Journal of Antimicrobial Chemotherapy (1992) 29, 639-647
In-vitro activity of tosnfloxadn, a new qninolone antibacterial agent M. A. Cooper, J. M. Andrews and R. Wise*
The in-vitro activity of tosufloxacin (A-61827) was compared with that of temafloxacin, ciprofloxacin and selected members of other groups of antimicrobial agents, against 684 recent distinct clinical isolates and strains with known mechanisms of resistance. Against members of the Enterobacteriaceac, ciprofloxacin was slightly more active than tosufloxacin, which was more active than temafloxacin. The M I Q , of tosufloxacin for all species of Enterobacteriaceae, Psetidomonas aeruginosa and Acinetobacter spp. was < 1 mg/L. Tosufloxacin was slightly more active than temafloxacin, and four to eight fold more active than ciprofloxacin, against the Gram-positive species tested. The MIC*, of tosufloxacin for Staphylococcus aureus was 0-12 mg/L, and for Streptococcus pneumoniae was 0 5 mg/L. All strains of Neisseria spp., Haemophilus inftuenzae and Moraxella catarrhalis were inhibited by tosufloxacin at a concentration of < 0-12 mg/L. Tosufloxacin was the most active quinolone against the anaerobic organisms tested. Cross resistance between quinclones was seen, but not between quinolones and other groups of antimicrobials. The protein binding of tosufloxacin across a range of concentrations averaged 60%. Human serum at a concentration of 70% decreased the bactericidal activity of tosufloxacin by about four-fold.
Introduction
Tosufloxacin (A-61827) is the tosylate salt of A-60969 (T-3262), and was developed to improve the solubility of the parent compound (Barry & Jones, 1989). Previous studies have compared the in-vitro activity of A-60969 with that of other quinolones (Fujimaki et al., 1988), ceftazidime, imipenem and gentamicin (Espinoza et al., 1988), and that of tosufloxacin with other quinolones (Fernandes et al., 1988; Takahata, Otsuki & Nishino, 1988; Barry & Jones, 1989). Only ciprofloxacin was shown in these studies to have greater activity than tosufloxacin or its parent compound against enteric pathogens and Pseudomonas aeruginosa, while tosufloxacin was consistently the most active quinolone tested against Gram-positive species. In this study we have compared the invitro activity of tosufloxacin with that of ciprofloxacin, temafloxacin, gentamicin, cefuroxime or cefaclor and, where appropriate, erythromycin or penicillin. We have also evaluated the effect of human serum and urine at various pHs on the activity of tosufloxacin, and measured the degree of protein binding in human serum. 'Corresponding author. 0305-7453/92/060639+09 $02.00
639 © 1992 The Britiih Society for Anumicrobu] Chemotherapy
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Department of Medical Microbiology, Dudley Road Hospital, Birmingham B18 7QH, UK
640
M. A- Cooper et aL
Materials and methods
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A total of 684 distinct bacterial strains was tested, of which 643 were recent clinical isolates, 26 were well-characterized /Mactamase producers, and the remainder (IS) had been shown previously to have reduced susceptibility to quinolones. The latter 15 strains were isolated from clinical trials or were laboratory mutants (kindly provided by L. Piddock). The following antimicrobials were obtained from the indicated sources: tosufloxacin from Lederle Laboratories, Gosport, UK; tcmafloxacin and erythromycin from Abbott Laboratories, Chicago, USA; ciprofloxacin from Bayer AG, Wuppertal, Germany; gentamicin from Roussel Laboratories, Uxbridge, UK; cefuroxime from Glaxo Group Research, Greenford, UK; cefaclor from Eli Lilly, Basingstoke, UK; penicillin from SmithKline Beecham, Brentford, UK. MICs were determined with a routine agar plate dilution method. Overnight broth cultures were diluted to yield 104 cfu in each 100 fih inoculum; for selected organisms a comparison was made between the MICs obtained with inocula of 104 and 106 cfu. Todd-Hewitt broth (Oxoid, Basingstoke, UK) was used for the /?-haemolytic streptococci; Brain Heart Infusion broth (Oxoid), supplemented with nicotinamide adenine dinucleotide (NAD; Sigma, Poole, UK) 20 mg/L and 5% lysed horse blood (Oxoid), was used for Streptococcus pneumoniae, Haemophilus influenzae and Neisseria spp.; Clostridium spp. were grown in Wilkins-Chalgren broth (Oxoid), supplemented with 1 % Tween 80; Bacteroides spp. were grown in Wilkins-Chalgren broth supplemented with 0-25% sodium succinate; all remaining organisms were grown in Digest broth (Southern Group, Hither Green, UK). Iso-Sensitest agar (Oxoid), supplemented with a-p-nitrophenylglycerol (PNPG; BDH Chemicals, Atherton, UK), was used for all non-anaerobic organisms. This was further supplemented with NAD 20 mg/L and 5% horse blood for streptococci, H. influenzae and Neisseria spp. Wilkins-Chalgren agar, supplemented with PNPG 50 mg/L and 5% horse blood, was used for all anaerobes. The non-fastidious organisms were incubated in air for 18-24 h at 35-37°C; 6% CO2 was added to the atmosphere for the fastidious organisms. Anaerobes were incubated at 35-37°C for 48 h in an anaerobic cabinet (Don Whitley Scientific, Skipton, UK) with an atmosphere of 10% COj, 10% H2 and 80% N2. The MIC was defined as the antibiotic concentration at which no more than two colonies were visible. In the case of the higher inoculum, a slight haze of growth was ignored. The effect of human serum on the MICs and MBCs was studied with eight strains (two each of Escherichia coli, Klebsiella spp., P. aerugirtosa and Staphylococcus aureus), and the effect of urine at different pHs was studied with one strain (E. coli), by a method based on that of Pearson et al. (1980). Iso-Sensitest broth, alone or supplemented with 20% or 70% human serum (Bradsure Biologicals, Market Harborough, UK), or with fresh pooled human urine at pHs of 5-0, 60, 7-0 and 80, was used as the medium. Broth dilution MICs were performed with an initial inoculum 103 cfu/mL. All broths showing no visible growth were subculturcd on to antibiotic-free agar. The MBC was determined with a 99-9% kill as the endpoint. The protein binding of tosufloxacin at concentrations of 0-5, 3 and 10 mg/L was assessed by comparing drug concentrations, measured in distilled water and pooled human serum, after separation of bound drug from free drug by centrifugation at 2000 rpm for 10 min in 'Centriflo' units (Amicon, Danvers, MA, USA). Drug concen-
ID-THTO actirtry of toggfloxtdn
641
tration was measured with a microbiological assay; standards were prepared in phosphate buffer at pH 7 and the indicator organism was E. coli strain 4004 (Bayer). Results
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Table I shows the results obtained against the clinical isolates, excluding the known /Mactamase producers and quinolone-resistant mutants. Against the Enterobacteriaceae, tosufloxacin was between two- and 16-fold more active than temafloxacin, but was less active than ciprofloxacin by a factor of two- to eight-fold for all species, except Providencia spp. and Enterobacter spp. which were more susceptible to tosufloxacin. The quinoloncs were the most active agents tested against these organisms. Tosufloxacin was twice as active as temafloxacin and ciprofloxacin against Acinetobacter spp., and twice as active as temafloxacin, but only half as active as ciprofloxacin, against P. aeruginosa. Clinical isolates of Enterobacteriaceae and Acinetobacter spp. known to be resistant to fluoroquinolones (Table II) were, generally, equally-susceptible to ciprofloxacin and tosufloxacin, and less-susceptible to temafloxacin. Also shown in Table II are the results obtained for the genetically-defined quinolone-resistant mutants. There was a two-fold decrease in susceptibility to tosufloxacin with the gyrA and nalB mutants compared with the parent strain, and an eight-fold increase in susceptibility with the nalC mutant. A known enoxacin-resistant strain, isolated after a course of enoxacin therapy, was 16-fold less-susceptible than the pre-treatment strain to all of the quinolones tested. No cross-resistance between the quinolones and other classes of antibiotic was seen. The known /Mactamase producers (results not shown) or known gentamicin-resistant strains included in the clinical isolates were not quinolone-resistant. Against the Gram-positive cocci, tosufloxacin was two- to four-fold more active than temafloxacin, and four- to eight-fold more active than ciprofloxacin. Tosufloxacin was the most active agent tested against the staphylococci, but the /Mactam compounds and erythromycin were the most active agents against the streptococci. Against five strains each of Enterococcus faecalis (mode MIC of 0-25 mg/L), Enterococcus faecium (mode MIC of 0-5 mg/L), Streptococcus milleri (mode MIC of 0-12 mg/L) and Streptococcus mitts (mode MIC of 025 mg/L), tosufloxacin was the most active agent tested; it was twice as active as temafloxacin and two to four times as active as ciprofloxacin. All strains of H. influenza* tested were inhibited by tosufloxacin at < 0-015 mg/L; tosufloxacin had marginally better activity than the other two quinolones against this organism, while temafloxacin was marginally more active against Moraxella catarrhalis than tosufloxacin, which in turn was more active than ciprofloxacin. Temafloxacin was again the most active quinolone against Neisseria gonorrhoeae, with tosufloxacin more active than ciprofloxacin. All three agents were equally as active against Neisseria meningitidis. Tosufloxacin was four-fold more active than temafloxacin and ciprofloxacin against Clostridium spp., and four-fold more active than ciprofloxacin against Bacteroides fragilis. In general, increasing the inoculum from 104 cfu to 106 caused a change in MIC of one dilution or less for the organisms tested. The protein binding of tosufloxacin at 0-5 mg/L was 60%, at 3 mg/L was 63%, and at 10 mg/L was 57%.
642
M. A. Cooper et aL Table L MICs of tosufloxacin iil comparison with other antimicrobials
\" Rflniffrn
Antimicrobial
E. coli (57)
tosufloxacin tcmafloxacin dprofloxadn ccfuroxime cefaclor gentamicin tosufloxacin tcmafloxacin dprofloxacin ccfuroxime cefaclor gentamicin tosufloxacin tcmafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxadn cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxadn cefuroxime cefaclor gentamidn tosufloxadn temafloxadn dprofloxacin cefuroxime cefaclor gentamidn tosufloxadn temafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxacin
Kkbsiella spp. (39)
Proteus mirabilis (38)
Proteus vulgaris (9)
Proteus rettgeri (5)
Morganella morgani (24)
Serratia spp. (31) (25 Serratia marcescens) (6 Serratia liquefaciens)
Salmonella spp. (10)
Shigella sonnei (10)
MIQo (mg/L)
MIQo (mg/L)
O03 006 0-015 2 1 0-5 0-12 0-12 0-06 2 1 0-5 0-12 0-25 O03 1 1 1 — — —
O06 0-12 O06 4 1 1 2 1 8 4 0-5 0-5 1 0-12 2 2 2 — — —
— — — — —
— — — — —
— — 0-12 0-5 0015 32 > 128 1 0-25 0-5 0-12 > 128 > 128 1 0-06 0-12 CH)3 4 0-5 0-5 0-15 0-03 0-015
8
— — 0-5 1 006 128 > 128 2 1 2 0-5 > 128 > 128 32 (K>6 0-12 003
8
1 0-5 0-03 (H)6 0-015
Range (mg/L) O008->128 (M)15-> 128 0004-32 0-5-64 0-5-> 128 012-64 0015-> 128 O03->128 0008-16 025- > 128 05-128 025-128 003-4 006-4 O015-O5 025-16 05-64 025-8 006-025 012-1 0015-003 2 - > 128 16-> 128 012-1 006-025 012-05 O03-O5 006-64 025-32 012-2 0015-4 O015-8 O008-8 012-> 128 2 - > 128 025-2 012-> 128 012-> 128 006-64 16- > 128 128-> 128 05-128 0015-012 006-012 0015-003 2-8 05-8 05 0015-006 003-006 0008-0015
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(no.)
In-tftro actfcrfty of tonfloxcdn
643
Tabk Leonid Of ffflniffrn
(no.)
Providencia spp. (29)
Citrobacter spp. (9)
Acinetobacter spp. (28)
P. aeruginosa (29)
5. aureus (40) (including 11 MRSA)
Staphylococcus saprophyticus (28)
Group A streptococci (17)
cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin
MIC, (mg/L) 4 2 0-5 025 0-25 0-12 1 16 8 — — — — — — — — — — 0-12 012 0-25 32 32 1 0-25 0-5 0-12 > 128 > 128 1 0-06 0-25 0-5 0-25 1 0-25 O06 0-25 0-25 1 1 0-03 0-12 0-5 0-5 0-008 0-06 4
MIC* (mg/L) 16 64 05 05 2 1 16 128 64 — — — — — — — — — — — — 05 1 1 128 64 32 05 1 025 > 128 > 128 2 012 025 1 05 2 05 O12 O5 05 2 1 O06 O12 05 O5 O008 012 4
Range (mg/L) 2-16 1-64 025-1 O06-2 012-16 003-^ 012-64 0 5 - > 128 0 2 5 - > 128 006-4 012-32 O03-8 8 - > 128 8->128 05-4 0015-012 O03-O5 0008-006 025- > 128 0 5 - > 128 025-128 O008-> 128 0008-> 128 O008-> 128 025- > 128 O06-> 128 006- > 128 012-1 025-2 O06-O5 > 128 > 128 05-4 0008-025 O03-O5 012-4 006-2 012-8 012-1 0015-025 006-05 012-1 O03-2 025-2 0-03-> 128 006-025 025-05 025-05 0008-0015 006-012 2-4
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Enterobacter spp. (9)
Antimicrobial
644
M. A. Cooper etu. Table L contd
Organism (no.)
Group B streptococci (19)
H. influenxae (47)
M. catarrhalis (49)
N. meningitidis (9)
N. gonorrhoeae (34)
B. fragilU (27)
MIC, (mg/L)
erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor
006 O0008 025 05 05 003 05 16 006 O03 025 05 1 003 05 8 012 O015 0008 0015 O015 05 2 2 4 O03 O03 O03 05 025 025 025 2 — — — — — — . 0-004 O004 0-008 006 05 1 025 05 05 2 16 > 128
MIC* (mg/L) 2 0008 05 1 1 003 05 16 006 003 05 05 2 05 4 8 16 05 0015 0015 0015 2 8 2 4 O03 003 006 1 05 025 025 8 — — — — — — O008 0004 O015 025 8 4 2 1 1 4 > 128 > 128
Range (mg/L) O06-8 0008 025-05 05-1 05-1 O03 025-05 8-16 006-012 003 O03-O5 025-05 1-2 O008-4 025-64 4-16 O06-> 128 O008-1 0004-0015 0004-003 0004-003 0004-32 006-128 05-4 O03-8 0008-012 0015-003 003-006 025-8 012^4 006-025 O06-O5 O015-8 0004-0015 0008-0015 0008-0015 0015-006 012-1 025-0-5 0004-012 0004-012 O004-O5 0004-16 006-8 0004-^ O015-4 05-1 05-1 2-8 8 - > 128 > 128
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S. pneumoniae (29)
Antimicrobial
In-vttro acthrtty of tosofloxadn
645
Table I. contd Antimicrobial
(no.) Clostridaan spp. (17)
Range (mg/L)
MIC« (mg/L)
> 128 0-25 0-25 0-25 2 8 64
> 128 2 8 8 128 128 > 128
> 128 0-25-8 025-8 0-25-S 1-128 8-128 8 - > 128
Table IL MICs of temafloxacin, ciprofloxacin and temafloxacin for strains of Enterobacteriaceae and Acinetobacter spp. known to be resistant to fluoroquinolones, including strains with genetically-defined mechanisms of resistance Strain E. coli 1252 (norfloxacin-R) E. coli 1253 (norfloxacin-R) K. pneumoniae HI30 (norfloxacin-R) K. pneumoniae H132 (norfloxacin-R) K. pneumoniae HI86 (norfloxacin-R) M. morganii J327 (ciprofloxacin-R) Enterobacter cloacae K355 (norfloxacin-R) Enterobacter aerogenes K158 (norfloxacin-R) Acinetobacter spp K295 (norfloxacin-R) Acinetobacter spp. K298 (norfloxacin-R) E. co/ZKLl 6 (wild-type) E. coli 1203 (gyrA) E. coli 1204 (nalB) E. coli 1205 (.nalQ E. coli 1226 (enoxacin-sensitive) E. coli 1227 (enoxacin-R)
tosufloxacin
MIC(g/L) temafloxacin
ciprofloxacin
2 128 1 8 128 4 2 4 32 128 0-06 0-12 0-12 0-004 0-008 0-12
2 > 128 2 16 > 128 8 4 32 64 > 128 006 0-25 0-25 0-015 0015 0-25
2 32 2 8 > 16 8 1 8 32 > 128 0O15 0-12 003 0O04 0O08 0-12
R, Resistant. Table IIL Effect of serum on the activity of tosufloxacin
Strain E.coli 1331 E. coli 1332 KkbsieUa spp. H200 Klebsiella spp. H227 P. aeruginosa G344 P. aeruginosa G341 . S. aureus F361 S. aureus F408
Iso-Sensitest broth Iso-Scnsitest broth Iso-Sensitest broth +20% serum +70% serum MIC MBC MIC MBC MIC MBC (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 0015 0O3 0O6 0O6 0-25 0-5 0-25 0-12
0O3 006 0O6 0O6 0-25 1 0-25 0-5
0O3 0015 0-12 0O6 0-25 0-25 0-12 0-25
0O6 0O15 0-25 0-12 1 1 0-5 0-25
0-06 006 0-5 0-25 1 1 0-5 0-5
0-25 0-25 4 0-5 4 2 1 0-5
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gentamicin tosufloxacin temafloxacin riprofloxacin cefuroxime ccfaclor gentamicin
MIC* (mg/L)
646
M. A. Cooper rt «/.
Table III shows the effect of the addition of serum to the bactericidal activity of tosufloxacin. The addition of human serum to a concentration of 20% had no effect on MICs and MBCs overall, but the addition of human serum to a concentration of 70% increased the MICs for the organisms tested by a factor of two- to eight-fold, and increased the MBCs by a factor of one to 64-fold. This effect was most marked with Klebsiella spp. At pH 8, the MIC and MBC for E. coli 1331 were both 0-25 mg/L. This increased to 0-5 mg/L for both strains at pH 7, and to 2 mg/L for both strains at pH 6 and pH 5.
Our results show that tosufloxacin has a very broad spectrum of activity, which although slightly inferior to ciprofloxacin against P. aeruginosa and members of the Enterobacteriaceae, is greater than ciprofloxacin and temafloxacin against the Gram-positive and anaerobic species. Of particular note is the enhanced activity against staphylococci and S. pneumoniae when compared with the other quinolones. The results obtained were, in general, in agreement with those of other workers, but there were some exceptions. Most notably, other workers have found consistently that Klebsiella spp. and Enterobacter spp. show greater susceptibility to tosufloxacin (or T-3262) than our results indicate; thus, for Klebsiella pneumoniae, a tosufloxacin MIC*, of 0-06 mg/L (Fernandes et al., 1988) and T-3262 MICV of 0-1 mg/L (Fujimaki et al., 1988), 0-06 mg/L (Espinoza et al., 1988) and 0-1 mg/L (Takahata et al., 1988) have been reported, compared with our tosufloxacin result of 1 mg/L for Klebsiella spp. Similarly, other workers have found lower ciprofloxacin MICs while the results for Enterobacter spp. show a similar discrepancy. Other discrepancies involve Providencia spp. and Serratia spp.; some workers have found these species to be more susceptible to the quinolones (Fernandes et al., 1988), while other workers report these organisms to be much less susceptible than our results suggest (Fujimaki et al., 1988; Takahata et al., 1988). The reason for these differences is not known; it may reflect the different degrees of quinolone usage around the world, leading to genuine variability in the susceptibility of these organisms from country to country, or it may be due to methodological differences giving a false impression of variations in susceptibility. Tosufloxacin is more highly protein-bound than other quinolones, and this was reflected in the decrease in activity with increased amounts of serum in the culture media. As with other quinolones, the activity of tosufloxacin was greatest at alkaline pH. Early human pharmacokinetic studies of oral tosufloxacin gave mean peak serum levels of 1-2 mg/L after 300 mg, 1-9 mg/L after 600 mg, and 2-8 mg/L after 900 mg, with a serum half-life of 5-6-6-9 h (Kinzig et al., 1991). Consideration of these data with our in-vitro susceptibility results indicates that tosufloxacin may be a useful addition to the fluoroquinolone family, especially against the Gram-positive and anaerobic bacteria which are seen as the weakness of those quinolones currently available.
Acknowledgements We thank P. Brock and S. Lockhart of Lederle Laboratories for their advice and financial support.
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Discussion
In-rttro acthrfty of tosnfloxsdn
647
References
{Received 5 November 1991; revised version accepted 30 January 1992)
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Barry, A. L. & Jones, R. N. (1989). In-vitro activities of temafloxacin, tosufloxacin (A-61827) and five other fluoroquinolone agents. Journal of Antimicrobial Chemotherapy 23, 527-35. Espinoza, A. M., Chin, N-X., Novelli, A. & Neu, H. C. (1988). Comparative in vitro activity of a new fluorinated 4-quinolone, T-3262 (A-60969). Antimicrobial Agents and Chemotherapy 32, 663-70. Fernandes, P. B., Chu, D. T. W., Swanson, R. N., Ramer, N. R., Hanson, C. W., Bower, R. R. el al. (1988). A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 32, 27-32. Fujimaki, K., Noumi, T., Saikawa, I., Inoue, M. & Mitsuhashi, S. (1988). In vitro and in vivo antibacterial activities of T-3262, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 32, 827-33. Kinzig, M., Kuye, O., Sorgel, F., Greene, D. S., Muth, P., Batra, V. K. et al. (1991). Pharmacolrinetics of rising single doses of tosufloxacin. In Proceedings of the Seventeenth International Congress of Chemotherapy, Berlin. 1991. Abstract 380. Pearson, R. D., Steigbigel, R. T., Davis, H. T. & Chapman, S. W. (1980). Method of reliable determination of minimal lethal antibiotic concentrations. Antimicrobial Agents and Chemotherapy 18, 699-708. Takahata, M., Otsulri, M. & Nishino, T. (1988). In-vitro and in-vivo activities of T-3262, a new pyridone carboxylic acid. Journal of Antimicrobial Chemotherapy 24, 143-54.
In-vitro activity of tosnfloxadn, a new qninolone antibacterial agent M. A. Cooper, J. M. Andrews and R. Wise*
The in-vitro activity of tosufloxacin (A-61827) was compared with that of temafloxacin, ciprofloxacin and selected members of other groups of antimicrobial agents, against 684 recent distinct clinical isolates and strains with known mechanisms of resistance. Against members of the Enterobacteriaceac, ciprofloxacin was slightly more active than tosufloxacin, which was more active than temafloxacin. The M I Q , of tosufloxacin for all species of Enterobacteriaceae, Psetidomonas aeruginosa and Acinetobacter spp. was < 1 mg/L. Tosufloxacin was slightly more active than temafloxacin, and four to eight fold more active than ciprofloxacin, against the Gram-positive species tested. The MIC*, of tosufloxacin for Staphylococcus aureus was 0-12 mg/L, and for Streptococcus pneumoniae was 0 5 mg/L. All strains of Neisseria spp., Haemophilus inftuenzae and Moraxella catarrhalis were inhibited by tosufloxacin at a concentration of < 0-12 mg/L. Tosufloxacin was the most active quinolone against the anaerobic organisms tested. Cross resistance between quinclones was seen, but not between quinolones and other groups of antimicrobials. The protein binding of tosufloxacin across a range of concentrations averaged 60%. Human serum at a concentration of 70% decreased the bactericidal activity of tosufloxacin by about four-fold.
Introduction
Tosufloxacin (A-61827) is the tosylate salt of A-60969 (T-3262), and was developed to improve the solubility of the parent compound (Barry & Jones, 1989). Previous studies have compared the in-vitro activity of A-60969 with that of other quinolones (Fujimaki et al., 1988), ceftazidime, imipenem and gentamicin (Espinoza et al., 1988), and that of tosufloxacin with other quinolones (Fernandes et al., 1988; Takahata, Otsuki & Nishino, 1988; Barry & Jones, 1989). Only ciprofloxacin was shown in these studies to have greater activity than tosufloxacin or its parent compound against enteric pathogens and Pseudomonas aeruginosa, while tosufloxacin was consistently the most active quinolone tested against Gram-positive species. In this study we have compared the invitro activity of tosufloxacin with that of ciprofloxacin, temafloxacin, gentamicin, cefuroxime or cefaclor and, where appropriate, erythromycin or penicillin. We have also evaluated the effect of human serum and urine at various pHs on the activity of tosufloxacin, and measured the degree of protein binding in human serum. 'Corresponding author. 0305-7453/92/060639+09 $02.00
639 © 1992 The Britiih Society for Anumicrobu] Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Florida Atlantic University on November 7, 2014
Department of Medical Microbiology, Dudley Road Hospital, Birmingham B18 7QH, UK
640
M. A- Cooper et aL
Materials and methods
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A total of 684 distinct bacterial strains was tested, of which 643 were recent clinical isolates, 26 were well-characterized /Mactamase producers, and the remainder (IS) had been shown previously to have reduced susceptibility to quinolones. The latter 15 strains were isolated from clinical trials or were laboratory mutants (kindly provided by L. Piddock). The following antimicrobials were obtained from the indicated sources: tosufloxacin from Lederle Laboratories, Gosport, UK; tcmafloxacin and erythromycin from Abbott Laboratories, Chicago, USA; ciprofloxacin from Bayer AG, Wuppertal, Germany; gentamicin from Roussel Laboratories, Uxbridge, UK; cefuroxime from Glaxo Group Research, Greenford, UK; cefaclor from Eli Lilly, Basingstoke, UK; penicillin from SmithKline Beecham, Brentford, UK. MICs were determined with a routine agar plate dilution method. Overnight broth cultures were diluted to yield 104 cfu in each 100 fih inoculum; for selected organisms a comparison was made between the MICs obtained with inocula of 104 and 106 cfu. Todd-Hewitt broth (Oxoid, Basingstoke, UK) was used for the /?-haemolytic streptococci; Brain Heart Infusion broth (Oxoid), supplemented with nicotinamide adenine dinucleotide (NAD; Sigma, Poole, UK) 20 mg/L and 5% lysed horse blood (Oxoid), was used for Streptococcus pneumoniae, Haemophilus influenzae and Neisseria spp.; Clostridium spp. were grown in Wilkins-Chalgren broth (Oxoid), supplemented with 1 % Tween 80; Bacteroides spp. were grown in Wilkins-Chalgren broth supplemented with 0-25% sodium succinate; all remaining organisms were grown in Digest broth (Southern Group, Hither Green, UK). Iso-Sensitest agar (Oxoid), supplemented with a-p-nitrophenylglycerol (PNPG; BDH Chemicals, Atherton, UK), was used for all non-anaerobic organisms. This was further supplemented with NAD 20 mg/L and 5% horse blood for streptococci, H. influenzae and Neisseria spp. Wilkins-Chalgren agar, supplemented with PNPG 50 mg/L and 5% horse blood, was used for all anaerobes. The non-fastidious organisms were incubated in air for 18-24 h at 35-37°C; 6% CO2 was added to the atmosphere for the fastidious organisms. Anaerobes were incubated at 35-37°C for 48 h in an anaerobic cabinet (Don Whitley Scientific, Skipton, UK) with an atmosphere of 10% COj, 10% H2 and 80% N2. The MIC was defined as the antibiotic concentration at which no more than two colonies were visible. In the case of the higher inoculum, a slight haze of growth was ignored. The effect of human serum on the MICs and MBCs was studied with eight strains (two each of Escherichia coli, Klebsiella spp., P. aerugirtosa and Staphylococcus aureus), and the effect of urine at different pHs was studied with one strain (E. coli), by a method based on that of Pearson et al. (1980). Iso-Sensitest broth, alone or supplemented with 20% or 70% human serum (Bradsure Biologicals, Market Harborough, UK), or with fresh pooled human urine at pHs of 5-0, 60, 7-0 and 80, was used as the medium. Broth dilution MICs were performed with an initial inoculum 103 cfu/mL. All broths showing no visible growth were subculturcd on to antibiotic-free agar. The MBC was determined with a 99-9% kill as the endpoint. The protein binding of tosufloxacin at concentrations of 0-5, 3 and 10 mg/L was assessed by comparing drug concentrations, measured in distilled water and pooled human serum, after separation of bound drug from free drug by centrifugation at 2000 rpm for 10 min in 'Centriflo' units (Amicon, Danvers, MA, USA). Drug concen-
ID-THTO actirtry of toggfloxtdn
641
tration was measured with a microbiological assay; standards were prepared in phosphate buffer at pH 7 and the indicator organism was E. coli strain 4004 (Bayer). Results
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Table I shows the results obtained against the clinical isolates, excluding the known /Mactamase producers and quinolone-resistant mutants. Against the Enterobacteriaceae, tosufloxacin was between two- and 16-fold more active than temafloxacin, but was less active than ciprofloxacin by a factor of two- to eight-fold for all species, except Providencia spp. and Enterobacter spp. which were more susceptible to tosufloxacin. The quinoloncs were the most active agents tested against these organisms. Tosufloxacin was twice as active as temafloxacin and ciprofloxacin against Acinetobacter spp., and twice as active as temafloxacin, but only half as active as ciprofloxacin, against P. aeruginosa. Clinical isolates of Enterobacteriaceae and Acinetobacter spp. known to be resistant to fluoroquinolones (Table II) were, generally, equally-susceptible to ciprofloxacin and tosufloxacin, and less-susceptible to temafloxacin. Also shown in Table II are the results obtained for the genetically-defined quinolone-resistant mutants. There was a two-fold decrease in susceptibility to tosufloxacin with the gyrA and nalB mutants compared with the parent strain, and an eight-fold increase in susceptibility with the nalC mutant. A known enoxacin-resistant strain, isolated after a course of enoxacin therapy, was 16-fold less-susceptible than the pre-treatment strain to all of the quinolones tested. No cross-resistance between the quinolones and other classes of antibiotic was seen. The known /Mactamase producers (results not shown) or known gentamicin-resistant strains included in the clinical isolates were not quinolone-resistant. Against the Gram-positive cocci, tosufloxacin was two- to four-fold more active than temafloxacin, and four- to eight-fold more active than ciprofloxacin. Tosufloxacin was the most active agent tested against the staphylococci, but the /Mactam compounds and erythromycin were the most active agents against the streptococci. Against five strains each of Enterococcus faecalis (mode MIC of 0-25 mg/L), Enterococcus faecium (mode MIC of 0-5 mg/L), Streptococcus milleri (mode MIC of 0-12 mg/L) and Streptococcus mitts (mode MIC of 025 mg/L), tosufloxacin was the most active agent tested; it was twice as active as temafloxacin and two to four times as active as ciprofloxacin. All strains of H. influenza* tested were inhibited by tosufloxacin at < 0-015 mg/L; tosufloxacin had marginally better activity than the other two quinolones against this organism, while temafloxacin was marginally more active against Moraxella catarrhalis than tosufloxacin, which in turn was more active than ciprofloxacin. Temafloxacin was again the most active quinolone against Neisseria gonorrhoeae, with tosufloxacin more active than ciprofloxacin. All three agents were equally as active against Neisseria meningitidis. Tosufloxacin was four-fold more active than temafloxacin and ciprofloxacin against Clostridium spp., and four-fold more active than ciprofloxacin against Bacteroides fragilis. In general, increasing the inoculum from 104 cfu to 106 caused a change in MIC of one dilution or less for the organisms tested. The protein binding of tosufloxacin at 0-5 mg/L was 60%, at 3 mg/L was 63%, and at 10 mg/L was 57%.
642
M. A. Cooper et aL Table L MICs of tosufloxacin iil comparison with other antimicrobials
\" Rflniffrn
Antimicrobial
E. coli (57)
tosufloxacin tcmafloxacin dprofloxadn ccfuroxime cefaclor gentamicin tosufloxacin tcmafloxacin dprofloxacin ccfuroxime cefaclor gentamicin tosufloxacin tcmafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxadn cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxadn cefuroxime cefaclor gentamidn tosufloxadn temafloxadn dprofloxacin cefuroxime cefaclor gentamidn tosufloxadn temafloxacin dprofloxacin cefuroxime cefaclor gentamicin tosufloxadn temafloxacin dprofloxacin
Kkbsiella spp. (39)
Proteus mirabilis (38)
Proteus vulgaris (9)
Proteus rettgeri (5)
Morganella morgani (24)
Serratia spp. (31) (25 Serratia marcescens) (6 Serratia liquefaciens)
Salmonella spp. (10)
Shigella sonnei (10)
MIQo (mg/L)
MIQo (mg/L)
O03 006 0-015 2 1 0-5 0-12 0-12 0-06 2 1 0-5 0-12 0-25 O03 1 1 1 — — —
O06 0-12 O06 4 1 1 2 1 8 4 0-5 0-5 1 0-12 2 2 2 — — —
— — — — —
— — — — —
— — 0-12 0-5 0015 32 > 128 1 0-25 0-5 0-12 > 128 > 128 1 0-06 0-12 CH)3 4 0-5 0-5 0-15 0-03 0-015
8
— — 0-5 1 006 128 > 128 2 1 2 0-5 > 128 > 128 32 (K>6 0-12 003
8
1 0-5 0-03 (H)6 0-015
Range (mg/L) O008->128 (M)15-> 128 0004-32 0-5-64 0-5-> 128 012-64 0015-> 128 O03->128 0008-16 025- > 128 05-128 025-128 003-4 006-4 O015-O5 025-16 05-64 025-8 006-025 012-1 0015-003 2 - > 128 16-> 128 012-1 006-025 012-05 O03-O5 006-64 025-32 012-2 0015-4 O015-8 O008-8 012-> 128 2 - > 128 025-2 012-> 128 012-> 128 006-64 16- > 128 128-> 128 05-128 0015-012 006-012 0015-003 2-8 05-8 05 0015-006 003-006 0008-0015
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(no.)
In-tftro actfcrfty of tonfloxcdn
643
Tabk Leonid Of ffflniffrn
(no.)
Providencia spp. (29)
Citrobacter spp. (9)
Acinetobacter spp. (28)
P. aeruginosa (29)
5. aureus (40) (including 11 MRSA)
Staphylococcus saprophyticus (28)
Group A streptococci (17)
cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin
MIC, (mg/L) 4 2 0-5 025 0-25 0-12 1 16 8 — — — — — — — — — — 0-12 012 0-25 32 32 1 0-25 0-5 0-12 > 128 > 128 1 0-06 0-25 0-5 0-25 1 0-25 O06 0-25 0-25 1 1 0-03 0-12 0-5 0-5 0-008 0-06 4
MIC* (mg/L) 16 64 05 05 2 1 16 128 64 — — — — — — — — — — — — 05 1 1 128 64 32 05 1 025 > 128 > 128 2 012 025 1 05 2 05 O12 O5 05 2 1 O06 O12 05 O5 O008 012 4
Range (mg/L) 2-16 1-64 025-1 O06-2 012-16 003-^ 012-64 0 5 - > 128 0 2 5 - > 128 006-4 012-32 O03-8 8 - > 128 8->128 05-4 0015-012 O03-O5 0008-006 025- > 128 0 5 - > 128 025-128 O008-> 128 0008-> 128 O008-> 128 025- > 128 O06-> 128 006- > 128 012-1 025-2 O06-O5 > 128 > 128 05-4 0008-025 O03-O5 012-4 006-2 012-8 012-1 0015-025 006-05 012-1 O03-2 025-2 0-03-> 128 006-025 025-05 025-05 0008-0015 006-012 2-4
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Enterobacter spp. (9)
Antimicrobial
644
M. A. Cooper etu. Table L contd
Organism (no.)
Group B streptococci (19)
H. influenxae (47)
M. catarrhalis (49)
N. meningitidis (9)
N. gonorrhoeae (34)
B. fragilU (27)
MIC, (mg/L)
erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin penicillin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor gentamicin erythromycin tosufloxacin temafloxacin ciprofloxacin cefuroxime cefaclor
006 O0008 025 05 05 003 05 16 006 O03 025 05 1 003 05 8 012 O015 0008 0015 O015 05 2 2 4 O03 O03 O03 05 025 025 025 2 — — — — — — . 0-004 O004 0-008 006 05 1 025 05 05 2 16 > 128
MIC* (mg/L) 2 0008 05 1 1 003 05 16 006 003 05 05 2 05 4 8 16 05 0015 0015 0015 2 8 2 4 O03 003 006 1 05 025 025 8 — — — — — — O008 0004 O015 025 8 4 2 1 1 4 > 128 > 128
Range (mg/L) O06-8 0008 025-05 05-1 05-1 O03 025-05 8-16 006-012 003 O03-O5 025-05 1-2 O008-4 025-64 4-16 O06-> 128 O008-1 0004-0015 0004-003 0004-003 0004-32 006-128 05-4 O03-8 0008-012 0015-003 003-006 025-8 012^4 006-025 O06-O5 O015-8 0004-0015 0008-0015 0008-0015 0015-006 012-1 025-0-5 0004-012 0004-012 O004-O5 0004-16 006-8 0004-^ O015-4 05-1 05-1 2-8 8 - > 128 > 128
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S. pneumoniae (29)
Antimicrobial
In-vttro acthrtty of tosofloxadn
645
Table I. contd Antimicrobial
(no.) Clostridaan spp. (17)
Range (mg/L)
MIC« (mg/L)
> 128 0-25 0-25 0-25 2 8 64
> 128 2 8 8 128 128 > 128
> 128 0-25-8 025-8 0-25-S 1-128 8-128 8 - > 128
Table IL MICs of temafloxacin, ciprofloxacin and temafloxacin for strains of Enterobacteriaceae and Acinetobacter spp. known to be resistant to fluoroquinolones, including strains with genetically-defined mechanisms of resistance Strain E. coli 1252 (norfloxacin-R) E. coli 1253 (norfloxacin-R) K. pneumoniae HI30 (norfloxacin-R) K. pneumoniae H132 (norfloxacin-R) K. pneumoniae HI86 (norfloxacin-R) M. morganii J327 (ciprofloxacin-R) Enterobacter cloacae K355 (norfloxacin-R) Enterobacter aerogenes K158 (norfloxacin-R) Acinetobacter spp K295 (norfloxacin-R) Acinetobacter spp. K298 (norfloxacin-R) E. co/ZKLl 6 (wild-type) E. coli 1203 (gyrA) E. coli 1204 (nalB) E. coli 1205 (.nalQ E. coli 1226 (enoxacin-sensitive) E. coli 1227 (enoxacin-R)
tosufloxacin
MIC(g/L) temafloxacin
ciprofloxacin
2 128 1 8 128 4 2 4 32 128 0-06 0-12 0-12 0-004 0-008 0-12
2 > 128 2 16 > 128 8 4 32 64 > 128 006 0-25 0-25 0-015 0015 0-25
2 32 2 8 > 16 8 1 8 32 > 128 0O15 0-12 003 0O04 0O08 0-12
R, Resistant. Table IIL Effect of serum on the activity of tosufloxacin
Strain E.coli 1331 E. coli 1332 KkbsieUa spp. H200 Klebsiella spp. H227 P. aeruginosa G344 P. aeruginosa G341 . S. aureus F361 S. aureus F408
Iso-Sensitest broth Iso-Scnsitest broth Iso-Sensitest broth +20% serum +70% serum MIC MBC MIC MBC MIC MBC (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) (mg/L) 0015 0O3 0O6 0O6 0-25 0-5 0-25 0-12
0O3 006 0O6 0O6 0-25 1 0-25 0-5
0O3 0015 0-12 0O6 0-25 0-25 0-12 0-25
0O6 0O15 0-25 0-12 1 1 0-5 0-25
0-06 006 0-5 0-25 1 1 0-5 0-5
0-25 0-25 4 0-5 4 2 1 0-5
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gentamicin tosufloxacin temafloxacin riprofloxacin cefuroxime ccfaclor gentamicin
MIC* (mg/L)
646
M. A. Cooper rt «/.
Table III shows the effect of the addition of serum to the bactericidal activity of tosufloxacin. The addition of human serum to a concentration of 20% had no effect on MICs and MBCs overall, but the addition of human serum to a concentration of 70% increased the MICs for the organisms tested by a factor of two- to eight-fold, and increased the MBCs by a factor of one to 64-fold. This effect was most marked with Klebsiella spp. At pH 8, the MIC and MBC for E. coli 1331 were both 0-25 mg/L. This increased to 0-5 mg/L for both strains at pH 7, and to 2 mg/L for both strains at pH 6 and pH 5.
Our results show that tosufloxacin has a very broad spectrum of activity, which although slightly inferior to ciprofloxacin against P. aeruginosa and members of the Enterobacteriaceae, is greater than ciprofloxacin and temafloxacin against the Gram-positive and anaerobic species. Of particular note is the enhanced activity against staphylococci and S. pneumoniae when compared with the other quinolones. The results obtained were, in general, in agreement with those of other workers, but there were some exceptions. Most notably, other workers have found consistently that Klebsiella spp. and Enterobacter spp. show greater susceptibility to tosufloxacin (or T-3262) than our results indicate; thus, for Klebsiella pneumoniae, a tosufloxacin MIC*, of 0-06 mg/L (Fernandes et al., 1988) and T-3262 MICV of 0-1 mg/L (Fujimaki et al., 1988), 0-06 mg/L (Espinoza et al., 1988) and 0-1 mg/L (Takahata et al., 1988) have been reported, compared with our tosufloxacin result of 1 mg/L for Klebsiella spp. Similarly, other workers have found lower ciprofloxacin MICs while the results for Enterobacter spp. show a similar discrepancy. Other discrepancies involve Providencia spp. and Serratia spp.; some workers have found these species to be more susceptible to the quinolones (Fernandes et al., 1988), while other workers report these organisms to be much less susceptible than our results suggest (Fujimaki et al., 1988; Takahata et al., 1988). The reason for these differences is not known; it may reflect the different degrees of quinolone usage around the world, leading to genuine variability in the susceptibility of these organisms from country to country, or it may be due to methodological differences giving a false impression of variations in susceptibility. Tosufloxacin is more highly protein-bound than other quinolones, and this was reflected in the decrease in activity with increased amounts of serum in the culture media. As with other quinolones, the activity of tosufloxacin was greatest at alkaline pH. Early human pharmacokinetic studies of oral tosufloxacin gave mean peak serum levels of 1-2 mg/L after 300 mg, 1-9 mg/L after 600 mg, and 2-8 mg/L after 900 mg, with a serum half-life of 5-6-6-9 h (Kinzig et al., 1991). Consideration of these data with our in-vitro susceptibility results indicates that tosufloxacin may be a useful addition to the fluoroquinolone family, especially against the Gram-positive and anaerobic bacteria which are seen as the weakness of those quinolones currently available.
Acknowledgements We thank P. Brock and S. Lockhart of Lederle Laboratories for their advice and financial support.
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Discussion
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647
References
{Received 5 November 1991; revised version accepted 30 January 1992)
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Barry, A. L. & Jones, R. N. (1989). In-vitro activities of temafloxacin, tosufloxacin (A-61827) and five other fluoroquinolone agents. Journal of Antimicrobial Chemotherapy 23, 527-35. Espinoza, A. M., Chin, N-X., Novelli, A. & Neu, H. C. (1988). Comparative in vitro activity of a new fluorinated 4-quinolone, T-3262 (A-60969). Antimicrobial Agents and Chemotherapy 32, 663-70. Fernandes, P. B., Chu, D. T. W., Swanson, R. N., Ramer, N. R., Hanson, C. W., Bower, R. R. el al. (1988). A-61827 (A-60969), a new fluoronaphthyridine with activity against both aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 32, 27-32. Fujimaki, K., Noumi, T., Saikawa, I., Inoue, M. & Mitsuhashi, S. (1988). In vitro and in vivo antibacterial activities of T-3262, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 32, 827-33. Kinzig, M., Kuye, O., Sorgel, F., Greene, D. S., Muth, P., Batra, V. K. et al. (1991). Pharmacolrinetics of rising single doses of tosufloxacin. In Proceedings of the Seventeenth International Congress of Chemotherapy, Berlin. 1991. Abstract 380. Pearson, R. D., Steigbigel, R. T., Davis, H. T. & Chapman, S. W. (1980). Method of reliable determination of minimal lethal antibiotic concentrations. Antimicrobial Agents and Chemotherapy 18, 699-708. Takahata, M., Otsulri, M. & Nishino, T. (1988). In-vitro and in-vivo activities of T-3262, a new pyridone carboxylic acid. Journal of Antimicrobial Chemotherapy 24, 143-54.
