Abstracts

MICROBIOLOGY

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

Atividade in vitro de minociclina sobre microrganismos superinfectantes isolados de pacientes com periodontite crônica

Luciana Fernandes de Oliveira^I; Antonio Olavo Cardoso Jorge^II; Silvana Soléo Ferreira dos Santos^III

^IMaster's in Periodontology

^IIProfessor and Chair of Microbiology and Immunology Research and Graduate Studies Division, Department of Dentistry

^IIIAssistant Professor of Microbiology and Immunology, Department of Dentistry – University of Taubaté

ABSTRACT

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 eficaz 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 suficiente 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 factors¹⁴. 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 inflammation¹¹.

Enterobacteria, Pseudomonas, Staphylococcus and Candida have been collected from periodontal pockets of chronic periodontitis subjects²³ and are described as superinfectious microorganisms¹⁹. 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 superinfection²⁷.

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. Eighty-four (84) strains were included, Enterobacteriaceae (n = 25), Staphylococcus spp. (n = 25), Pseudomonas aeruginosa (n = 9), Candida spp. (n = 25).

Initially the microorganisms were transferred to Brain Heart Infusion broth (BHI, Difco, Detroit, USA) and inoculated in selective media. MacConkey 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 duplicate¹⁶.

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 × 10⁸ 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 surface¹⁶. 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 MIC₅₀ and MIC₉₀ variation of minocycline for Staphylococcus aureus between 0.25 and 8 µg/mL. Gales, Jones³ (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, Jones³ (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 infections¹³. Ichimiya et al.⁵ (1994) found MIC values for Pseudomonas between 1.56 µg/mL and 3.13 µg/mL. Gales, Jones³ (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.

Satomi²¹ (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/mL²⁹ concentration was detected, representing, in the present study, a MIC able to inhibit 97% of the superinfectious microorganisms in the first 24 hours.

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 (MIC₅₀ 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:

ACKNOWLEDGMENTS

The authors thank Dr. Ivan Balducci, from the State University of São Paulo (UNESP) at São José dos Campos, for his valuable statistical data analysis. They also wish to thank Cristiane Yumi Koga-Ito for revising this work.

Received for publication on Aug 18, 2005

Sent for alterations on Dec 09, 2005

Accepted for publication on May 19, 2006

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