Synergic activity, for anaerobes, of trovafloxacin with clindamycin or metronidazole: chequerboard and time–kill methods

Lois M. Edniea, Kim L. Creditoa, Mayuree Khantiponga, Michael R. Jacobsb and Peter C. Appelbauma,*

Departments of Pathology (Clinical Microbiology), a Hershey Medical Center, Hershey, PA 17033 and b Case Western Reserve University, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chequerboard titrations were used to test the activity of trovafloxacin, alone and in combination with clindamycin or metronidazole, against 156 Gram-positive or Gram-negative anaerobes, including 47 Bacteroides fragilis group, 36 Prevotella spp., 26 fusobacteria, 21 peptostreptococci and 26 clostridia. MIC50/MIC90 values (mg/L) of each drug alone against all 156 strains were: trovafloxacin, 0.5/1; clindamycin, 0.25/2; metronidazole, 1/2. Synergy (FIC indices <= 0.5) was seen in two strains with trovafloxacin plus clindamycin, and seven with trovafloxacin plus metronidazole. All other combinations were additive (FIC indices >0.5–2.0); no antagonism (FIC indices >4.0) was seen. In addition, synergy was tested by time–kill methodology for each of the above combinations against 12 Gram-positive or Gram-negative strains. Results indicated that synergy (defined as a >= 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 Bacteroides fragilis, Bacteroides 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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anaerobes are established causes of serious human infections, especially in debilitated hosts. Although infections caused by members of the Bacteroides fragilis group occur most commonly, infections caused by other Gram-negative anaerobic bacilli, as well as by Gram-positive cocci and clostridia, are increasingly encountered. The susceptibility spectrum of clinically isolated anaerobes is changing: Although ß-lactamase production, and concomitant resistance to ß-lactams, is the rule in the B. fragilis group, both phenomena are increasingly encountered in non-B. fragilis group Bacteroides, Prevotella, Porphyromonas and Fusobacterium spp. ß-Lactamase production has also been described in some non-Clostridium perfringens species of clostridia. Metronidazole resistance, apart from being the rule among anaerobic Gram-positive non-spore-forming bacilli (which most often prove to be microaerophiles), has been reported in peptostreptococci, non-C. perfringens clostridia and members of the B. fragilis group. Additionally, clindamycin resistance is not unusual among anaerobic Gram-negative bacilli.1

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 trovafloxacin–metronidazole 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, time–kill methodology was also used to test possible synergy with the above combinations against 12 anaerobes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria and antimicrobials

All anaerobic strains were recent clinical isolates (1994–1997) 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 time–kills 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 (Pharmacia–Upjohn, 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 IGo) 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 (0.06–16.0 mg/L) dispensed alone in the first row, and clindamycin (0.004–8.0 mg/L) or metronidazole (0.25–4.0 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 1–2 x 106 cfu/mL with a multipoint inoculator (1–2 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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Table I. MICs (mg/L) of drugs alone for 156 anaerobic strains tested
 

FIC indices were interpreted as synergic if values were <=0.5, additive/indifferent if >0.5–4 and antagonistic if >4.0.13

Time–kill determinations

Twelve strains were tested by time–kill 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 1–5 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. Time–kills yielded additive results when the number of surviving organisms in the combination was 0–1.9 log10 cfu/mL below the starting inoculum3,4,13 Synergy time–kill 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results of MIC studies are presented in Table IGo. MIC50/ MIC90 values (mg/L) of each drug alone against all 156 strains were: trovafloxacin, 0.5/1; clindamycin, 0.25/2; metronidazole, 1/2. Chequerboard testing (Table IIGo) showed that synergic FIC indices of <=0.5 were seen in two B. fragilis group strains with trovafloxacin–clindamycin and in seven strains (five B. fragilis and two fusobacteria) with trovafloxacin–metronidazole. All other combinations were additive (FIC indices >0.5–2.0, with none >2.0); no antagonistic FICs >4.0 were seen.


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Table II. Results of chequerboard titrationsa
 
Because chequerboard assays are less discriminatory than time–kill tests in determining synergy, and because previous studies using aerobic organisms have shown that all strains shown to be synergic by chequerboard methods were also synergic as judged by time–kill (but not vice versa),1418 we elected to screen all 156 strains by chequerboard methods, and then test 12 representative strains which had additive FIC indices by time–kill. Strains chosen for time–kill synergy assays were selected to represent a spectrum of clinically significant anaerobe species.

Table IIIGo presents results of time–kill 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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Table III. Comparison of synergy testing by chequerboard and time–kill methodologies
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The anti-anaerobic activity of trovafloxacin has been well established, both by us and by other workers.2,11 However, MICs vary between studies; some differences may be ascribed to differences in techniques.2,11,19,20 Activity of trovafloxacin at a recently established susceptibility breakpoint of 2.0 mg/L21 is not universal.2,11,19,20 It has also been established that trovafloxacin is bactericidal at the MIC after 48 h.3

Studies by us and other workers have established that time–kill 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 time–kill.

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.


    Notes
 
* Correspondence address. Department of Pathology, Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA. Tel: +1-717-531-5113; Fax: +1-717-531-7953; E-mail: pappelbaum{at}psghs.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Appelbaum, P. C., Spangler, S. K. & Jacobs. M. R. (1993). Susceptibility of 539 Gram-positive and Gram-negative anaerobes to new agents, including RP 59500, biapenem, trospectomycin and piperacillin/tazobactam. Journal of Antimicrobial Chemotherapy 32, 223–31.[Abstract]

2 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1994). Activity of CP 99,219 compared with those of ciprofloxacin, grepafloxacin, metronidazole, cefoxitin, piperacillin and piperacillin–tazobactam against 489 anaerobes. Antimicrobial Agents and Chemotherapy 38, 2471–6.[Abstract]

3 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Time–kill study of the activity of trovafloxacin compared with ciprofloxacin, sparfloxacin, metronidazole, cefoxitin, piperacillin and piperacillin/tazobactam against six anaerobes. Journal of Antimicrobial Chemotherapy 39, Suppl. B, 23–7.[Abstract/Free Full Text]

4 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1997). Bactericidal activity of DU-6859a compared to activities of three quinolones, three ß-lactams, clindamycin, and metronidazole against anaerobes as determined by time–kill methodology. Antimicrobial Agents and Chemotherapy 41, 847–9.[Abstract]

5 . Jones, R. N. & Erwin, M. E. (1992). In vitro activity of CP-74667 compared to four other fluoroquinolones. Diagnostic Microbiology and Infectious Diseases 15, 531–6.[ISI][Medline]

6 . Eliopoulos, G. M., Klimm, K., Eliopoulos, C. T., Ferraro, M. J. & Moellering, R. C. (1993). In vitro activity of CP-99,219, a new fluoroquinolone, against clinical isolates of gram-positive bacteria. Antimicrobial Agents and Chemotherapy 37, 366–70.[Abstract]

7 . Gooding, B. & Jones, R. N. (1993). In vitro antimicrobial activity of CP-99,219, a novel azabicyclo-naphthyridone. Antimicrobial Agents and Chemotherapy 37, 349–53.[Abstract]

8 . Neu, H. C. & Chin, N.-X. (1994). In vitro activity of the new fluoroquinolone CP-99,219. Antimicrobial Agents and Chemotherapy 38, 2615–22.[Abstract]

9 . Child, J., Andrews, J., Boswell, F., Brenwald, N. & Wise, R. (1995). The in-vitro activity of CP 99,219, a new naphthyridone antimicrobial agent: a comparison with fluoroquinolone agents. Journal of Antimicrobial Chemotherapy 35, 869–76.[Abstract]

10 . Fass, R. J., Barnishan, J., Solomon, M. C. & Ayers, L. W. (1996). In vitro activities of quinolones, ß-lactams, tobramycin, and trimethoprim–sulfamethoxazole against nonfermentative gramnegative bacilli. Antimicrobial Agents and Chemotherapy 40, 1412–8.[Abstract]

11 . Wexler, H. M., Molitoris, E., Molitoris, D. & Finegold, S. M. (1996). In vitro activities of trovafloxacin against 557 strains of anaerobic bacteria. Antimicrobial Agents and Chemotherapy 40, 2232–5.[Abstract]

12 . Visalli, M. A., Bajaksouzian, S., Jacobs, M. R. & Appelbaum, P. C. (1998). Synergistic action of trovafloxacin with other agents against gram-positive and -negative organisms. Diagnostic Microbiology and Infectious Diseases 30, 61–4.[ISI][Medline]

13 . Eliopoulos, G. M. & Moellering, R. C. (1996). In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 330–96. Williams and Wilkins, Baltimore, MD.

14 . Cappelletty, D. M. & Rybak, M. J. (1996). Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 40, 677–83.[Abstract]

15 . Bajaksouzian, S., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1996). Antipneumococcal activities of cefpirome and cefotaxime, alone and in combination with vancomycin and teicoplanin, determined by checkerboard and time–kill methods. Antimicrobial Agents and Chemotherapy 40, 1973–6.[Abstract]

16 . Bajaksouzian, S., Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1997). Activities of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with amikacin, against acinetobacters as determined by checkerboard and time–kill studies. Antimicrobial Agents and Chemotherapy 41, 1073–6.[Abstract]

17 . Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1998). Determination of activities of levofloxacin, alone and combined with gentamicin, ceftazidime, cefpirome, and meropenem, against 124 strains of Pseudomonas aeruginosa by checkerboard and time–kill methodology. Antimicrobial Agents and Chemotherapy 42, 953–5.[Abstract/Free Full Text]

18 . Visalli, M. A., Jacobs, M. R. & Appelbaum, P. C. (1998). Activities of three quinolones, alone and in combination with extended-spectrum cephalosporins or gentamicin, against Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 42, 2002–5.[Abstract/Free Full Text]

19 . Hecht, D. W. & Osmolski, J. R. (1996). Comparison of activities of trovafloxacin (CP 99,219) and five other agents against 585 anaerobes with use of three media. Clinical Infectious Diseases 23, Suppl. 1, S44–50.[ISI][Medline]

20 . Falagas, M. E., McDermott, L. & Snydman, D. R. (1997). Effect of pH on in vitro antimicrobial susceptibility of the Bacteroides fragilis group. Antimicrobial Agents and Chemotherapy 41, 2047–9.[Abstract]

21 . National Committee for Clinical Laboratory Standards. (1997). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Fourth Edition: Approved Standard M11-A4. NCCLS, Villanova, PA.

Received 15 June 1999; returned 17 September 1999; revised 12 November 1999; accepted 14 December 1999