of otloxacin) and sparfloxacin against 152 S. aureus isolates, 104 of which were ... superior killing effect compared with DU-6859a and sparfloxacin, whereas.
Clin. Drug Invest. 1997 Oct; 14 (4): 330-336
PHARMACODYNAMICS
1173-2563/97/ool0-0330/S03. 50/0
© Adis International limited. All rig hts reserved
Comparative In Vitro Activity and Killing Effect of Trovafloxacin, DV-6859a, Levofloxacin and Sparfloxacin against Staphylococcus aureus Focus on Methicillin-Resistant Isolates E.f. Giamarellos-Bourboulis, P. Grecka, I. Galani and H. Giamarellou 1st Department of Propedeutic Medicine, Athens Medical School, Athens, Greece
Summary
The predominance of quinolone resistance among methicillin-resistant Staphylococcus aureus (MRS A) isolates in the nosocomial environment led to the in vitro comparison of trovafloxacin (CP-99,219) and DU-6859a (which are novel f1uoroquinolone compounds) with levofloxacin (the L-geometric isomer of otloxacin) and sparfloxacin against 152 S. aureus isolates, 104 of which were MRSA. At 2 mg/L trovafloxacin and DU-6859a inhibited 92.3 and 77.9% of MRSA isolates, respectively, possessing lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. than levotloxacin and sparfloxacin, which inhibited 38.5 and 36.5% of isolates, respectively. In contrast, in the methicillin-susceptible population all tested tluoroquinolones had an equal intrinsic activity. Trovafloxacin possessed a superior killing effect compared with DU-6859a and sparfloxacin, whereas Ievofloxacin was the least bactericidal quinolone. Indeed, the effect of trovafloxacin was mainly expressed at a quinolone concentration of 2 x MIC and after 24 hours of growth. However, in levofloxacin- and sparfloxacin-resistant MRS A isolates , DU-6859a had a superior killing effect compared with trovatloxacin. Trovafloxacin had a killing effect in only 20% of DU-6859a-, levofloxacin- and sparfloxacin-resistant isolates. Our findings support trovatloxacin and DU-6859a as highly active in vitro antistaphylococcal agents, especially in MRSA, which merit further clinical investigation.
The incidence of fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) in Western European countries ranges between 55 and 96% of isolates,[IJ and renders the treatment of infections caused by these microorganisms a therapeutic chaIIengePJ Novel quinolones are characterised by their in vitro potency against Gram-positive cocciPl
Trovafloxacin (CP-99,219), with the chemical structure 7-(azabicycIo[3.I.O]-hexyl)-naphthyridone,[41 and DU-6859a, with the chemical structure 7[(7S)-7 -amino-5-azaspiro [2,4] heptan-5-yl]-8chloro-6-fluoro-l-[( 1R,2S)-2-fluoro-l-cyclopropyl] -1,4-dihydro-4-oxo-3-quinoline carboxylic acid sesquihydratep,61 are new fluoroquinolones possessing high potency against MRSA isolates.
In Vitro Activity of Newer Fluoroquinolones
Levofloxacin constitutes the L-geometric isomer of ofloxacin, being 8 to 128 times more potent than D-ofloxacin for S. aureus, and it was the first quinolone to be described as active against ciprofloxacin-resistant MRSA.l7 1 Sparfloxacin has been reported to be more active than ciprofloxacin and ofloxacin against MRSA.[81 The purpose of the present study was the in vitro comparison of trovafloxacin and DU-6859a with levofloxacin and sparfloxacin against both MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates, and the evaluation of the overtime bactericidal effect of the four newer fluoroquinolones on ciprofloxacin-resistant MRSA.
Material and Methods Detection of Methicillin Resistance
One hundred and four fresh MRSA isolates were collected from different patients and from the following sources: pus (92), bronchial secretions (9), cerebrospinal fluid (2) and blood (I). Resistance to methicillin was determined after plating single colonies of a fresh S. aureus isolate onto MuellerHinton agar (Oxoid Ltd, London, England) with 6 mg/L of incorporated oxacillin (Sigma Co., St Louis, Missouri, USA) along with 4% NaCI. Development of colonies after 18 hours of incubation at 30°C (Forma Scientific, Ohio, USA) was indicative of resistance to methicillin.l 91 The accuracy of the oxacillin screen plate test l91 in defining methicillin resistance was confirmed for the above strains by using a DNA probe according to Archer and Pennell.l lOI The probe used was a 1.1 kilobase 8g1 Il-Xbal fragment internal to the penicillin-binding protein (PBP) 2a structural gene carried to a pUC 18 plasmid vector, both labelled with digoxigenin-dUTP (Boehringer Mannheim, Indianapolis, Indiana, USA), and hybridised probes were immunodetected with an alkaline phosphatase conjugated anti-digoxigenin antibody. Susceptibility Testing on MRSA Isolates
Trovafloxacin was provided by Pfizer Inc. (Groton, Connecticut, USA) as an amorphous crys© Adis International Limited. All rights reserved.
331
talline powder, DU-6859a and levofloxacin by Daiichi Pharmaceutical Co. (Tokyo, Japan), and sparfloxacin by Rh6ne-Poulenc Rorer (Paris, France) as yellowish crystalline powders. Minimum inhibitory concentrations (MICs) to trovafloxacin, DU-6859a, levofloxacin and sparfloxacin were determined by a microdilution technique at volumes of 0.1 ml and those to ciprofloxacin, oxacillin, imipenem, erythromycin, c1indamycin, gentamicin, rifampicin and vancomycin were determined on ready-made microdilution plates (Sensititre Ltd, West Sussex, England). Colonies from an overnight agar culture were suspended in Mueller-Hinton broth (BBL, Becton Dickinson, Cockeysville, Maryland, USA) to reach the turbidity of a 0.5 McFarland standard. They were incubated for I hour at 35°C and diluted to 5 x 105 colony-forming units (cfu)/ml to be applied for MIC determination. The following antimicrobial concentrations were tested: trovafloxacin, DU-6859a, levofloxacin and sparfloxacin 0.03 to 64 mg/L; ciprofloxacin 0.5 to 4 mg/L, oxacillin and imipenem 0.25 to 32 mg/L; erythromycin and c1indamycin 0.12 to 16 mg/L; gentamicin 0.25 to 32 mg/L; rifampicin 0.5 to 4 mg/L; and vancomycin 0.5 to 64 mg/L. MICs to oxacillin were performed in the presence of 2% NaCI. MICs were determined as the lowest antimicrobial concentration limiting visible bacterial growth after an 18-hour incubation at 35°C (Forma Scientific, Ohio, USA), and susceptibility interpretation was based on the following National Committee for Clinical Laboratory Standards criteria:[II] ciprofloxacin and oxacillin 2 mg/L; imipenem 4 mg/L; erythromycin and clindamycin 0.5 mg/L; gentamicin 4 mg/L; rifampicin I mg/L and vancomycin 4 mg/L. For trovafloxacin, DU-6859a, levofloxacin and sparfloxacin a concentration of 2 mg/L was considered as their breakpoint according to their achievable mean serum levels in vivo.l 12 - 15 ] Minimum bactericidal concentrations (MBCs) were performed after plating a 50111 volume of all clear wells onto blood agar (BBL, Becton Dickinson, Cockeysville, Maryland, USA), and they were measured as the lowest antimicrobial concentraClin. Drug Invest. 1997 Oct: 14 (4)
Giamarellos-Bourboulis et al.
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tion permitting 99.9% of killing of the applied inoculum. S. aureus ATCC 29213 (methicillin susceptible) was used as a control strain. Susceptibility Testing on MSSA Isolates
Forty-eight fresh S. aureus pus isolates derived from different patients were tested and their susceptibility to methicillin was evaluated by the above oxacillin spread plate test. MICs-MBCs to trovafloxacin, DU-6859a, levofloxacin and sparfloxacin were determined as above. Killing Effect of Newer Fluoroquinolones on MRSA
Twenty-nine ciprofloxacin-resistant MRSA isolates were exposed over time to concentrations of I x MIC and 2 x MIC of the tested fluoroquinolones. The strains were selected so that their MBC value was either equal to or two-fold the MIC. Among the 29 isolates, II susceptible to all four quinolones (MIC range ~0.03 to 0.25 mg/L) were exposed to all four of them; 8 resistant to levofloxacin and sparfloxacin and susceptible to trovafloxacin and DU-6859a (MIC: 0.06 to 0.5 mg/L) were exposed to trovafloxacin and DU-6859a; 10 resistant to DU-6859a, levofloxacin and sparfloxacin but susceptible to trovafloxacin (MIC: ~0.03 to I mg/L) were exposed to trovafloxacin. Therefore, a total of 140 killing-curves were performed. Prepared tubes per tested strain contained the applied fluoroquinolone at 1 x MIC or 2 x MIC, a 5 x 105 cfu/ml log-phase inoculum and MuellerHinton broth (Oxoid Ltd, London, England) at a 10ml final volume. A growth control tube without added antimicrobial was applied per tested strain. All tubes were incubated at 37°C in a water bath and bacterial growth was measured in each tube at standard time intervals (0 time, 3, 5 and 24 hours of growth). The latter was performed by four consecutive I: 10 dilutions of a 100111 aliquot of each tube into Mueller-Hinton broth and by plating a I 00111 aliquot of each dilution onto blood agar. This procedure avoided any antimicrobial carryover ef© Adis International Limited. All rights reserved.
fect and had a lower detection limit of 10 cfu/ml. A value greater than or equal to a 3 log 10 decrease of viable cell counts at a specific time of growth compared with the growth control was considered as an adequate killing effect.[16] For colonies surviving after the 24-hour fluoroquinolone exposure, MICs were redetermined as described above. Values greater than or equal to a four-fold MIC increase compared with the MIC of the parent strain were evaluated as resistance development whenever the new MIC value surpassed the susceptibility breakpoint. Statistical Analysis
Statistical analysis of results was performed by the X2 test (p < 0.05).
Results Susceptibility Testing on MRSA Isolates
The Iysates of all isolates being characterised as methicillin-resistant by the oxacillin screen plate test were hybridised by the PBP2a gene-identical probe. 96 (92.3%), 81 (77.9%),40 (38.5%) and 38 (36.5%) of the tested strains were found to be susceptible to trovafloxacin, DU-6859a, levofloxacin and sparfloxacin, respectively (p < 0.001 between trovafloxacin and the three other quinolones). Their comparative activity to that of 8 other anti staphylococcal agents is demonstrated in table I. It should be mentioned that 86 (83.5%) and 64 (61.5%) MRS A isolates were found to be susceptible at a concentration of less than or equal to 1 mg/L of trovafloxacin and DU-6859a, respectively. Sixty-six strains were found to be resistant to sparfloxacin. Among these isolates 62 (93.9%),47 (71.2%) and 15 (22.7%) were inhibited by trovafloxacin, DU-6859a and levofloxacin, respectively (p < 0.00 I). Among the 64 levofloxacinresistant isolates 62 (96.9%),41 (64.1%) and 13 (20.3%) were found to be susceptible to trovafloxacin, DU-6859a and sparfloxacin, respectively (p < 0.00 I), whereas among the 23 DU-6859aresistant isolates 18 (78.3%), 2 (8.7%) and 4 (17.4%) Clin. Drug Invest. 1997 Oct; 14 (4)
In Vitro Activity of Newer Fluoroquinolones
--------------------------------------------------
were inhibited by trovafloxacin, levofloxacin and sparfloxacin, respectively (p < 0.00 I). 51 strains were resistant to both sparfloxacin and levofloxacin, 46 (90.2%) and 35 (68.6%) being inhibited by trovafloxacin and DU-6859a, respectively (p < 0.05). Susceptibility Testing on MSSA Isolates
Trovafloxacin, DU-6859a, levofloxacin and sparfloxacin inhibited 47 (97.9%), 46 (95.8%), 46 (95.8%) and 46 (95 .8%) of the tested isolates, respectively (NS). MICso to MIC 90 of the above fluoroquinolones were $0.03 to 0. 12, $0.03 to 0.25, $0.03 to 0.5 and 110.03 to 0.06 mg/L, respectively, whereas MBXso to MBC 90 were $0.03 to
333
0.12, $0.03 to 0.25, $0.03 to 4 and $0.03 to 0.5 mg/L, respectively. Killing Effect of Newer Fluoroquinolones on MRSA
The comparative killing effect of the tested compounds on 11 isolates susceptible to all four of them is shown in table II. Among colonies surviving after the 24-hour quinolone exposure, 11.1 % became resistant to levofloxacin, a phenomenon not observed with the three other tested compounds, whose MICs remained the same. The timekill curves of one strain exposed to all four fluoroquinolones are shown in figure I. Regrowth was observed at 24 hours by 1 x MIC to 2 x MIC of trovafloxacin, DU-6859a, levofloxacin and
Table I. Susceptibility patterns of 104 methicillin-resistant Staphylococcus aureus isolates exposed to 12 antistaphylococcal agents Antimicrobial agent"
Range of MICs (mg/L)
MICso
Trovafloxacin (2)
5:0.03 - ~64
0.25
DU-6859a (2)
5:0.03 - ~64
% inhibited
MIC90
MBCso
MBC90
2
0.5
8
92.3
16
77.9
8
Levofloxacin (2)
5:0.03-~4
4
16
4
32
38.5
Sparfloxacin (2)
5:0.03 -
~64
4
16
4
~4
36.5
Giprofloxacin (2)
5:0.5 -
~4
~4
~4
~4
~4
8.7
~32
~32
~32
~32
0.0 18.3
Oxacillin (2)
8 - ~32
Imipenem (4)
5:0.25 - ~32
~32
~32
~32
~32
Erythromycin (0.5)
5:0.12 - ~ 16
~ 16
~ 16
~ 16
216
6.7
Clindamycin (0.5)
5:0.12 -
~ 16
~ 16
~ 16
~ 16
~16
11.5
Gentamicin (4)
5:0.25 - ~32
~32
~32
~32
~32
13.5
Rifampicin (I)
5:0.5 - ~4
~4
~4
~4
~4
28.8
Vancomycin (4)
5:0.5 - 4
a
100.0
Figures in parentheses indicate the susceptibility breakpoint of the antimicrobial agent.
Abbreviations: MBC = minimum bactericidal concentration; MIG = minimum inhibitory concentration.
Table II. Comparative over-time killing effect of four newer fluoroquinolones on 11 ciprofloxacin-resistant methicillin-resistant Staphylococcus aureus isolates Time (h)
Number of strains being killed by trovafloxacin 1 xMIC
3
1
DU-6859a 2xMIC
levofloxacin
sparfloxacin
1 xMIC
2xMIC
1 x MIC
2xMIG
1 xMIG
2xMIC
3
3
3
1
1
2
3
5
5
11
3
6
1
1
6
8
24
9
10
4
8
3
3
5
9
Abbreviation: MIG = minimum inhibitory concentration.
© Adis Inte rnational Limited. All rights reserved.
Clin. Drug Invest. 1997 Oct: 14 (4)
334
Giamarellos-Bourboulis e/ al.
• •
o
Trovafloxacin
DU - 6859a
~~
7
6 5
ro
3
OJ
2
III .0
:0 III ':;;
'0 ci
.s 0
Oi
~~
4
~u
+-------------~~
r-__~----------------~
Sparfloxacin
7
Levofloxacin
~
r-
~~
6
0
-'
Control 1 xMIC 2 x MIC
5 4
3 2 +--.-----.-----.~ r-~~--.-----.-----.-~ ~
0
3
24 0
5
3
5
24
Time (h) Fig. 1. Killing curves of one methicillin-resistant Staphylococcus aureus isolate exposed to trovafloxacin, DU-6859a, levofloxacin and sparfloxacin . Minimum inhibitory concentrations (MICs) of trovafloxacin , DU-6859a, levofloxacin and sparfloxacin of that isolate were SO.03, 0.06, s O.03 and s O.03 mg/L, respectively.
Table III. Killing effect of trovafloxacin and DU-6859a for methicillin-resistant Staphylococcus aureus (MRSA) isolates resistant to other fluoroquinolones Time (h)
No. of MRSA isolates resistant to levofloxacin and sparfloxacin killed by/no. of tested MRSA strains
No. of MRSA isolates resistant to DU-6859a, levofloxacin and sparfloxacin killed by/no. of tested MRSA strains
trovafloxacin
DU-6859a
trovafloxacin
1 x MIC
1 xMIC
2 x MIC
2xMIC
1 x MIC
2 x MIC
1/10
2/10
1/8
3 5
1/8
24
1/8
1/10
4/8 1/8
2/8
Abbreviation: MIC = minimum inhibitory concentration .
sparfloxacin in I to 2, 5 to 2, 3 to 3 and 6 to 4 isolates, respectively. The comparative killing effect of trovafloxacin and DU-6859a on 8 MRSA isolates resistant to both levofloxacin and sparfloxacin is shown in table Ill. Among colonies surviving after a 24-hour exposure to I x MIC and 2 x MIC of DU-6859a, 25 % and 37.5%, respectively, became resistant. © Adis International Limited. All rights reserved.
Resistance was observed for 12.5 % of colonies surviving after a 24-hour 2 x MIC trovafloxacin exposure. The killing effect of trovafloxacin on 10 MRSA isolates resistant to the other three tested quinolones is also shown in table III. Resistance development was found in 10% and 10% of surviving isolates after a 24-hour I x MIC and 2 x MIC exposure to trovafloxacin, respectively. C lin. Drug Invest. 1997 Oct; 14 (4)
III Vitro Activity of Newer Fluoroquinolones
-------------------------------------------
Discussion Emergence of qui no lone-resistant MRSA nosocomial isolates in Western European countries renders infections caused by these strains a serious threat,111 and the need for newer compounds active against MRS A is obvious. We compared the in vitro activity of trovafloxacin with that of DU-6859a, levofloxacin and sparfloxacin against a variety of MRSA isolates of nosocomial origin. Even though other studies evaluating the in vitro potential of newer fluoroquinolones on S. aureus exist in the literature,13-7] the present study is the only one to our knowledge encompassing such a large number of intrinsic resistant MRSA isolates coding for the PBP 2a gene,IIOI and that investigated such a large number of ciprofloxacin-resistant MRS A isolates (table I). With regard to MRS A isolates, trovafloxacin possessed a superior in vitro intrinsic activity compared with the three other compounds, characterised by lower MIC and MBC values, whereas it inhibited 92.3 % of the tested isolates (table I). DU-6859a inhibited 77.9% of the MRSA isolates with lower MICs than those of levofloxacin and sparfloxacin. The two latter compounds possessed an equal in vitro intrinsic activity inhibiting approximately 38% of MRSA strains, whereas ciprofloxacin was the tenth most active compound on MRSA inhibiting only 8.7% of isolates. Our results are in accordance with studies showing trovafloxacin to inhibit approximately 90% of MRSA isolates with similar MIC90 values to those in our study.14.17 1 However, other authors have described much higher M1Cso and MIC 90 values for trovafloxacin against ciprofloxacinresistant MRSA,1 18 1 which might be explained by the limited number of S. aureus strains applied in these studies. MIC 90 values for levofloxacin in the present study were the same as those reported by other investigators,119 1 while lower MIC 90 values of DU-6859a and sparfloxacin for ciprofloxacinsusceptible MRSA have been reported compared with the values determined in our ciprofloxacinresistant population.IS.6.201 © Adis International Limited. All rights reserved.
335
Importantly, trovafloxacin and DU-6859a demonstrated a very promising activity towards levofloxacin- and sparfloxacin-resistant isolates, inhibiting 90.2 and 68.6% of them, respectively. It has also been observed that novel fluoroquinolones are bactericidal agents possessing MBC values equal to or twice the MICs (table I), an important quinolone characteristic.13l Even though the emphasis was directed towards MRSA in this present study, all four tested fluoroquinolones retained the same activity for MSSA as proposed by other authors. IS ,17. 19 1 Emphasis was also placed on the killing effect of the four tested f1uoroquinolones at a I x and 2 x MIC concentration. The MICs of MRSA isolates ranged between ~0.03 and I mg/L, so that the tested concentrations were either lower or equal to the mean 2 mglL fluoroquinolone serum level,l12-ISI allowing for results to be relevant to clinical conditions. Trovafloxacin possessed a superior bactericidal activity compared with DU-6859a and sparfloxacin, whereas all three compounds were superior to levofloxacin (table II). For all fluoroquinolones such an effect was maximised when the applied concentration was 2 x MIC and after 24 hours of growth. These characteristics of the quinolone killing activity were consistent with reported findings on the time-kill effect of trovafloxacin on Enterococcus Spp.,141 as well as with those of DU-6859a and levofloxacin on MRSA.l6.71 To our knowledge the over-time killing effect of trovafloxacin has not been reported by other authors for MRSA. However, the obtained killing results for MRSA isolates with various quinolone phenotypes of resistance differ significantly (table III). Against levofloxacin- and sparfloxacinresistant MRSA, DU-6859a was superior to trovafloxacin , whereas trovafloxacin was found to be bactericidal against only 10 to 20% of DU-6859a-, levofloxacin- and sparfloxacin-resistant strains. Results for the in vitro over-time bactericidal activity of trovafloxacin and DU-6859a on levoClin. Drug Invest. 1997 Oct; 14 (4)
Giamarellos-Bourboulis et al.
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floxacin- and sparfloxacin-resistant MRSA have not been reported elsewhere to our knowledge. Importantly, 37.5% of levofloxacin- and sparfloxacin-resistant isolates became resistant to DU-6859a after a 24-hour exposure.
Conclusion The present study revealed trovafloxacin and DU-6859a to be highly active newer quinolones against S. aureus and especially against nosocomial MRSA. Both drugs were also characterised by a high bactericidal activity against levofloxacin- and sparfloxacin-susceptible MRSA, but with moderate bactericidal activity against levofloxacin- and sparfloxacin-resistant MRSA. Further studies are necessary to establish the clinical applications of these newer fluoroquinolones for infections dominated by MRSA.
Acknowledgements We are grateful to Dr Gordon L. Archer (Medical College of Virginia, Division of Infectious Diseases and Department of Microbiology/Immunology) for his kind donation of the PBP-2a-identical gene probe. The data appearing in this paper were presented as part of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 1995, San Francisco [abstract FI85].
References I. Goldstein FW, Acar JF. Epidemiology of quinolone resistance: Europe and North and South America. Drugs 1995; 49 Suppl. 2: 36-42 2. Giamarellou H. Activity of quinolones against Gram-positive cocci: clinical features. Drugs 1995; 49 Suppl. 2: 58-66 3. Eliopoulos GM. In vitro activity of fluoroquinolones against Gram-positive bacteria. Drugs 1995; 49 Suppl. 2: 48-57 4. Coque TM, Singh KY, Murray BE. Comparative in vitro activity of the new fluoroquinolone trovatloxacin (CP-99,219) against Gram-positive cocci. J Antimicrob Chemother 1996; 37 (5): 1011-6 5. Korten Y, Tomayako JF, Murray BE. Comparative in vitro activity of DU-6859a, a new fluoroquinolone agent, against Gram-positive cocci. Antimicrob Agents Chemother 1994; 38 (3): 611-5 6. Nakane T, Iyobe S, Sato K, et al. In vitro antibacterial activity of DU-6859a, a new fluoroquinolone. Antimicrob Agents Chemother 1995; 39 (12): 2822-6
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7. Patel JA, Pachucki CT, Lentino JR. Synergy of levofloxacin (L-ofloxacin) and oxacillin against quinolone-resistant Staphylococcus au reus measured by the time-kill method. Antimicrob Agents Chemother 1993; 37 (2): 339-41 8. Baquero F, Canton R. In vitro activity of sparfloxacin in comparison with currently available antimicrobials against respiratory tract pathogens. J Antimicrob Chemother 1996; 37 Suppl. A: 1-18 9. Hindler J. Tests to detect oxacillin (methicillin)-resistant staphylococci with an oxacillin screen plate. In: Isenberg HD, editor. Clinical microbiology procedures handbook. Washington DC: American Society for Microbiology 1992; 5.5.1-7 10. Archer GL, Pennell E. Detection of methicillin resistance in staphylococci by using a DNA probe. Antimicrob Agents Chemother 1990; 34 (9): 1720-4 II. Woods GL, Washington JA. Antibacterial susceptibility tests: dilution and disk diffusion methods. In: Murray PR, Baron EJ, Pfaller MA, et ai, editors. Manual of clinical microbiology. 6th ed. Washington DC: American Society for Microbiology, 1995; 1327-41 12. Teng R, Harris SC, Nix DE, et al. Pharmacokinetics and safety of trovatloxacin (CP-99,219), a new quinolone antibiotic, following administration of single oral doses to healthy male volunteers. J Antimicrob Chemother 1995 ; 36 (2): 385-94 13. Nakashima M, Yematsu J, Kosuge K, et al. Pharmacokinetics and tolerance of DU-6859a, a new fluoroquinolone, after single and multiple oral doses in healthy volunteers. Antimicrob Agents Chemother 1995; 39 (I): 170-4 14. Child J, Mortiboy D, Andrews JM, et al. Open-label crossover study to determine pharmacokinetics and penetration of two dose regimens of levofloxacin into inflammatory fluid. Antimicrob Agents Chemother 1995; 39 (12): 2749-51 15. Montay G. Pharmacokinetics of sparfloxacin in healthy volunteers: a review. J Antimicrob Chemother 1996; 37 Suppl. A: 27-39 16. Hindler 1. Tests to assess bactericidal activity. In: Isenberg HD, editor. Clinical microbiology procedures handbook. Washington DC: American Society for Microbiology, 1992: 5.16.1421 17. Gooding BB, Jones RN. In vitro antimicrobial activity of CP99,2 I 9, a novel azabicyclo-naphthyridone. Antimicrob Agents Chemother 1993; 37 (2): 349-53 18. Child J, Andrews J, Boswell F, et al. The in vitro activity of CP 99,219, a new naphthyridone antimicrobial agent: a comparison with fluoroquinolone agents. J Antimicrob Chemother 1995; 35 (6): 869-76 19. Zhang YY, Wang F, Zhang J, et al. In vitro antibacterial activity of levofloxacin. Drugs 1995; 49 Suppl. 2: 274-5 20. Parade lis AG, Delidou K, Grigoriadou A, et al. The antimicrobial activity of sparfloxacin, a new quinolone. Drugs 1995; 49 Suppl. 2: 238-9
Correspondence and reprints: Prof. H. Giamarellou, 1st Department of Propedeutic Medicine, Laiko General Hospital, 17, Agiou Thoma Street, Athens 115 27, Greece.
Clin. Drug Invest. 1997 Oct: 14 (4)
