Journal of Antimicrobial Chemotherapy (1992) 30, 625-^32
The bactericidal activity of sparfloxacin C. S. Lewis', I. Morrissey* and J. T. Smith*
Sparfloxacin was found to display a biphask response against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis in nutrient broth. Its optimum bactericidal concentration was found to be identical for all three species which contrasts with other clinically available fluoroquinolones that are more active against E. coli than against staphylococci. Bacterial protein and RNA synthesis as well as cell division were not found to be essential for all the lethality of sparfloxacin, which hence displays bactericidal mechanism B. However, sparfloxacin was unable to kill bacteria in absence of oxygen.
Introduction Sparfloxacin (AT-4140), 5-amino-cyclopropyl-6, 8-difluoro-7 (cis-3,5-dimethyl-l piperazinyl)-4-oxoquinolone-3-carboxylic acid is a new difluorinated quinolone antibacterial agent that exhibits low minimum inhibitory concentrations (MICs) against a wide range of both Gram-negative and Gram-positive clinical isolates (Kojima, Inoue & Mitsuhashi, 1989; Chaudry et al., 1990; Cooper et al., 1990). In vitro, sparfloxacin displays a greater activity against staphylococci than either ciprofloxacin or ofloxacin (Chaudry et al., 1990). This may suggest an advantage in the use of sparfloxacin as both ciprofloxacin and ofloxacin are not as effective against staphylococcal infections as they are against infections due to Gram-negative pathogens (Smith et al., 1990). The rate of kill of the 4-quinolones is dependent on certain factors and protein synthesis, RNA synthesis and bacterial cell division are essential for the bactericidal activity of older 4-quinolones, such as nalidixic acid (Smith, 1984; Smith & Lewin, 1988). One explanation for this is that the older 4-quinolones kill bacteria by a single mechanism, termed A, for which bacterial protein and RNA synthesis as well as cell division are prerequisites (Dietz, Cook & Goss, 1966; Smith, 1984; Smith & Lewin, 1988). However, all the modern fluorinated 4-quinolones investigated so far are active against non-dividing bacteria (Zeilcr & Grohe, 1984; Ratcliffe & Smith, 1985; Smith, 1989; Lewin & Amyes, 1989; Lewin, Amyes & Smith, 1989; Lewin & Amyes, 1990) most probably due to the possession of an extra bactericidal mechanism in addition to A (Zeiler & Grohe, 1984). The modern fluorinated 4-quinolones can be classified into groups based upon the conditions required for kill. The first group includes ciprofloxacin, ofloxacin (Smith & Lewin, 1988), DR-3355 (Lewin & Amyes, 1989), lomefloxacin (Lewin et al., 1989), 0305-7453/92/110625+08 $08.00/0
62S © 1992 The Britith Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
'Department of Medical Microbiology. University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG;bMicrobiology Section, Department of Pharmaceutics, The School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK
626
C S. Lewtn ef a*.
Materials and methods Bacterial strains E. coli KL16 (Smith, 1984), 5. epidermidis SK360 (Lewin & Smith, 1988) and S. aureus E3T (Tennent et al., 1988) were used in this study. These strains were chosen because of their previous use to establish the fundamental bactericidal mechanisms of other 4-quinolone antibacterials. These strains were not, however, of recent clinical isolation. Despite this the 4-quinolones do not generally give different bacteriological responses when recent clinical isolates are compared with laboratory strains, unlike the situation with other antibiotics (Finch & Gabbay, 1986; Smith, 1989). The strains were kept on drug-free nutrient agar plates which were sub-cultured every ten days. Colonies taken from drug-free nutrient agar plates were used to prepare the overnight cultures used in all experiments. Antibacterial preparation Sparfloxacin (Rhone-Poulenc, France) was dissolved in 05 M NaOH (0-02 g/L) before being made up in sterile distilled water. Ciprofloxacin (Bayer, UK), chloramphenicol (Parke-Davis, UK) and rifampicin (Ciba, UK) were dissolved in sterile distilled water. Drug solutions for anaerobic experiments were prepared acrobically and then equilibrated ovenight in an anaerobic cabinet before use. The drug solutions were such that the addition of 10 /JL to the medium gave appropriate drug concentration. Determination of antibacterial effects of sparfloxacin under aerobic conditions The killing activity of sparfloxacin at OK) 15 to 90mg/L was determined in nutrient broth (Oxoid, UK) at 37°C over a 3 h period using an initial inoculum size of 106— 107 cfu/mL. Survival was estimated by serial dilution in nutrient broth followed by
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
pcfloxacin and flcroxacin (Lcwin & Amyes, 1990) which possess a second bactericidal mechanism termed B, that requires neither bacterial protein or RNA synthesis nor cell division for its activity. The second group contains norfloxacin and enoxacin which while bactericidal against non-dividing bacteria still require bacterial protein and RNA synthesis to be able to kill bacteria (Ratdiffe & Smith, 1985; Zeiler & Grohe, 1984). This mechanism has been termed C (Ratcliffe & Smith, 1985). Oxygen also appears to be required for the bactericidal activity of some 4-quinolones. Ciprofloxacin and ofloxacin are unable to kill bacteria under anaerobic conditions although bacterial multiplication is still inhibited (Morrissey, Lewin & Smith, 1990). In contrast PD 127,391 (CI-960) is still bactericidal in the absence of oxygen (Morrissey & Smith, 1990). The majority of previous studies on sparfloxacin have concentrated on MIC tests (Kojima et al., 1989; Cooper et al., 1990). However, determination of the minimum inhibitory concentration does not provide any information on the optimum bactericidal concentration (OBQ of a 4-quinolone, defined as the concentration at which bacterial kill is maximal. Therefore, the conditions required for sparfloxacin to kill E. coli, S. aureus and S. epidermidis were studied. Piddock & Zhu (1991) have studied sparfloxacin kill against both Gram-positive and Gram-negative bacteria, but did not study the conditions required for kill.
Tbe bactericidal actirfty of sparfloxadn
627
viable counting on nutrient agar as previously described (Lewin et al., 1989). The rate of kill of bacteria treated with sparfloxadn in nutrient broth or in phosphate-bufferedsaline (PBS) was measured at 30 min intervals over a 4 h period by viable counts on nutrient agar as previously described (Lewin & Smith, 1988). Determination of antibacterial effects of sparfloxadn under anaerobic conditions
Results The bactericidal activity of sparfloxadn at a range of concentrations from 0015 to 90 mg/L against E. coli, S. aurevs and S. epidermidis in nutrient broth over 3 h showed a biphasic effect, characteristic of the bactericidal activity of the 4-quinolones (Figure 1). Survival of bacteria decreased with increasing sparfloxadn concentration until the OBC of 0-9 mg/L for all three spedes was reached and then survival increased progressively as the concentration rose above the OBC. Although the OBC was identical for all spedes investigated the level of kill observed at that concentration varied. Sparfloxadn was ten-fold less active against S. aureus and 100-fold less active against S. epidermidis than against E. coli (Figure 1). These results are in broad agreement with those of Piddock & Zhu (1991). At its OBC, sparfloxadn was still significantly bactericidal against E. coli in nutrient broth even in the presence of a bacteriostatic concentration of chloramphenicol, an inhibitor of protein synthesis, or rifampidn, an inhibitor of RNA synthesis (Figure 2). As rifampidn is bacteriddal to staphylococd it could not be used to investigate the bacteriddal mechanisms of sparfloxadn against this genus. However, this problem does not apply to chloramphenicol. It can be seen from Figure 3 that the addition of a bacteristatic concentration of chloramphenicol did not completely abolish the bacteriddal activity of sparfloxadn at its OBC against S. aureus and similar results were obtained with S. epidermidis (results not shown). However, the addition of chloramphenicol antagonized the lethality of sparfloxadn against the staphylococd much more than against E. coli (Figures 2 and 3). Less than 1% of the E. coli survived after
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Aliquots (4-9 mL) of thioglycollate USP medium (Oxoid, UK) in 0-5 oz bottles were rendered anaerobic by heat sterilization, transferred into an anaerobic cabinet (Don Whitley Anaerobe Chamber mkll, Don Whitley Ltd., UK) and left to equilibrate for at least 48 h at 37°C before use to ensure total anaerobirity. Any bottles containing media that was still oxygenated, i.e. where the resazurin indicator was pink, were rejected immediately. Biological indicators for anaerobidty were not used because they were considered to not be discriminatory enough to indicate whether conditions are suffidently anaerobic to antagonize 4-quinolone loll (Morrissey & Smith, 1992). Drug solutions were left in the anaerobic cabinet overnight to equilibrate and then added as required. After 15 min the reaction mixtures were inoculated with 0-1 mL of an anaerobic overnight culture of bacteria in the thioglycollate USP medium prepared as before to give initially 10*-107cfu/mL. Duplicate bottles were prepared for each incubation time, so that at time zero, one set of bottles was removed and immediately aerated with air and incubated aerobically at 37°C to act as aerobic controls. At 30 min intervals percentage survival was estimated by the same technique used for the experiments performed under aerobic conditions.
C S. Lewta et uL
£28 1000
10
0-1 Sport toxoctn
Figure 1. Survival o f f . coli KLI6 ( • ) , S. aureus E3T ( • ) and 5. epidermitBs SK360 (O) after exposure to gparfloxacin for 3 h in nutrient broth at 37°C.
4 h exposure to sparfloxacin in the presence of chloramphenicol (Figure 2) but 25% of the staphylococci survived after 4 h exposure to both these drugs (Figure 3). The bactericidal activity of sparfloxacin was then investigated in phosphate buffered saline (PBS). The 4-quinolone was able to kill E. coli, S. aureus and S. epidermidis 100
OOCH
60
120 180 Time (rrtn)
240
Figure 2. Survrval o f f . coli K.L16 in nutrient broth at 3TC with sparfloxacin (09 mg/L) (D). sparfloxacin (09mg/L) and chloramphenicol (20 mg/L) ( • ) , cparfloxacin (09 mg/L) and rifampicin (160 mg/L) ( • ) , chloramphenicol (20 mg/L) or rifampicin (160 mg/L) (O)-
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
0-01
The bactericidal activity of sparfioxadn
60
120 180 Tim* (mln)
240
Flgnre 3. Survival of 5. aurtus E3T in nutrient broth at 37°C with iparfloucin (09mg/L) ( • ) , sparfloxacin (0-9 mg/L) and chlorampbenicol (20 mg/L) ( • ) , chloramphenicol (20 mg/L) (O)-
under these conditions although the bactericidal activity of sparfloxacin was weaker against the staphylococci than against E. coli (results not shown). The bactericidal activity of sparfloxacin was then investigated in the absence of oxygen. At its OBC, sparfloxacin was unable to kill E. coli KL16 in USP thioglycollate broth under anaerobic conditions, but under aerobic conditions sparfloxacin was bactericidal in UPS thioglycollate broth (Figure 4). Similar results were found using S. aureus (results not shown). S. epidermidis was unable to grow sufficiently under anaerobic conditions for the bactericidal activity of sparfloxacin to be tested.
60
120 Tim* (mln) Fhjwc 4. Survival of E. coli KL16 in USP thiogtycollate broth at 37°C with sparfloxacin (09 mg/L) under aerobic (O) and anaerobic ( • ) conditions.
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
0-1
629
630
C S. Lewto et aL Table. Comparisons of the OBCs of 4-quinolones (mg/L) Drug Sparfloxacin Ciprofloxacin DR-3355 Ofloxacin Norfloxacin Fleroxacin Enoxacin
S. aureus E3T
S. epidermidis SK360 E. coli KL16
09 3 3 5 30 IS 30
0-9 3 3 5 30 15 30
0-9 0-15
0-5 1-5 1-5 3
The bactericidal activity of sparfloxacin against E. coli KL16, S. aureus E3T and S. epidermidis SK360 exhibited a biphasic response common to all other 4-quinolones so far investigated (Dietz et al., 1966; Smith, 1984; Zeiler & Grohc, 1984; Lewin et al., 1989; Smith et al., 1990). The optimal concentration for the bactericidal activity of sparfloxacin was 0-9 mg/L for all three species. This is unusual as the OBCs of the other 4-quinolones tend to be higher against staphylococci than against E. coli (Table). Although sparfloxacin was slightly less active in terms of OBC against E. coli than some of the clinically available 4-quinolones it was more active than these drugs against staphylococci (Table). Sparfloxacin, in common with the other modern fluorinated 4-quinolones that have been investigated, displayed another bactericidal mechanism in addition to bactericidal mechanism A. This appears to be mechanism B as sparfloxacin was able to kill bacteria in the absence of protein or of RNA synthesis. It is interesting to note that sparfloxacin was less bactericidal against staphylococci than against E. coli when mechanism A was abolished suggesting that the mechanism B displayed by the drug against staphylococci was weaker than its mechanism B against E. coli. This seems to correlate well with the observation that the bactericidal activity of sparfloxacin at the OBC was lower against the staphylococci than against E. coli. Although sparfloxacin was able to kill bacteria in the absence of protein and RNA synthesis it did require oxygen for its lethality as it was unable to kill bacteria under anaerobic conditions in contrast to the data described by Cooper et al. (1991). However, although Cooper et al. used an anaerobic medium (Wilkens & Chalgren) for their MIC tests, and also for the tests of bactericidal activity with Bacteroides fragilis a specific anaerobic medium was not used for the bactericidal activity tests with E. coli, which were done with Isosensitest broth that may have contained sufficient traces of oxygen to sustain a bactericidal effect. Hence sparfloxacin would appear to resemble ciprofloxacin or ofloxacin rather than PD127, 391 which can kill bacteria under anaerobic conditions (Morrissey & Smith, 1990; Morrissey et al., 1990). In conclusion, sparfloxacin does not require protein synthesis, RNA synthesis or bacterial cell division to kill Gram-negative or Gram-positive bacteria although oxygen does appear to be essential for its lethality. Despite being less bactericidal against E. coli KL16 than ciprofloxacin, which is the most active clinically-available 4-quinolone, sparfloxacin was more active against staphylococci. This enhanced activity against staphylococci may be significant because, at present, the 4-quinolones are not always successful in resolving staphylococcal infections (Smith et al., 1990).
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Discussion
The bactericidal actfrtty of sparfloxadn
631
Acknowledgements We are grateful to the Scottish Home and Health Department for financial support for C. S. Lewin and to Rhone-Poulenc for providing us with sparfloxadn. We are also grateful to Daiichi Research Institute, Tokyo, Japan, for the funding of a post-doctoral fellowship for I. Morrissey. References
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Chaudhry, A. Z., Knapp, C. C , Sierra-Madero, J. & Washington, J. A. (1990). Antistaphylococcal activities of sparfloxacin (CI-978; AT-4140), ofloxacin, and dprofloxadih Antimicrobial Agents and Chemotherapy 34, 1843-5. Cooper, M. A., Andrews, J. M., Ashby, J. P., Matthews, R. S. & Wise, R. (1990). In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent Journal of Antimicrobial Chemotherapy 26, 667-76. Cooper, M. A., Andrews, J. M. & Wise, R. (1991). Bactericidal activity of sparfloxacin and dprofloxacin under anaerobic conditions. Journal of Antimicrobial Chemotherapy 28, 399-406. Deitz, W. H., Cook, T. M. & Goss, W. A. (1966). Mechanism of action of nalidixic add on Escherichia coli. HI. Conditions required for lethality. Journal of Bacteriology 91, 768-73. Finch, R. G. & Gabbay, F. J. (1986). The 4-quinolones, in vitro activity, pharmacolrinetic behaviour and in vivo considerations. In Quinolones—Their future in Clinical Practise. International Congress Symposium Series No. 104, 17-27, Royal Sodety of Medicine Services, London, New York. Kojima, T., Inoue, M. & Mitsuhashi, S. (1989). In vitro activity of AT-4140 against clinical bacterial isolates. Antimicrobial Agents and Chemotherapy 33, 1980-8. Lewin, C. S. & Amyes, S. G. B. (1989). The bactericidal activity of DR-3355, an optically active isomer of ofloxacin. Journal of Medical Microbiology 30, 227-31. Lewin, C. S. & Aymes, S. G. B. (1990). Conditions required for the bacteriddal activity of fleroxacin and pcfloxarin against Escherichia coli KL16. Journal of Medical Microbiology 32, 83-6. Lewin, C. S., Amyes, S. G. B. & Smith, J. T. (1989). Bacteriddal activity of enoxadn and lomefloxadn against Escherichia coli KL16. European Journal of Clinical Microbiology and Infectious Diseases 8, 731-3. Lewin, C. S. & Smith, J. T. (1988). Bacteriddal mechanisms of ofloxacin. Journal of Antimicrobial Chemotherapy 22, Suppl. C, 1-8. Morrissey, I., Lewin, C. S. & Smith, J. T. (1990). The influence of oxygen upon bacteriddal potency. In The Quinolones: Anti-bacterial Agents in Vitro (Crumplin, G. C , Ed.), pp. 23-36. Springer-Verlag, London. Morrissey, I. & Smith, J. T. (1990). PD-127,391-2: a 4-quinolone bacteriddal under anaerobic conditions. In Program and Abstracts of the Thirtieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta. GA. 1990. Abstract 377, p. 149. American Sodety for Microbiology, Washington, DC. Morrissey, I. & Smith, J. T. (1992). Absence of bacteriddal activity of sparfloxadn and dprofloxacin under anaerobic conditions. Journal of Antimicrobial Chemotherapy 29, 589-90. Piddock, L. J. V. & Zhu, M. (1991). Mechanism of actions of sparfloxacin against, and mechanisms of resistance on Gram negative and Gram positive bacteria. Antimicrobial Agents and Chemotherapy 35, 2423-7. Ratcliffe, N. T. & Smith, J. T. (1985). Norfloxadn has a novel bacteriddal mechanism unrelated to that of other 4-quinolones. Journal of Pharmacy and Pharmacology 37, Suppl., 92P. Smith, J. T. (1984). Awakening the slumbering potential of the 4-quinolone antibacterials. Pharmaceutical Journal 233, 299-305. Smith, J. T. (1989). Clinical impact of in vitro data with newer quinolones. Journal of Antimicrobial Chemotherapy 24, 662-3. Smith, J. T. & Lewin, C. S. (1988). Chemistry and mechanisms of action of the quinolone antibacterials. In The Quinolones (Andriole, V. T., Ed.), pp. 23-82, Academic Press, London.
632
C S. Lewta et mL
Smith, S. M., Eng, R. H. K., Bais, P., Fan-Havard, P. &. Tecson-Tumang, F. (1990). Epidemiology of dprofloxarin resistance among patients with mcthicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 26, S67-72. Tennent, J. M., Young, H.-K., Lyon, B. R., Amyes, S. G. B. & Skurray, R. A. (1988). Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylocoed. Journal of Medical Microbiology 26, 67-73. Zeiler, H. J. & Grohe, K. (1984). The in vitro and in vivo activity of ciprofloxacin. European Journal of Clinical Microbiology 3, 339-43. (Received 4 February 1992; revised version accepted 29 June 1992)
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
The bactericidal activity of sparfloxacin C. S. Lewis', I. Morrissey* and J. T. Smith*
Sparfloxacin was found to display a biphask response against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis in nutrient broth. Its optimum bactericidal concentration was found to be identical for all three species which contrasts with other clinically available fluoroquinolones that are more active against E. coli than against staphylococci. Bacterial protein and RNA synthesis as well as cell division were not found to be essential for all the lethality of sparfloxacin, which hence displays bactericidal mechanism B. However, sparfloxacin was unable to kill bacteria in absence of oxygen.
Introduction Sparfloxacin (AT-4140), 5-amino-cyclopropyl-6, 8-difluoro-7 (cis-3,5-dimethyl-l piperazinyl)-4-oxoquinolone-3-carboxylic acid is a new difluorinated quinolone antibacterial agent that exhibits low minimum inhibitory concentrations (MICs) against a wide range of both Gram-negative and Gram-positive clinical isolates (Kojima, Inoue & Mitsuhashi, 1989; Chaudry et al., 1990; Cooper et al., 1990). In vitro, sparfloxacin displays a greater activity against staphylococci than either ciprofloxacin or ofloxacin (Chaudry et al., 1990). This may suggest an advantage in the use of sparfloxacin as both ciprofloxacin and ofloxacin are not as effective against staphylococcal infections as they are against infections due to Gram-negative pathogens (Smith et al., 1990). The rate of kill of the 4-quinolones is dependent on certain factors and protein synthesis, RNA synthesis and bacterial cell division are essential for the bactericidal activity of older 4-quinolones, such as nalidixic acid (Smith, 1984; Smith & Lewin, 1988). One explanation for this is that the older 4-quinolones kill bacteria by a single mechanism, termed A, for which bacterial protein and RNA synthesis as well as cell division are prerequisites (Dietz, Cook & Goss, 1966; Smith, 1984; Smith & Lewin, 1988). However, all the modern fluorinated 4-quinolones investigated so far are active against non-dividing bacteria (Zeilcr & Grohe, 1984; Ratcliffe & Smith, 1985; Smith, 1989; Lewin & Amyes, 1989; Lewin, Amyes & Smith, 1989; Lewin & Amyes, 1990) most probably due to the possession of an extra bactericidal mechanism in addition to A (Zeiler & Grohe, 1984). The modern fluorinated 4-quinolones can be classified into groups based upon the conditions required for kill. The first group includes ciprofloxacin, ofloxacin (Smith & Lewin, 1988), DR-3355 (Lewin & Amyes, 1989), lomefloxacin (Lewin et al., 1989), 0305-7453/92/110625+08 $08.00/0
62S © 1992 The Britith Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
'Department of Medical Microbiology. University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG;bMicrobiology Section, Department of Pharmaceutics, The School of Pharmacy, University of London, Brunswick Square, London WC1N 1AX, UK
626
C S. Lewtn ef a*.
Materials and methods Bacterial strains E. coli KL16 (Smith, 1984), 5. epidermidis SK360 (Lewin & Smith, 1988) and S. aureus E3T (Tennent et al., 1988) were used in this study. These strains were chosen because of their previous use to establish the fundamental bactericidal mechanisms of other 4-quinolone antibacterials. These strains were not, however, of recent clinical isolation. Despite this the 4-quinolones do not generally give different bacteriological responses when recent clinical isolates are compared with laboratory strains, unlike the situation with other antibiotics (Finch & Gabbay, 1986; Smith, 1989). The strains were kept on drug-free nutrient agar plates which were sub-cultured every ten days. Colonies taken from drug-free nutrient agar plates were used to prepare the overnight cultures used in all experiments. Antibacterial preparation Sparfloxacin (Rhone-Poulenc, France) was dissolved in 05 M NaOH (0-02 g/L) before being made up in sterile distilled water. Ciprofloxacin (Bayer, UK), chloramphenicol (Parke-Davis, UK) and rifampicin (Ciba, UK) were dissolved in sterile distilled water. Drug solutions for anaerobic experiments were prepared acrobically and then equilibrated ovenight in an anaerobic cabinet before use. The drug solutions were such that the addition of 10 /JL to the medium gave appropriate drug concentration. Determination of antibacterial effects of sparfloxacin under aerobic conditions The killing activity of sparfloxacin at OK) 15 to 90mg/L was determined in nutrient broth (Oxoid, UK) at 37°C over a 3 h period using an initial inoculum size of 106— 107 cfu/mL. Survival was estimated by serial dilution in nutrient broth followed by
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
pcfloxacin and flcroxacin (Lcwin & Amyes, 1990) which possess a second bactericidal mechanism termed B, that requires neither bacterial protein or RNA synthesis nor cell division for its activity. The second group contains norfloxacin and enoxacin which while bactericidal against non-dividing bacteria still require bacterial protein and RNA synthesis to be able to kill bacteria (Ratdiffe & Smith, 1985; Zeiler & Grohe, 1984). This mechanism has been termed C (Ratcliffe & Smith, 1985). Oxygen also appears to be required for the bactericidal activity of some 4-quinolones. Ciprofloxacin and ofloxacin are unable to kill bacteria under anaerobic conditions although bacterial multiplication is still inhibited (Morrissey, Lewin & Smith, 1990). In contrast PD 127,391 (CI-960) is still bactericidal in the absence of oxygen (Morrissey & Smith, 1990). The majority of previous studies on sparfloxacin have concentrated on MIC tests (Kojima et al., 1989; Cooper et al., 1990). However, determination of the minimum inhibitory concentration does not provide any information on the optimum bactericidal concentration (OBQ of a 4-quinolone, defined as the concentration at which bacterial kill is maximal. Therefore, the conditions required for sparfloxacin to kill E. coli, S. aureus and S. epidermidis were studied. Piddock & Zhu (1991) have studied sparfloxacin kill against both Gram-positive and Gram-negative bacteria, but did not study the conditions required for kill.
Tbe bactericidal actirfty of sparfloxadn
627
viable counting on nutrient agar as previously described (Lewin et al., 1989). The rate of kill of bacteria treated with sparfloxadn in nutrient broth or in phosphate-bufferedsaline (PBS) was measured at 30 min intervals over a 4 h period by viable counts on nutrient agar as previously described (Lewin & Smith, 1988). Determination of antibacterial effects of sparfloxadn under anaerobic conditions
Results The bactericidal activity of sparfloxadn at a range of concentrations from 0015 to 90 mg/L against E. coli, S. aurevs and S. epidermidis in nutrient broth over 3 h showed a biphasic effect, characteristic of the bactericidal activity of the 4-quinolones (Figure 1). Survival of bacteria decreased with increasing sparfloxadn concentration until the OBC of 0-9 mg/L for all three spedes was reached and then survival increased progressively as the concentration rose above the OBC. Although the OBC was identical for all spedes investigated the level of kill observed at that concentration varied. Sparfloxadn was ten-fold less active against S. aureus and 100-fold less active against S. epidermidis than against E. coli (Figure 1). These results are in broad agreement with those of Piddock & Zhu (1991). At its OBC, sparfloxadn was still significantly bactericidal against E. coli in nutrient broth even in the presence of a bacteriostatic concentration of chloramphenicol, an inhibitor of protein synthesis, or rifampidn, an inhibitor of RNA synthesis (Figure 2). As rifampidn is bacteriddal to staphylococd it could not be used to investigate the bacteriddal mechanisms of sparfloxadn against this genus. However, this problem does not apply to chloramphenicol. It can be seen from Figure 3 that the addition of a bacteristatic concentration of chloramphenicol did not completely abolish the bacteriddal activity of sparfloxadn at its OBC against S. aureus and similar results were obtained with S. epidermidis (results not shown). However, the addition of chloramphenicol antagonized the lethality of sparfloxadn against the staphylococd much more than against E. coli (Figures 2 and 3). Less than 1% of the E. coli survived after
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Aliquots (4-9 mL) of thioglycollate USP medium (Oxoid, UK) in 0-5 oz bottles were rendered anaerobic by heat sterilization, transferred into an anaerobic cabinet (Don Whitley Anaerobe Chamber mkll, Don Whitley Ltd., UK) and left to equilibrate for at least 48 h at 37°C before use to ensure total anaerobirity. Any bottles containing media that was still oxygenated, i.e. where the resazurin indicator was pink, were rejected immediately. Biological indicators for anaerobidty were not used because they were considered to not be discriminatory enough to indicate whether conditions are suffidently anaerobic to antagonize 4-quinolone loll (Morrissey & Smith, 1992). Drug solutions were left in the anaerobic cabinet overnight to equilibrate and then added as required. After 15 min the reaction mixtures were inoculated with 0-1 mL of an anaerobic overnight culture of bacteria in the thioglycollate USP medium prepared as before to give initially 10*-107cfu/mL. Duplicate bottles were prepared for each incubation time, so that at time zero, one set of bottles was removed and immediately aerated with air and incubated aerobically at 37°C to act as aerobic controls. At 30 min intervals percentage survival was estimated by the same technique used for the experiments performed under aerobic conditions.
C S. Lewta et uL
£28 1000
10
0-1 Sport toxoctn
Figure 1. Survival o f f . coli KLI6 ( • ) , S. aureus E3T ( • ) and 5. epidermitBs SK360 (O) after exposure to gparfloxacin for 3 h in nutrient broth at 37°C.
4 h exposure to sparfloxacin in the presence of chloramphenicol (Figure 2) but 25% of the staphylococci survived after 4 h exposure to both these drugs (Figure 3). The bactericidal activity of sparfloxacin was then investigated in phosphate buffered saline (PBS). The 4-quinolone was able to kill E. coli, S. aureus and S. epidermidis 100
OOCH
60
120 180 Time (rrtn)
240
Figure 2. Survrval o f f . coli K.L16 in nutrient broth at 3TC with sparfloxacin (09 mg/L) (D). sparfloxacin (09mg/L) and chloramphenicol (20 mg/L) ( • ) , cparfloxacin (09 mg/L) and rifampicin (160 mg/L) ( • ) , chloramphenicol (20 mg/L) or rifampicin (160 mg/L) (O)-
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
0-01
The bactericidal activity of sparfioxadn
60
120 180 Tim* (mln)
240
Flgnre 3. Survival of 5. aurtus E3T in nutrient broth at 37°C with iparfloucin (09mg/L) ( • ) , sparfloxacin (0-9 mg/L) and chlorampbenicol (20 mg/L) ( • ) , chloramphenicol (20 mg/L) (O)-
under these conditions although the bactericidal activity of sparfloxacin was weaker against the staphylococci than against E. coli (results not shown). The bactericidal activity of sparfloxacin was then investigated in the absence of oxygen. At its OBC, sparfloxacin was unable to kill E. coli KL16 in USP thioglycollate broth under anaerobic conditions, but under aerobic conditions sparfloxacin was bactericidal in UPS thioglycollate broth (Figure 4). Similar results were found using S. aureus (results not shown). S. epidermidis was unable to grow sufficiently under anaerobic conditions for the bactericidal activity of sparfloxacin to be tested.
60
120 Tim* (mln) Fhjwc 4. Survival of E. coli KL16 in USP thiogtycollate broth at 37°C with sparfloxacin (09 mg/L) under aerobic (O) and anaerobic ( • ) conditions.
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
0-1
629
630
C S. Lewto et aL Table. Comparisons of the OBCs of 4-quinolones (mg/L) Drug Sparfloxacin Ciprofloxacin DR-3355 Ofloxacin Norfloxacin Fleroxacin Enoxacin
S. aureus E3T
S. epidermidis SK360 E. coli KL16
09 3 3 5 30 IS 30
0-9 3 3 5 30 15 30
0-9 0-15
0-5 1-5 1-5 3
The bactericidal activity of sparfloxacin against E. coli KL16, S. aureus E3T and S. epidermidis SK360 exhibited a biphasic response common to all other 4-quinolones so far investigated (Dietz et al., 1966; Smith, 1984; Zeiler & Grohc, 1984; Lewin et al., 1989; Smith et al., 1990). The optimal concentration for the bactericidal activity of sparfloxacin was 0-9 mg/L for all three species. This is unusual as the OBCs of the other 4-quinolones tend to be higher against staphylococci than against E. coli (Table). Although sparfloxacin was slightly less active in terms of OBC against E. coli than some of the clinically available 4-quinolones it was more active than these drugs against staphylococci (Table). Sparfloxacin, in common with the other modern fluorinated 4-quinolones that have been investigated, displayed another bactericidal mechanism in addition to bactericidal mechanism A. This appears to be mechanism B as sparfloxacin was able to kill bacteria in the absence of protein or of RNA synthesis. It is interesting to note that sparfloxacin was less bactericidal against staphylococci than against E. coli when mechanism A was abolished suggesting that the mechanism B displayed by the drug against staphylococci was weaker than its mechanism B against E. coli. This seems to correlate well with the observation that the bactericidal activity of sparfloxacin at the OBC was lower against the staphylococci than against E. coli. Although sparfloxacin was able to kill bacteria in the absence of protein and RNA synthesis it did require oxygen for its lethality as it was unable to kill bacteria under anaerobic conditions in contrast to the data described by Cooper et al. (1991). However, although Cooper et al. used an anaerobic medium (Wilkens & Chalgren) for their MIC tests, and also for the tests of bactericidal activity with Bacteroides fragilis a specific anaerobic medium was not used for the bactericidal activity tests with E. coli, which were done with Isosensitest broth that may have contained sufficient traces of oxygen to sustain a bactericidal effect. Hence sparfloxacin would appear to resemble ciprofloxacin or ofloxacin rather than PD127, 391 which can kill bacteria under anaerobic conditions (Morrissey & Smith, 1990; Morrissey et al., 1990). In conclusion, sparfloxacin does not require protein synthesis, RNA synthesis or bacterial cell division to kill Gram-negative or Gram-positive bacteria although oxygen does appear to be essential for its lethality. Despite being less bactericidal against E. coli KL16 than ciprofloxacin, which is the most active clinically-available 4-quinolone, sparfloxacin was more active against staphylococci. This enhanced activity against staphylococci may be significant because, at present, the 4-quinolones are not always successful in resolving staphylococcal infections (Smith et al., 1990).
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Discussion
The bactericidal actfrtty of sparfloxadn
631
Acknowledgements We are grateful to the Scottish Home and Health Department for financial support for C. S. Lewin and to Rhone-Poulenc for providing us with sparfloxadn. We are also grateful to Daiichi Research Institute, Tokyo, Japan, for the funding of a post-doctoral fellowship for I. Morrissey. References
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
Chaudhry, A. Z., Knapp, C. C , Sierra-Madero, J. & Washington, J. A. (1990). Antistaphylococcal activities of sparfloxacin (CI-978; AT-4140), ofloxacin, and dprofloxadih Antimicrobial Agents and Chemotherapy 34, 1843-5. Cooper, M. A., Andrews, J. M., Ashby, J. P., Matthews, R. S. & Wise, R. (1990). In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent Journal of Antimicrobial Chemotherapy 26, 667-76. Cooper, M. A., Andrews, J. M. & Wise, R. (1991). Bactericidal activity of sparfloxacin and dprofloxacin under anaerobic conditions. Journal of Antimicrobial Chemotherapy 28, 399-406. Deitz, W. H., Cook, T. M. & Goss, W. A. (1966). Mechanism of action of nalidixic add on Escherichia coli. HI. Conditions required for lethality. Journal of Bacteriology 91, 768-73. Finch, R. G. & Gabbay, F. J. (1986). The 4-quinolones, in vitro activity, pharmacolrinetic behaviour and in vivo considerations. In Quinolones—Their future in Clinical Practise. International Congress Symposium Series No. 104, 17-27, Royal Sodety of Medicine Services, London, New York. Kojima, T., Inoue, M. & Mitsuhashi, S. (1989). In vitro activity of AT-4140 against clinical bacterial isolates. Antimicrobial Agents and Chemotherapy 33, 1980-8. Lewin, C. S. & Amyes, S. G. B. (1989). The bactericidal activity of DR-3355, an optically active isomer of ofloxacin. Journal of Medical Microbiology 30, 227-31. Lewin, C. S. & Aymes, S. G. B. (1990). Conditions required for the bacteriddal activity of fleroxacin and pcfloxarin against Escherichia coli KL16. Journal of Medical Microbiology 32, 83-6. Lewin, C. S., Amyes, S. G. B. & Smith, J. T. (1989). Bacteriddal activity of enoxadn and lomefloxadn against Escherichia coli KL16. European Journal of Clinical Microbiology and Infectious Diseases 8, 731-3. Lewin, C. S. & Smith, J. T. (1988). Bacteriddal mechanisms of ofloxacin. Journal of Antimicrobial Chemotherapy 22, Suppl. C, 1-8. Morrissey, I., Lewin, C. S. & Smith, J. T. (1990). The influence of oxygen upon bacteriddal potency. In The Quinolones: Anti-bacterial Agents in Vitro (Crumplin, G. C , Ed.), pp. 23-36. Springer-Verlag, London. Morrissey, I. & Smith, J. T. (1990). PD-127,391-2: a 4-quinolone bacteriddal under anaerobic conditions. In Program and Abstracts of the Thirtieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta. GA. 1990. Abstract 377, p. 149. American Sodety for Microbiology, Washington, DC. Morrissey, I. & Smith, J. T. (1992). Absence of bacteriddal activity of sparfloxadn and dprofloxacin under anaerobic conditions. Journal of Antimicrobial Chemotherapy 29, 589-90. Piddock, L. J. V. & Zhu, M. (1991). Mechanism of actions of sparfloxacin against, and mechanisms of resistance on Gram negative and Gram positive bacteria. Antimicrobial Agents and Chemotherapy 35, 2423-7. Ratcliffe, N. T. & Smith, J. T. (1985). Norfloxadn has a novel bacteriddal mechanism unrelated to that of other 4-quinolones. Journal of Pharmacy and Pharmacology 37, Suppl., 92P. Smith, J. T. (1984). Awakening the slumbering potential of the 4-quinolone antibacterials. Pharmaceutical Journal 233, 299-305. Smith, J. T. (1989). Clinical impact of in vitro data with newer quinolones. Journal of Antimicrobial Chemotherapy 24, 662-3. Smith, J. T. & Lewin, C. S. (1988). Chemistry and mechanisms of action of the quinolone antibacterials. In The Quinolones (Andriole, V. T., Ed.), pp. 23-82, Academic Press, London.
632
C S. Lewta et mL
Smith, S. M., Eng, R. H. K., Bais, P., Fan-Havard, P. &. Tecson-Tumang, F. (1990). Epidemiology of dprofloxarin resistance among patients with mcthicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 26, S67-72. Tennent, J. M., Young, H.-K., Lyon, B. R., Amyes, S. G. B. & Skurray, R. A. (1988). Trimethoprim resistance determinants encoding a dihydrofolate reductase in clinical isolates of Staphylococcus aureus and coagulase-negative staphylocoed. Journal of Medical Microbiology 26, 67-73. Zeiler, H. J. & Grohe, K. (1984). The in vitro and in vivo activity of ciprofloxacin. European Journal of Clinical Microbiology 3, 339-43. (Received 4 February 1992; revised version accepted 29 June 1992)
Downloaded from http://jac.oxfordjournals.org/ at Michigan State University on June 29, 2015
