| Objective:The study aims to provide scientific basis of antibiotic treatment for chronic periodontitis by isolating and culturing predominant anaerobes from chronic periodontitis during treatment, performing antibiotic susceptibility test and detecting drug resistant genes of the strains. It also aims to know the relationship between the virulence of Prevotella nigrescens and clinical symptoms.Methods:1. Twelve chronic periodontitis patients, including 6 males and 6 females, were enrolled in this study. Forty-one chronic periodontitis samples were collected and cultured on selective and non-selective culture media. After verifying bacteria tolerance of oxygen for 2-3 times, strict anaerobes were isolated from primary culture. The strains were then identified by 16S rRNA method.2. The antibiotic susceptibility test to 9 antibiotic, including ceftriaxone, cephradine, cefixime, amoxicillin, clindamycin, roxithromycin, metronidazole, doxycycline and imipenem, were performed by agar dilution method, according to the Clinical Laboratory Standards Institution(CLSI 2012).3. The presence of drug resistance genes of ermF, tetQ, nim and cfxA were detected by PCR assays.4. According to the results of 16S rRNA identification of molecular biology techniques, dorsal abscess formation in mice is used to preliminary analyze the virulence characteristics of Prevotella nigrescens.Results:1. A total of 84 different bacterial colonies were isolated and identified. Sixty-eight strains were of Prevotella genus.2. All the Prevotella species were sensitive to imipenem. The resistance rates of ceftriaxone, cephradine, cefixime were 2.94%,4.41% and 2.94%. The resistance rates of clindamycin, roxithromycin, amoxicillin were 22.06%,17.64% and 8.82%. The resistance rates of metronidazole and doxycycline resistance rates were 4.88% and 2.94%.3. In Prevotella species,73.53% strains were detected with cfxA gene and 58.82% amoxycillin-resistant strains were detected with ermF gene. Before treatment,73.33% Prevotella bacteria containd the resistance genes and the resistance rates was 1.47%; after treatment,97.37% Prevotella bacteria contained the resistance genes and resistance rates raised to 52.63%.4. Five Prevotella strains were harmful compared with the negative control. Compared with the positive control strain Prevotella melaninogenica ATCC25845, five strains showed different virulent properties in mouse dorsal abscess formation experiments. The clinical symptoms were positive related to the virulent properties.Conclusion:1. In our research, Prevotella species were the most frequently isolated bacteria from chronic periodontitis. As the continuity of treatment, the types of predominant anaerobes decreased.2. Our results alarm high resistant rate of clindamycin and roxithromycin, which makes its use unacceptable for the empiric therapy of chronic periodontitis. Meanwhile, the concern of reduced susceptibility to penicillins and metronidazole should be raised. Therefore, before useing antibiotics, antibiotic sensitivity should be tested, in order to reduce the generation of drug-resistant bacteria.3. The prevalence of resistant genes was differences. The detection rate of cfxA gene and ermF gene were high in resistant Prevotella species. After medication in patients, the predominant anaerobes appeared different levels of drug resistance. The original resistance genes were related to the evolution of drug resistance4. Primary virulence analysis showed virulence was associated to severity, treatment and clinical symptoms in patients with chronic periodontitis. |
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