Objectives: Aggregatibacter actinomycetemcomitans is major putative bacterial pathogen in human periodontitis, particularly in aggressive periodontitis in younger-aged individuals. Systemic administration of certain antibiotics in combination have been demonstrated to exert synergistic antimicrobial effects against A. actinomycetemcomitans, and to markedly enhance elimination or suppression of A. actinomycetemcomitans from the subgingival dental plaque microbiome of periodontitis patients beyond that attained by periodontal mechanical debridement/surgery. However, studies on antibiotic synergisms against periodontal A. actinomycetemcomitans were conducted over 20 years ago, and only involved subgingival clinical isolates of the organism from periodontitis patients in Europe. Since temporal and geographic changes in antimicrobial susceptibility are documented among periodontitis-associated microorganisms, it is not known whether or not antibiotic synergisms against periodontal A. actinomycetemcomitans are present today among clinical isolates of the organism as recovered from periodontitis patients in the United States. As a result, the purpose of the present study was to assess the potential in vitro antimicrobial synergisms between amoxicillin plus metronidazole, between ciprofloxacin plus metronidazole, and between spiramycin and metronidazole, against clinical periodontal isolates of A. actinomycetemcomitans of United States origin. Methods: Standardized cell suspensions, equivalent to a 1.0 McFarland turbidity standard, were prepared with four fresh clinical isolates of A. actinomycetemcomitans, each recovered from the subgingival microbiota of United States periodontitis subjects, and plated onto to the surfaces of 150-mm diameter culture plates containing Haemophilus test medium. After drying, antibiotic-impregnated, quantitative, gradient diffusion strips (MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, ciprofloxacin, spiramycin, and metronidazole were placed apart from each other onto the inoculated Haemophilus test medium surfaces, so that two test antibiotics per plate were employed against each A. actinomycetemcomitans clinical isolate for antibiotic susceptibility testing of individual antibiotic drugs. After 48 hours incubation in air + 5% CO2, individual MIC values for each antibiotic against A. actinomycetemcomitans were read in ug/ml at the point where the edge of the bacterial inhibition ellipse intersected with the MIC Test Strip, with the occurrence of antibiotic resistance among the A. actinomycetemcomitans clinical isolates determined using Haemophilus species antibiotic resistance breakpoint concentrations established by the United States Clinical Laboratory Standards Institute (CLSI) and the Société Francaise de Microbiologie. In vitro synergy testing of amoxicillin plus metronidazole, ciprofloxacin plus metronidazole, and spiramycin plus metronidazole was performed after determination of individual antibiotic MIC testing by placing MIC Test Strips for each of the two test antibiotics per combination in a cross formation onto the surfaces of A. actinomycetemcomitans-inculated Haemophilus test medium agar so that there was a 90° angle between the two antibiotic strips at the point where their individual MIC values against A. actinomycetemcomitans intersected on their respective MIC interpretive scales. After 48 hours incubation in air + 5% CO2, the MIC values for each antibiotic in combination against the A. actinomycetemcomitans clinical isolates was read in ug/ml where the edge of the bacterial inhibition ellipses intersected with each of the MIC Test Strips. For each of the three antibiotic combinations tested in vitro against the four A. actinomycetemcomitans clinical isolates, the fractional inhibitory concentration (FIC) index was calculated per each antibiotic in combination. FIC index values of less than or equal to 0.5 indicated the presence of synergistic antimicrobial effects of the antibiotic combination against the test bacterial clinical isolates, whereas FIC index values of greater than 0.5, but less than or equal to 4.0 indicated indifference, and FIC values greater than 4.0 represented the presence of in vitro antimicrobial antagonism between the two antibiotics in combination. Results: All of the four A. actinomycetemcomitans clinical isolates were susceptible in vitro to amoxicillin alone (all MIC values actinomycetemcomitans clinical isolates were resistant in vitro to metronidazole alone, with MIC values of resistant strains ranging between 24.0-48.0 ug/ml (resistance breakpoint threshold MIC value >16 ug/ml), and all were resistant in vitro to spiramycin alone, with MIC values of > 32.0 ug/ml exhibited by each of the four tested periodontal A. actinomycetemcomitans strains. In synergy testing, markedly lower MIC values were found for amoxicillin and metronidazole, as well as with ciprofloxacin and metronidazole, when tested in combination together as compared to being tested alone. FIC index values for the combination of amoxicillin plus metronidazole were all actinomycetemcomitans clinical isolates tested. Similarly, FIC index values for the combination of ciprofloxacin plus metronidazole were all actinomycetemcomitans clinical isolates tested. FIC index values for the combination of spiramycin plus metronidazole were > 0.5 for three of the periodontal A. actinomycetemcomitans strains, ranging from 0.875-2.0, which is indicative of an indifferent in vitro antimicrobial interaction between spiramycin and metronidazole against the three periodontal A. actinomycetemcomitans clinical isolates. The FIC index value of the combination of spiramycin plus metronidazole against a single periodontal A. actinomycetemcomitans clinical isolate was actinomycetemcomitans clinical isolate. Conclusions: Amoxicillin and ciprofloxacin individually were active against all A. actinomycetemcomitans clinical periodontal isolates, whereas most or all strains were resistant to metronidazole and spiramycin by themselves. Antimicrobial synergism was found for the combinations of amoxicillin plus metronidazole, and for ciprofloxacin plus metronidazole, against all four periodontal A. actinomycetemcomitans clinical isolates of United States origin. Spiramycin plus metronidazole generally failed to exhibit antimicrobial synergism against periodontal A. actinomycetemcomitans. These findings confirm and extend European synergism studies conducted in mid-1990s on antibiotic synergisms against periodontal A. actinomycetemcomitans, and are the first to demonstrate antibiotic synergism against United States periodontal A. actinomycetemcomitans clinical isolates. Additional research studies are needed to determine whether these in vitro synergistic effects of amoxicillin plus metronidazole, and of ciprofloxacin plus metronidazole, also occur in vivo and significantly enhance subgingival elimination or suppression of subgingival A. actinomycetemcomitans. / Oral Biology
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/1195 |
| Date | January 2012 |
| Creators | Faucher, Joanie |
| Contributors | Rams, Thomas E., Suzuki, Jon, 1947-, Whitaker, Eugene J. |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 44 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/1177, Theses and Dissertations |
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