In vitro minocycline activity on superinfecting microorganisms isolated from chronic periodontitis patients

Chronic periodontitis is the most common type of periodontitis and it is associated with various species of microorganisms. Enteric rods, Pseudomonas, Staphyloccocus and Candida have been retrieved from periodontal pockets of patients with chronic periodontitis and correlated to cases of superinfection. Local or systemic antibiotic therapy is indicated to reinforce the effects of the conventional mechanical therapy. Minocycline has been suggested as one of the most effective drugs against periodontal pathogens. The aim of this work was to evaluate the minimal inhibitory concentration (MIC) of minocycline on superinfecting microorganisms isolated from the periodontal pocket and the oral cavity of individuals with chronic periodontitis. Isolates of Enterobacteriaceae (n = 25), Staphylococcus spp. (n = 25), Pseudomonas aeruginosa (n = 9) and Candida spp. (n = 25) were included in the study. Minimal inhibitory concentrations (MIC) of minocycline were determined using the Müeller-Hinton agar dilution method. Staphylococcus spp. isolates were the most sensitive to minocycline with a MIC of 8 μg/mL, followed by Enterobacteriaceae with a MIC of 16 μg/mL. The concentration of 16 μg/mL inhibited 96% of Candida spp. isolates. The MIC for 88.8% of the isolates of Pseudomonas aeruginosa was 128 μg/mL. A concentration of 1,000 μg/mL was not enough to inhibit 100% of the tested isolates. DESCRIPTORS: Periodontitis; Minocycline; Minimum inhibitory concentration. RESUMO: Periodontite crônica é a forma mais comum de periodontite e está associada a diversas espécies de microrganismos. Enterobactérias, Pseudomonas, Staphyloccocus e Candida têm sido recuperados de bolsas periodontais de indivíduos com periodontite crônica e implicados em casos de superinfecção. A terapia antimicrobiana local ou sistêmica pode ser utilizada para reforçar os efeitos da terapia mecânica convencional, e a minociclina tem sido sugerida como antimicrobiano efi caz frente a periodontopatógenos. O objetivo deste trabalho foi avaliar a concentração inibitória mínima (CIM) de minociclina sobre microrganismos superinfectantes isolados de bolsas periodontais e cavidade bucal de indivíduos com periodontite crônica. Foram utilizadas 84 cepas de microrganismos, incluindo Enterobacteriaceae (n = 25), Staphylococcus spp. (n = 25), Pseudomonas aeruginosa (n = 9) e Candida spp. (n = 25). A CIM foi determinada pelo método de diluição em ágar Müeller-Hinton. Staphylococcus foram os microrganismos mais sensíveis a minociclina com CIM de 8 μg/mL, seguidos por Enterobacteriaceae com CIM de 16 μg/mL. Entre as espécies de Candida, 96% foram inibidas na concentração de 16 μg/mL. Para 88,8% das cepas de Pseudomonas aeruginosa a CIM foi de 128 μg/mL. A concentração de 1.000 μg/mL não foi sufi ciente para inibir 100% das cepas testadas. DESCRITORES: Periodontite; Minociclina; Concentração inibitória mínima.


INTRODUCTION
Periodontitis is a multi-factorial disease and dental biofilm is considered the initiator of periodontal disease 4,7,10,24 .However, the manifestation and progression of periodontitis are influenced by a wide variety of factors, including subject characteristics, social and behavioral factors, systemic factors, genetic factors, microbial composition of dental biofilm and other emerging risk factors 14 .Several hundred recognized species of microorganisms, and many more that have yet to be identified, inhabit the gingival crevice.However, it has been shown that only few species play a significant role in the etiology of periodontal dieseases.This evidence is largely based on epidemiologic data, the ability of a microorganism to produce disease when inoculated in animals, and the capacity to produce virulence factors.Therefore, the mere presence of putative periodontopathogens in the gingival crevice is not sufficient to initiate or cause periodontal inflammation 11 .
Enterobacteria, Pseudomonas, Staphylococcus and Candida have been collected from periodontal pockets of chronic periodontitis subjects 23 and are described as superinfectious microorganisms 19 .Such microorganisms present virulence factors that contribute to their action on the periodontal tissues, as leukotoxins and colagenases produced by Staphylococcus, the production of endotoxin (LPS) by enteric bacilli, and exotoxin A produced by Pseudomonas 15,17,25 .
Minocycline is an antimicrobial drug with large spectrum of activity that has been used as adjunct to periodontal treatment, through systemic or local administration, due to its efficacy on periodontopathogens 1,29 .Its antifungal effect has been already referred to when used alone or in association with other antimicrobial drugs 8,18,22,28 .However, an antibiotic therapy of large spectrum can promote the development of resistant or opportunistic pathogens, and consequent superinfection 27 .
The purpose of this in vitro study was to assess the minimum inhibitory concentration (MIC) of minocycline on Enterobacteriaceae, Pseudomonas, Staphylococcus and Candida isolates from periodontal pockets and oral cavities of chronic periodontitis patients.

MATERIALS AND METHODS
All subjects included in the present study signed an Informed Consent which was previously approved by the Institutional Committee on Research Involving Humans (Protocol of the University of Taubaté Ethics Committee #035/02).
The microorganisms included in the study belonged to the Culture Collection of the University of Taubaté (CCUT) and were isolated during previous studies from periodontal pockets and oral cavities of chronic periodontitis patients 9,12,20  Initially the microorganisms were transferred to Brain Heart Infusion broth (BHI, Difco, Detroit, USA) and inoculated in selective media.MacCo-nkey agar (Difco, Detroit, USA) was used for Enterobacteriaceae and Pseudomonas aeruginosa; Baird-Parker agar (Difco, Detroit, USA), for Staphylococcus spp.; and Sabouraud agar (Difco, Detroit, USA), for Candida spp.The plates were incubated at 37°C for 24 h.
The minimum inhibitory concentration (MIC) of minocycline was determined using the method of dilution in Müeller-Hinton Agar (Difco, Detroit, USA), in duplicate 16 .
The antimicrobial agent (minocycline, Deg import, Italy, batch 0202009CX2) was sterilized by filtration, using 0.22 μm Millipore membrane and 0.1 ml of each dilution was added to the Müeller-Hinton Agar media at 50°C.Plate series were prepared containing from 0.25 to 256 μg/mL of minocycline in sequential dilutions multiple of two, plus 340, 500 and 1,000 μg/mL concentrations.
Each microbial sample was suspended in 10 ml of saline (0.9% NaCl) until a density corresponding to 3 × 10 8 cells/mL was obtained (tube #1 of McFarland scale), inoculated with the aid of Steers replicator, and the plates were incubated at 37°C for 24 h.Readings were performed every 24 h.Media without the addition of minocycline was used as positive control.
Readings were performed by observing the presence or absence of microbial growth on the agar surface 16 .Descriptive statistical analysis was used to interpret the results.

RESULTS
All tested microorganisms developed in the media without addition of minocycline (control group) after incubation for 24 h at 37°C.
Enterobacteriaceae (n = 25) presented a MIC between 4 and 16 μg/mL and strains of Staphylococcus spp.(n = 25), between 0.25 and 8 μg/mL.Among the tested Pseudomonas aeruginosa (n = 9) isolates, 88.89% showed a MIC between 64 and 128 μg/mL, whereas 55.56% (cumulated percentage) were inhibited at 64 μg/mL and one strain was resistant at the 1,000 μg/mL concentration level.Fungi of the Candida genus (n = 25) showed a MIC between 0.25 and 16 μg/mL after 24 h of incubation at 37°C, except for one strain that grew at the 1,000 μg/mL concentration level.
MIC absolute and cumulated frequencies, as well as the cumulated percentages, for all tested microorganisms are shown on Table 1.The absolute frequency corresponds to the number of strains inhibited in each concentration.

DISCUSSION
The results obtained in the present study for Staphylococcus spp.agree with those presented previously by Trzcinski et al. 26 (2000), who described MIC 50 and MIC 90 variation of minocycline for Staphylococcus aureus between 0.25 and 8 μg/mL.Gales, Jones 3 (2000) found that the MIC variation for minocycline for Staphylococcus aureus was 0.06 to 8 μg/mL, and Fluit et al. 2 (2001), between 1 and 4 μg/mL.However, Wilson et al. 30 (1991) presented different results with MIC varying from 6 to 128 μg/mL.This fact could be justified by Trzcinski et al. 26 (2000) who found TetM proteins (known to confer resistance to all tetracyclines including minocycline) in Staphylococcus aureus samples, explaining the development of resistance to minocycline by these isolates.
Our data showed that the MIC for Enterobacteriaceae varied between 4 and 16 μg/mL while Gales, Jones 3 (2000) observed a variation between 0.25 and 8 μg/mL.The presentation of data by concentrations > (greater than) or < (smaller than) prevents a clear comparison of the MIC obtained in the present study with that of other results, because > 8 μg/mL may represent a great variety of results.According to Ikeda et al. 6 (1999) the MIC for E. coli was 0.78 μg/mL.Although strains of the same species demonstrate variation concerning sensitivity to minocycline, 16 μg/mL was enough to eliminate all isolates belonging to the Enterobacteriaceae family tested in the present study as well as in those mentioned in the literature 3,6 .
Pseudomonas aeruginosa is one of the most resistant bacteria to antimicrobial agents and is one of the main causes of hospital infections 13 .Ichimiya et al. 5 (1994) found MIC values for Pseudomonas between 1.56 μg/mL and 3.13 μg/mL.Gales, Jones 3 (2000) observed MIC variation between 0.25 μg/mL and > 8 μg/mL.The present work's results demonstrated greater resistance to minocycline by these microorganisms, with MIC variation between 64 μg/mL and > 1,000 μg/mL.However, the incubation period in the study by Ichimiya et al. 5 (1994) was 16 h, which may have caused the variation in the results.Although the antibiotic therapy may eliminate microorganisms, these authors described that sub-inhibitory concentrations of minocycline may suppress the adhesion and expression of Pseudomonas spp.virulence factors.
Satomi 21 (1987) observed that after minocycline gel application (1,000 μg), a 130 μg/mL subgingival concentration was reached in the first hour, replaced by a 3.4 μg/mL concentration after 72 hours; such concentration would inhibit between 42.8 and 47.6% of the superinfectious microorganisms tested in this research.Fourteen days after the 1,000 μg minocycline application by a slow release device, a 340 μg/mL 29  In spite of minocycline being identified as an antibacterial substance, its antifungal action was observed in several studies 8,18,22,30 .The MIC of minocycline for Candida spp.observed by Schierholz et al. 22 (1999) was 256 to 512 μg/mL, after 18 h of incubation at 37°C.Wilson et al. 30 (1991) reported a MIC of 128 to 256 μg/mL after ten days of incubation.In the present work, Candida showed a MIC of 0.25 to > 1,000 μg/mL (MIC 50 4 μg/mL), after 24 h of incubation at 37°C.
The literature shows that minocycline is effective against the majority of periodontopathogens in low concentrations, but for superinfecting microorganisms such efficacy could not be clearly shown by the present work, because such microorganisms presented a MIC variation from 0.25 to > 1,000 μg/mL, and even the higher concentrations tested were not enough to inhibit 100% of the strains.

CONCLUSIONS
After analysis of the results, we concluded that: a) Staphylococcus was the microorganism most sensitive to minocycline, presenting a MIC value of 8 μg/mL, followed by Enterobacteriaceae, with a MIC of 16 μg/mL.b) Ninety six percent of the Candida spp.isolates were inhibited by concentrations up to 16 μg/mL.c) For 88.8% of the Pseudomonas aeruginosa strains, the MIC was 128 μg/mL.d) The 1,000 μg/mL concentration was not enough to inhibit 100% of the strains tested.