Departments of Pathology (Clinical Microbiology), a Hershey Medical Center, Hershey, PA 17033 and b Case Western Reserve University, Cleveland, OH 44106, USA
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Abstract |
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Introduction |
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Commercially available quinolones such as ciprofloxacin, ofloxacin, fleroxacin, pefloxacin, enoxacin, lomefloxacin, sparfloxacin and grepafloxacin, are inactive or marginally active against anaerobes, with MICs either higher than, or clustering around, breakpoints.24 Trovafloxacin is a new quinolone with a broad spectrum of activity against Gram-positive and Gram-negative aerobic and anaerobic bacteria.2,511 A recent preliminary study12 has indicated possible synergy between trovafloxacin clindamycin and trovafloxacinmetronidazole against B. fragilis and C. perfringens. In order to cast further light on these findings, this study employed chequerboard titration to examine the activity of trovafloxacin, alone and in combination with clindamycin or metronidazole, against 156 Gram-positive or Gram-negative anaerobes. Additionally, timekill methodology was also used to test possible synergy with the above combinations against 12 anaerobes.
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Materials and methods |
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All anaerobic strains were recent clinical isolates (19941997) which had been identified by standard procedures and kept frozen in double-strength skimmed milk (Difco Laboratories, Detroit, MI, USA) at 70°C until use. Before testing, strains were subcultured three times on to enriched blood agar plates. Throughout the study, strains were tested for purity by Gram stain and colonial morphology. Chequerboard titrations were performed on all 156 strains, and synergy timekills on 12 strains, chosen to represent a spectrum of species encountered in clinical practice. Gram-positive non-spore-forming bacilli were excluded from the study because of their metronidazole resistance and their rarity in clinically significant infections. Antimicrobials were obtained from their manufacturers as follows: trovafloxacin (Pfizer, New York, NY, USA), clindamycin (PharmaciaUpjohn, Kalamazoo, MI, USA), metronidazole (Sigma Inc., St Louis, MO, USA).
MIC and chequerboard titration assays
MIC and chequerboard titration assays were performed13 on 156 strains (Table I) in frozen microtitre trays (Micromedia Systems, Inc., Cleveland, OH, USA) using Brucella broth (BBL Microbiology Systems, Cockeysville, MD, USA) with 5% lysed sheep blood, 1 mg/L vitamin K and 5 mg/L haemin, with trovafloxacin (0.0616.0 mg/L) dispensed alone in the first row, and clindamycin (0.0048.0 mg/L) or metronidazole (0.254.0 mg/L) alone in the first column. Inocula were prepared by suspending growth from blood agar plates in sterile Brucella broth to a density of a 0.5 McFarland standard, and diluted 1 in 10 to produce a final inoculum of 12 x 106 cfu/mL with a multipoint inoculator (12 x 105 cfu/well). Trays were incubated for 48 h at 35°C in an anaerobic glove box (Coy Laboratory Products, Ann Arbor, MI, USA). Standard quality control strains were included with each run. Fractional inhibitory concentrations (FICs) were calculated as follows:
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FIC indices were interpreted as synergic if values were 0.5, additive/indifferent if >0.54 and antagonistic if >4.0.13
Timekill determinations
Twelve strains were tested by timekill according to methods developed in our laboratory3,4 as follows. A suspension equal to a 1 McFarland standard was made by suspending approximately five colonies from Brucella plates into prereduced Brucella broth in a 15 x 45 mm borosilicate screw-capped tube with 13-425 screw-thread open top screw caps (Baxter Diagnostics, Inc., McGaw Park, IL, USA) and 13 mm Teflon-faced rubber septa (Fisher Scientific, Inc., Pittsburgh, PA, USA). A 100 µL aliquot was then delivered by syringe into a similar tube containing 5 mL prereduced Brucella broth. All inocula were prepared in the glove box. Tubes were then removed from the glove box and incubated in a shaking water bath at 35°C for 24 h. Viability counts were then done in the glove box to obtain similar starting inocula (see below).
Empty tubes (as above) were then filled on the bench top by syringe with 2.7 mL prereduced Brucella broth with additives (5% lysed horse blood, 5 mg/L haemin, 1 mg/L vitamin K), 1 mL antibiotic dilutions (each drug tested alone, and trovafloxacin in combination with clindamycin or metronidazole) prepared in Brucella broth, 200 µL Oxyrase solution (Oxyrase Inc., Mansfield, OH, USA) and 100 µL inoculum (as above), after carefully expelling all air from all syringes. Controls without antibiotic were included in each run. Initial inocula were 15 x 106 cfu/mL (confirmed by prior viability counts).3,4
For each of the three drugs tested alone, the MIC and concentrations one dilution above and three dilutions below the MIC were tested. Combinations were tested at the MIC and one or two dilutions below the MIC of each drug. Viability counts were performed after incubation at 35°C in a shaking water bath at 0, 6, 12, 24 and 48 h by incubating plates inside the glove box at 35°C for 48 h. Drug carryover was addressed by dilution, as described previously.3,4 Synergy13 was defined as a 2 log10 decrease in cfu/mL between the combination and its most active constituent after 48 h3,4 and the number of surviving organisms in the presence of the combination was
2 log10 cfu/mL below the starting inoculum. Timekills yielded additive results when the number of surviving organisms in the combination was 01.9 log10 cfu/mL below the starting inoculum3,4,13 Synergy timekill tests were done in duplicate and yielded identical results. Atmosphere inside the glove box for all tests consisted of 85% N2, 10% CO2 and 10% H2.
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Results |
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Table III presents results of timekill experiments. All combinations yielded additive FICs with the chequerboard method. Results indicated that synergy (
2 log10 decrease in cfu/mL at 48 h compared with the more active drug alone) was found between trovafloxacin at or below the MIC and both clindamycin and metronidazole at or below the MIC in one strain each of B. fragilis, B. thetaiotaomicron, Prevotella intermedia, Fusobacterium varium, Peptostreptococcus asaccharolyticus and Clostridium bifermentans. Synergy between trovafloxacin (
MIC) and metronidazole alone was seen in one strain each of Bacteroides distasonis, Prevotella bivia, Fusobacterium mortiferum, P. asaccharolyticus and C. bifermentans. In many cases of synergy, including those at the trovafloxacin MIC, regrowth after 48 h, which was commonly seen with trovafloxacin alone, was inhibited, and 99.9% killing was observed with the combination after 48 h, but not with trovafloxacin alone. No strains gave synergy between trovafloxacin and clindamycin without giving synergy between trovafloxacin and metronidazole. Lower rates of synergy were found at earlier time periods (data not shown). No attempt was made to subculture clones from regrowth after 48 h with trovafloxacin alone, in order to determine whether resistant mutants were being selected.
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Discussion |
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Studies by us and other workers have established that timekill testing (which detects bactericidal activity) is more discriminatory than the chequerboard method (which only detects bacteriostatic activity) for synergy testing of a wide variety of Gram-positive and Gram-negative aerobic bacteria, including pneumococci and Gram-negative non-fermenters.1418 The current study detected the same phenomenon for anaerobes, where negligible synergy (but no antagonism) was found with the chequerboard method, while significant synergy, reproducible by duplicate testing, was found in the small number of anaerobes tested by timekill.
The NCCLS21 has established trovafloxacin anaerobe breakpoints of 2.0 mg/L (susceptible), 4.0 mg/L (intermediate) and
8.0 mg/L (resistant). Although trovafloxacin is active against most anaerobes at
2.0 mg/L, MICs of some strains cluster around the susceptible breakpoint. Results of this study, which require confirmation by testing of larger numbers of strains, indicate that combination of trovafloxacin with clindamycin or metronidazole yields trovafloxacin MICs which are well below the susceptible breakpoint. Additionally, regrowth with trovafloxacin alone which was sometimes seen after 48 h did not occur in synergic combinations. Further testing of clones which regrew after 48 h with trovafloxacin alone, as well as clinical studies will also be necessary to validate the significance of these in vitro findings.
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Notes |
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References |
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Received 15 June 1999; returned 17 September 1999; revised 12 November 1999; accepted 14 December 1999