In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 µg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.

Resistance profile survey of 50 periodontal strains of Actinobacillus actinomyectomcomitans.

BACKGROUND: Antibiotic resistance has been increasingly described among bacterial species colonizing periodontal pockets, particularly in Prevotella and Porphyromonas spp. strains producing beta-lactamases, and frequently associated with resistance to tetracycline and erythromycin. These resistance genes may be carried on motile genetic elements, or transposons, capable of interspecies and intergeneric transmission among bacterial strains colonizing a same ecological niche. The aim of this prospective study was to determine the resistance profile of Actinobacillus actinomycetemcomitans and the prevalence of A. actinomycetemcomitans strains producing beta-lactamases in periodontal pockets. METHODS: Fifty strains of A. actinomycetemcomitans were isolated from 42 patients with adult periodontitis. No patient had periodontal or antibiotic therapy in the previous 6 months. Bacterial samples were collected from periodontal pockets > or =5 mm, appropriately diluted, inoculated onto selective medium (chocolate blood agar with bacitracin 75 microg/ml and vancomycin 5 microm/ml) and incubated for 5 days at 37 degrees C in air with 5% CO2. After conventional identification, susceptibility testing to 11 antibiotics was performed by the broth dilution method, in trypticase soy broth supplemented with yeast extract, hemin, and 0.1% NaHCO3 to maintain microaerophilic conditions in the microtitration plate wells by CO2 formation. RESULTS: No strain demonstrated resistance to amoxicillin, amoxicillin-clavulanic acid combination, pristinamycin, or ciprofloxacin at the breakpoint, but 40% of the strains were slightly resistant to penicillin G, and 4% were resistant to erythromycin, 90% to spiramycin, 18% to clarythromycin, 4% to tetracycline, 72% to metronidazole, and 12% to ornidazole. Amoxicillin, followed by tetracycline and erythromycin, was the most effective antibiotic on A. actinomycetemcomitans. The phenotypic research of a beta-lactamase was negative for all the strains tested. CONCLUSIONS: In this work, most A. actinomycetemcomitans strains were resistant to metronidazole, but the amoxicillin-metronidazole association may be of interest against subgingival anaerobic and capnophilic mixed flora. Pristinamycin and ciprofloxacin appeared as effective alternative monotherapies against A. actinomycetemcomitans. The threat of beta-lactam antibiotic resistance related to beta-lactamase production is currently not a problem with A. actinomycetemcomitans as it has been reported in oral anaerobes.

beta-Lactamase producing bacteria in adult periodontitis.

In 23 untreated adult periodontitis patients, the occurrence of beta-lactamase producing periodontal bacteria was determined. In addition to non-selective isolation media, selective isolation and growth of beta-lactamase positive subgingival bacterial species was carried out on blood agar plates supplemented with amoxicillin and plates with amoxicillin+clavulanic acid. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Fusobacterium nucleatum, Bacteroides forsythus and Campylobacter rectus isolates from the non-selective medium were tested for beta-lactamase activity by a nitrocefin disk method (DrySlide) and by a laboratory chromogenic nitrocefin-based test. Isolates from the amoxicillin plates that were absent on the amoxicillin/clavulanic acid plates were identified and tested for beta-lactamase production. Based on the non-selective plates, six of 23 P. intermedia isolates, 2 of 19 B. forsythus isolates and 3 of 23 F. nucleatum isolates were beta-lactamase positive. The beta-lactamase positive species Prevotella loescheii, Prevotella buccae, Prevotella buccalis and Actinomyces spp were recovered from the selective amoxicillin plates. beta-Lactamase positive subgingival species were recovered from 17 of 23 patients (74%) but usually comprised low proportions of the subgingival microbiota (range < 0.01-15%). Comparison of the DrySlide test and the nitrocefin-based laboratory test revealed full agreement of test results. beta-Lactamase activity in whole subgingival plaque was detected in 12 patient samples (52%). It was concluded that beta-lactamase activity in subgingival bacteria in adult periodontitis is a common feature. However, since the majority of the samples showed only low-level enzymatic activity, the clinical relevance of this observation with regard to therapy with unprotected enzyme-susceptible beta-lactams is uncertain, though failure on the other hand, is difficult to rule out when a mechanism of resistance is present. The majority of beta-lactamase positive strains was found among species of the Prevotella genus.

Metronidazole susceptibility factors in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a facultative anaerobic bacterium that displays moderate susceptibility to metronidazole and this study was undertaken to identify the factors involved. A. actinomycetemcomitans appeared two to four times less susceptible to metronidazole when grown in air supplemented with 5% CO2 than under anaerobic conditions. Ferredoxin-linked pyruvate:oxidoreductase activity was absent but each strain exhibited nitroreductase activity which corresponded directly with uptake of metronidazole and susceptibility to the drug under anaerobic conditions but not in air supplemented with 5% CO2. Nitroreductase activity therefore appears responsible for the susceptibility of A. actinomycetemcomitans to metronidazole.