (16, 25) To inhibit these spirochetes there are varieties of antibiotic administrations available. Topical application of antibiotics can result in a more effective drug concentration than other administration techniques, and is the preferred method of delivery for treatment of periodontal disease. (23) Some spirochetes, however, may be able to avoid topical antibiotics until antibiotic concentrations fall below the minimum inhibitory concentration (MIC). Afterward, these spirochetes may relocate to the supragingival tissue and cause recurring periodontal diseases. This study investigated whether invasive spirochetes in the genus Treponema could avoid topical antibiotics by penetrating tissues.

MATERIALS AND METHODS

Bacterial strains, culture conditions, and growth conditions for mice: Three invasive species, T. denticola ST10 serovar D, T. socranskii subsp. buccale (ATCC 35534), T. maltophilum sp. nov. (ATCC 51939T), and one non-invasive species T. phagedenis subsp. biotype Reiter were used. (10,16,25) Bacteria were grown in 10 ml of NOS broth (ATCC medium #1357) and maintained at 37° C in a gas pack jar under anaerobic conditions. Primary cultures were subcultured twice before chamber experiments were conducted. C3H/HeNCrl female retired breeder mice were maintained and sacrificed in accordance with institutional guidelines. (10)

Antibiotics and Determination of Minimum Inhibitory Concentration (MIC): Tetracycline HCI (Boehringer Mannheim: Indianapolis, IN, cat. #109428) and Peridex (0.05% chlorhexidine rinse-Zila Pharmaceuticals Inc.; Phoenix AZ.) were the antibiotics used in this study. For each treponeme a minimum inhibitory concentration (MIC) needed to be measured so that each bacterial strain would be successfully inhibited. To determine MIC's, two-fold dilutions of media containing a concentration of 20 mg of tetracycline ml- 1 NOS were performed to a lowest concentration of 1.25 mg of tetracycline ml-1 NOS. To determine MICs for different bacterial densities, MICs were determined using ten-fold dilutions of a primary culture containing 10 (8) bacteria ml-l of media to a lowest density of 10 (4) bacteria ml-l of media. Spirochetes were exposed to each concentration of antibiotic at each density for 2 hours. Subcultures from the antibiotic were transferred to fresh, antibiotic-free media and incubated under anaerobic conditions at 37° C for 7 days. Viability was determined by observation of spirochetes growth and motility under dark-field microscopy. MICs for Peridex were determined using the same procedure.

CHAMBER ASSAYS:

Mice were asphyxiated using dry ice for 10 minutes. The abdominal wall covering the peritoneal cavity of each mouse was then excised under aseptic conditions. Excised tissues were kept in 0.85% saline, containing rifampin (40 mg ml-l), polymixin B. (800 units ml-1), and clindamycin (10 mg ml-l) for less than 30 minutes before they were used in chambers. Culture chambers were assembled by placing the piece of abdominal wall, between the halves of the dialysis cell to produce a tissue barrier chamber as described previous. (10)

After entry and exit side solutions were removed, to be sure that the chambers were intact and no leakage had occurred, 2 drops of sterile 0.4% of trypan blue and 2 ml 0.85% saline was added to the entry side, while 2 ml 0.85% saline was added to the exit side. Chambers were left for three days to verify that no blue color crossed from entry to exit side. Spectrophotometry analyses were performed with 2 hours exit fluid from each chamber, and a base wavelength was determined from a trypan blue solution high peak of 6742.

With each experimental chamber a control chamber without antibiotic was used to confirm spirochetes were able to grow under the same conditions. 1\vo additional controls were set up with each experiment. 1 ml of treponemes was inoculated into a tube of 10 ml NOS broth 1 (positive control) and into a second tube of 10 ml NOS broth 2 (negative control.) Tubes were incubated anaerobically at 37° C along with the entry and exit subcultures.

Treponema and antibiotics in tissue:
T. denticola was the only treponema successful in evading chlorhexidine. No treponeme was successful in evading tetracycline (Table 2). The observation that two of the invasive treponema were not able to evade the antibiotic suggests that chlorhexidine does infiltrate tissue. To further explore antibiotic penetration through tissue, two additional chambers were set up. These chambers were employed for the antibiotic inhibitory time and then entry and exit sides were inoculated with 15 ml bacteria ml-l NOS broth. Placed under anaerobic conditions for 7 days they were observed under dark-field microscopy.

Discussion

The purpose of this study was to determine whether spirochetes in murine peritoneal tissue could successfuly avoid topical antibiotics. 1\vo frequently-used antibiotics were employed; a bacteriostatic antiobiotic (tetracycline) and a bactericidal antibiotic (chlorhexidine). Treponema used were facultative anaerobes, and though they are found throughout the dentition, they have an extremely low oxygen tolerance. (24) Previous studies have found that oral spirochetes are not uniformly distributed within the oral flora and that they are commonly found with high density in plaque from subjects with periodontitis. (4,6,13-17,22)

Previous studies evaluated leakage of chambers by use of radiolabelling.IO,21 These studies evaluated passage of radiolabelled spirochetes through membranes by scintillation counting and by dark-field microscopy. For this study equipment necessary for a radiolabelling technique was not available, so spectrophotometry was used to analyze the integrity of the chamber.

The use of two additional chambers with just antibiotics employed for the antibiotic inhibitory time showed that tetracycline penetrated through tissue to the exit side whereas chlorhexidine did not penetrate through tissue to the exit side. Through the knowledge gained by studying r socranskii and r maltophilum chambers in this study, we know that chlorhexidine pentrates into tissue. We can presume along with previous unpublished results, (Riviere et al.) that T. denticola has a more active locomotion than either T. socranskii and T. maltophilum. Thus, T. denticola is able to progress into tissue and through to the exit side before chlorhexidine moves into tissue.

T. denticola populates areas of periodontal disease and can produce recurring inflammation and disease.(12,18,21) Previous research identified that a protein from Porphyromonas gingivalis promotes growth of T. denticola. (7) This gives rise to the possibility that other spirochetes which may evade topical antibiotics, including T. denticola, may also have proteins containing growth promoting factors for other pathogens that produce recurring periodontal diseases.

Kasuga et al. (5) showed that relationships between spirochetes P. gingivalis, Bacteroides forsythus and
T. denticola present in periodontal pockets shows greater bleeding on probing than when only one or two of these species are present. Detection of all three of these micro-organisms waS highly correlated with adult periodontitis. Noiri et al.(8) found that T. denticola and P. gingivalis were found in the "plaque-free zone," or the area where plaque organisms live within a biofilm covered with glycocalyx at the bottom of periodontal pockets. They hypothesized that some bacteria, including
T. denticola and P. gingivalis, were related to plaque-biofilm fonnation of subgingival plaque and possible periodontal disease.

Recent studies support the idea that there is a pathogenic relationship between periodontal diseases and endodontic infections. (9,19,20) Periodontal pathogens, including T. denticola and P. gingivalis, found in the endodontium often accompany endodontic infections.

Results of our study show evasion of chlorhexidine by invasive penetration through tissue by T. denticola. The presence of T. denticola was found to be prevalent with gingival inflammation in subjects with existing periodontitis. (13) Future research on growth promoting proteins from T. denticola, the "plaque-free zone," and the relationships with endodontic and periodontal diseases would produce a better understanding of how periodontal diseases occur and recur and how oral spirochetes are involved with relationships between periodontic-endodontic interrelationships.

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Corresponding author: George R. Riviere, DDS, Kathryn S. Smith, BS

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