Combined antimicrobial chemotherapy (fluoroquinolones and imidazoles) in the complex treatment of inflammatory diseases of the periodontal

Introduction

A special place in the complex treatment of chronic generalized periodontitis is played by antimicrobial therapy, which, according to most researchers, should be aimed at: elimination of acute inflammatory process (exacerbation); antimicrobial sanitation of periodontal tissues; changing the ratio between pathogenic (periodontopathogenic) and non-pathogenic (stabilizing) microflora towards the latter's side; prevention of systemic and local infectious complications, including those arising from invasive interventions; prevention of the development of superinfection, including fungal lesions; reducing the level of microbial "load" and thereby helping the body to fight infection [1—5].

Without undervaluing the role of personal and professional oral hygiene and local drug treatment in periodontal diseases, which are given a significant place in the modern literature [1, 6—8], it should be noted that mechanical (physical) treatment and local antimicrobial action does not allow to altogether remove periodontal pathogenic microbes from the tissues in the developed inflammatory process, primarily due to their intracellular localization and the formed multilayered microbial biofilm [3, 9].

Therefore, the combination of mechanical removal of microbes together with the systemic administration of antimicrobial chemotheraputics and antibiotics is considered the most effective method of complex treatment. the basis for conducting systemic antimicrobial therapy in the treatment of periodontitis is unstable and insufficient sanitation of periodontal pockets (periodontal complex tissues) from representatives of periodontal pathogenic species during professional hygiene and using only local antibacterial therapy [3, 5, 9—11].

Alongside this, a multiple resistance of some periodontal pathogens (P. gingivalis, A. actinomycetemcomitans, T. forsythia, P. intermedia, T. denticola, etc.) is noted to antimicrobial chemotherapy drugs [12—17]. In this regard, in addition to monotherapy (the appointment of a single drug), a combination of antibacterial chemotheraputics is widely used in mixed infections, which includes periodontitis, which makes it possible to cover the possible spectrum of resistance to antibiotics from the microorganisms [1, 2, 5, 10, 11].

As is known, in inflammatory periodontal diseases, the leading role in the occurrence and development of the process is assigned to the microbial biofilm, which contains periodontopathogenic species of the red and orange complex, according to S.S. Socransky (1997) [18]. Therefore, one of the requirements for antibiotics used in periodontology is the drug's ability to easily penetrate inside through the microbial biofilm's protective cover, affecting microbes in all phases of biofilm formation [3, 7, 11].

The group of medicament that penetrates the microbial biofilm includes, in particular, fluoroquinolones and 5-nitroimidazoles, which have high, although unequal, activity against the main periodontal pathogenic species. Besides, the combination of drugs of these groups has a synergistic effect [19—22].

In this regard, the work aimed to study the effect of a combination of a chemotherapy drug of the fluoroquinolone group (Ciprofloxacin) and a group of 5-nitroimidazole (Tinidazole) on the clinical manifestations and microbial composition of the biofilm in chronic generalized periodontitis.

Materials and methods

58 patients with exacerbation of chronic generalized periodontitis of moderate severity were examined. the diagnosis of CGP was made following the Russian Clinical recommendations (treatment protocols) for the diagnosis of periodontitis, 2014. All patients underwent professional hygiene following Clinical recommendations (protocols), if necessary — correction of occlusion.

In the group I (main) a combined drug of 500 mg of Ciprofloxacin with 600 mg of Tinidazole (Cifran СT) was prescribed, in the II group (comparison) — Amoxicillin with clavulanic acid (Amoxiclav). the duration of the course of antimicrobial therapy was 10—12 days.

To assess the condition of periodontal disease, we used the simplified Greene—Vermillion index of oral hygiene, the papillary-marginal-alveolar index in the modification of Parma (PMA) to assess the intensity of gingivitis, and the Mühlemann—Saxer papillary bleeding index (PBI), which allows us to judge the severity of inflammation.

The values of periodontal indices were determined in each patient three times: before professional hygiene and after 10—12 days (after the end of the antimicrobial chemotherapy course). When assessing the main parameters of the state of the periodontal complex tissues, the latest international recommendations on periodontics of the EFP/ORCA workshop were taken into account [23].

According to standard protocols, isolation and cultivation of the isolated strains were carried out after microbial culture before treatment and on the 12th day. After taking the material (biofilm) from the periodontal pocket, microbial culture was carried out. Primary inoculation for the isolation of gram-negative facultative anaerobic bacteria and the microaerophilic group's representatives was carried out on a growth medium M832 (HiMediaLabs, India) — anaerobic agar according to Wilkins—Chalgren with the addition of 5% blood.

The culture was placed in a thermostat at 37°C for 120 hours, after being placed in an anaerostat HiAnaerobic System Mark III (Himedia Labs, India). After obtaining a pure culture, identification was performed using the sets of Biochemical Identification TestKits (Himedia Labs, India). A modification of the serial dilution method was used to determine the sensitivity of the isolated strains to the studied films' action [3].

In the statistical processing of the results, the quantitative values are presented in the median (Me) and quartiles (Q25; Q75). the "K-means" clustering method was used to determine high and low values intervals. the Shapiro—Wilk criterion determined the correspondence of the sample distributions to the normality conditions. the Mann—Whitney test was used to estimate the difference in related indicators. Differences with a probability of differences p<0.05 were considered statistically significant.

Results

During the clinical examination after treatment, patients of both groups showed an improvement in periodontal tissues' clinical condition, which was reflected in a statistically significant decrease in index indicators (Table. 1), which approached the level of healthy individuals. the values of OHI-S, PMA, and PBI decreased in group I by 6.6, 3, and 4 times, respectively; in group II — 6, 2 and 1.6 times, respectively. Meanwhile, statistically significant differences were found between the groups in terms of changes in the PMA index, which characterizes the intensity of the inflammatory reaction (p=0.029): in the main group, its decrease was more pronounced than in the comparison group, as well as in the PBI bleeding index. There were no significant differences between the groups in terms of hygiene indices (p=0.507 and p=0.215, respectively).

Note: Differences before and after treatment and between groups after treatment are statistically significant (p<0.05)

To adequately characterize the effect of antimicrobial chemotherapy in the comparison groups on the pathogens and microbiocenosis of periodontal pockets, the total degree of microbial contamination, the frequency of detection of periodontal pathogens and residents, as well as fungi of the genus Candida were evaluated with an assessment of the degree of contamination with yeast fungi (Table 2).

Note: * statistically significant difference in the groups compared to the previous control period; ** statistically significant difference from group II indicators (p<0.05).

It was found that the total degree of microbial contamination (more than 10⁹ CFU/ml) corresponded to a high level of microbial contamination compared to the established norm (5—6 lg CFU/ml) [3].

Prior to the start of periodontal treatment, representatives of periodontal pathogenic species of the 1st order (at least one of P. gingivalis, T. forsythia, A. actinomycetemcomitans) were identified by PCR diagnostics in 100% of patients in both groups. Representatives of Candida spp. microbial culture were also determined in 13.8 and 17.2% of both groups' patients (the differences are not significant).

We drew attention to the fact that after periodontal treatment, against the background of systemic combined chemotherapy in 2 weeks, both groups observed a statistically significant decrease in the degree of microbial contamination to a normal level, and not only due to the eradication of periodontal pathogens, but also partially-residents (α-green streptococci and Corynebacteria). the frequency of detecting these crucial components of microbiocenosis decreased from 100% to 68.9 and 86.2% for group II and I, respectively. At the same time, there were significant differences in Candida spp., the indicator of which was 1.5 times higher in group II, and the frequency increased 2.4 times compared to group I.

In the control period after 1 month, the differences in the composition of microbiocenosis were partially levelled. However, in group I, the indicators were more favourable: the total microbial contamination was significantly lower, and the resident microbiota was detected in all treated patients, the frequency of detection of periodontal pathogens was 3 times lower, and yeast fungi — 1.7 times lower than in group II (p<0.05).

Thus, it can be argued that after 10—12 days of the systemic use of the antibiotic, a pronounced dysbiotic shift was observed with an increase in the population of yeast fungi, which after 1 month normalized in group I but persisted in group II.

As a clinical example, we can cite microbiological studies' results with data on the sensitivity of bacteria to antimicrobial chemotherapy drugs.

Clinical case 1

Patient G., 67 years old, with moderate exacerbation of CGP. Before treatment, the material was taken from the periodontal pocket for microbiological studies and determination of sensitivity to antibiotics. α-hemolytic streptococci (S. constellatus, S. intermedius), Corynebacteria (Corynebacterium spp.), in association with periodontal pathogenic species T. forsythia, P. gingivalis and A. actinomycetemcomitans, as well as enterobacteria in significant numbers, were isolated. Sensitivity to the studied drugs: Ciprofloxacin-highly sensitive, Tinidazole-sensitive (except for enterobacteria), Amoxicillin/clavulanate-highly sensitive.

After professional hygiene, Cifran CT is prescribed for 1 tablet 2 times a day for 12 days.

When re-examined, the degree of microbial contamination corresponds to the normal level (4·10⁵ CFU/ml), the microbiota is mainly represented by α-green streptococci (S. sanguis, S. salivarius) in association with C. albicans in a small amount (100 CFU/ml). Clinically, the inflammatory process was stopped.

Clinical case 2

Patient V., 52 years old, with moderate CGP. In the preliminary study, the periodontopathogenic flora representatives were identified — α-hemolytic streptococci (S. sanguis, S. intermedius), β-hemolytic streptococci, in association with periodontopathogenic species of pigment-forming bacteroids T. forsythia, A. actinomycetemcomitans and P. intermedia, fungi of the genus Candida. the bacterial microbiota's sensitivity to the studied antimicrobial drugs: Ciprofloxacin-highly sensitive, Tinidazole-highly sensitive, Amoxicillin/clavulanate sodium — highly sensitive.

After professional hygiene, Amoxiclav is prescribed 1 tablet 2 times a day for 12 days.

After the end of the course of antimicrobial chemotherapy, α-hemolytic streptococci were not detected; periodontal pathogenic bacteria's association is determined. T. forsythia Candida spp. in high amounts (3·10⁵ CFU/ml). Clinically, the inflammatory process signs remain but without the release of exudate from the periodontal pockets.

Discussion

In the first clinical case, when using Cifran CT, there was a complete rehabilitation of periodontal pockets. Periodontopathogens were not determined. In the second case, despite the sensitivity of the isolated species of periodontopathogens to Amoxicillin, the periodontopathogen of the 1st order — T. forsythia, which is prone to intracellular parasitism, was preserved, and the number of Candida spp. increased sharply, which indicates the development of dysbiosis. In recent years, researchers have found more and more evidence for the role of fungal or mycobiofilm formed by yeast fungi as a basis for colonization by other pathogens [25—27].

Experimental data obtained in our previous studies using scanning electron microscopy indicate that Ciprofloxacin concentration with Tinidazole (Cifran CT) approaching the MSC determines the complete destruction of the biofilm mantle and partial damage to the microbial cells of P. gingivalis [20]. This confirms the drug's active effect on the polymicrobial biofilm of the periodontium and indicates its high antimicrobial and antibiotic film effectiveness.

Probably, given the almost identical sensitivity of most periodontal pathogens to these drugs, this can be explained by the different ability of the drugs used to penetrate the microbial biofilm and affect its matrix. It should also be considered that all groups of obligate anaerobes colonizing the oral cavity (classes of bacteroids, fusobacteria, clostridium, etc.) are highly sensitive to Tinidazole [2, 3, 28].

The use of Amoxicillin with clavulanic acid (despite the relatively high sensitivity of many periodontopathogens) is apparently limited because it acts only on extracellularly located bacteria and bacteria in the upper layers of the biofilm. Simultaneously, the combined preparation of fluoroquinolone (Ciprofloxacin) with Tinidazole withal leads to the death of intracellularly located periodontopathogens and the destruction of the biofilm matrix.

Thus, summarizing the above material and literature data, we can conclude that for effective eradication of periodontal pathogenic species of microorganisms, it is necessary to overcome the following factors that determine the development of periodontal pathology:

• intracellular location of periodontopathogenic microbes [2, 12, 18];
• protective effect of microbial biofilm [7, 9, 14, 16 ,22];
• deep penetration of microbes into tissues (not available for mechanical and local antimicrobial action [5, 11];
• a short duration of action and a decrease in the concentration of the antimicrobial agent when applied topically due to dilution with oral fluid [2, 3, 6];
• decreased activity of local factors of mucosal immunity and activation of the proinflammatory cytokine cascade [12, 29—31].

Conclusion

Thus, after 2 weeks of the systemic use of antibacterial chemotherapy drugs, in the case of beta-lactams, a pronounced dysbiotic shift was observed with an increase in the population of yeast fungi, which persisted in group II after 1 month, while in group I (main) — normalized.

The inclusion of combined antimicrobial chemotherapy of Ciprofloxacin with Tinidazole (Cifran CT) in the algorithms of complex treatment of chronic generalized periodontitis increases the effectiveness of eradication of the periodontal pocket and periodontal complex tissues, both due to damage to the biofilm matrix and easier penetration into the biofilm (compared to beta-lactam antibiotics) and due to the synergistic effect of the drug components on the bacterial cells of pathogens themselves.