Journal of Antimicrobial Chemotherapy (1992) 29, 649-660
The in-vitro activity of two new qmnolones: rufloxacin and MF 961 R. Wise, J. M. Andrews, R. Matthews and M. Wobtenbohne
The in-vitro activity of two new quinolone antimicrobials, rufloxacin and MF 961, together with the dcsmethylated metabolite of rufloxacin (MF 922) were compared with other orally administered agents against 622 bacterial strains. Against Enterobacteriaceae and Pseudomonas aentginosa rufloxacin was generally active (MIQo 1-8 mg/L) with the exception of Klebsiella and Serratia spp. (MIC*, 32 mg/L and Enterobacter spp. (MlQo) 64 mg/L. The respiratory pathogens Haemophilus influenzae and Moraxella catarrhalis were susceptible to rufloxacin (MIC^ 05 and 1 mg/L respectively) but Streptococcus pneumoniae was less susceptible (MIC*, 32 mg/L). Staphylococcus attreus were susceptible to rufloxacin (MIC,, 2 mg/L). The rufloxacin metabolite MF 922 was generally as active as its parent. MF 961 was usually two-fold more active than rufloxacin. AU three compounds were four to 16 times less active than norfloxacin, but rufloxacin was as active or somewhat more active than norfloxacin against Staphylococcus spp. Any strains showing decreased susceptibility to other quinolones exhibited cross resistance to these new agents. The MBC of rufloxacin and MF 922 was within one dilution of the MIC and human serum had little effect upon the activity of both agents. The protein binding of rufloxacin and MF 922 at 1 and 10 mg/L were 55% and 63-8% and 30-3% and 32-6% respectively. The activity of rufloxacin against four strains of Chlamydia trachomatis and one strain of Chlamydia pneumoniae was determined. The MICs for C. trachomatis were 4-8 mg/L and 4 mg/L for C. pneumoniae. Introduction Rufloxacin (MF 934) is a new broad spectrum quinolone (Cecchetti et al., 1987) and has the formula 9-fluoro-10-(-methyl) piperazinyl-7-oxo-2, 3-dihydro-7H-pyrido-(l,2,3, de) (1,4) benzothiazine-6-carboxylic acid, the structure is shown in Figure 1. Rufloxacin undergoes a modest amount of metabolism in man and an ^-desmethylated derivative is found in urine (J. Kisicki, personal communication), which has been designated MF 922. In addition a molecular modification of rufloxacin has been produced (MF 961) which differs in that there is a fluoromethyl substituent in the benzothiazine ring (Figure 2).
COOH
Figure 1. The structure of rufloxacin (MF 934). 649 0305-7453/92/060649+12 $02.00/0 © 1992 The British Society for Antnnkrobud Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Department of Medical Microbiology, Dudley Road Hospital, Birmingham B18 7QH, UK
650
R.Wbetf aL
COOH
sv Figure 2. The itructure of MF 961.
Materials and methods
A total of 622 strains were studied, of which 589 were recent clinical isolates, eight were well characterized /Mactamase producers, 15 aminoglycoside resistant, and ten had been previously shown to have reduced sensitivity to the quinolones. The following antimicrobials of known potency were studied: rufloxacin, MF 961 and MF 922, obtained from Mediolanum Farmaceutici, Milan, Italy; cefuroxime, Glaxo Laboratories, Greenford, Middlesex, UK; norfloxacin, Merck Sharpe and Dohme, Hoddesdon, Herts., UK; penicillin, amoxycillin, davulanic acid and methicillin, Beechams, Brentford, UK; cefaclor and erythromycin, Eh" Lilly, Basingstoke, Hampshire, UK; ciprofloxacin, Bayer Pharmaceuticals, Newbury, UK. Minimum inhibitory concentrations (MICs) were determined by an agar plate dilution method (Wise, Ashby & Andrews, 1988). The final inoculum used was 104 cfu for all strains. For selected strains a comparison was made between results obtained with a final inocula of 104 and 10* cfu. IsoSensitest agar (pH 7-2, Oxoid, Basingstoke, UK) was used, supplemented with a 5% horse blood and 20 mg/L NAD (B.D.H. Ltd, Poole, Dorset) for fastidious organisms. For anaerobes Wilkins and Chalgren agar (Oxoid) with 5% horse blood was used. All plates were incubated in air for 18 h except for the following: Haemophilia inftuenzae and Neisseria spp. were incubated in air enriched with 6% CO2 for 18 h; anaerobes were incubated in an atmosphere containing 10% COj, 80% Nj, 10% H2 in an anaerobic cabinet (Don Whitley Ltd, Shipley, West Yorkshire, UK) for 48 h. The amoxycillin/clavulanate was tested in a ratio of 2: 1 and the susceptibilities were expressed in terms of the MIC to amoxycillin. The effect of human serum and urine on the MIC and MBC values of rufloxacin and MF 922 were studied with 8 strains (2 each of Staphylococcus aureus, Escherichia coli, Klebsiella sp., and Moraxella catarrhalis) by a method based on that of Pearson et al. (1980). In a microdilution system, Iso-Sensitest broth alone or supplemented with 20% or 70% human serum and pooled antibiotic free human urine at pH 5, 6, 7 and 8 were used as media, and broth dilution MICs were performed with an inoculum of 10*cfu/mL. All dilutions showing no visible growth after overnight incubation were subcultured on to antibiotic free agar, and the MBC was defined as a 99-9% kill as the endpoint
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
In this study we have compared the in-vitro activity of these two new agents and the desmethylated derivative of rufloxacin with those of norfloxacin, cefaclor, cefuroxime, amoxycillin and a 2: 1 mixture of amoxycillin/clavulanic acid. The antibacterial activity of rufloxacin against Chlamydia trachomatis and Chlamydia pneumoniae was also determined and compared to that of ciprofloxacin.
In-rttro activity of rafloudn and MF 961
651
Results Table I shows the activity of the different agents against the recent clinical isolates and the known /Mactamase producers. The activity of rufloxacin and its metabolite (MF 922) against the frequently encountered species of Enterobacteriaceae were remarkably similar and the activity of MF 961 was generally two-fold greater than that of rufloxacin. All three compounds were 4-16-times less active than norfloxacin, but were generally more active than the /Mactam antibiotics studied. If an individual strain was less susceptible to one of the quinolones it tended to be less susceptible to the others: an exception to this was an isolate of Providencia stuartii which was susceptible to 4 mg/L of rufloxacin, norfloxacin and MF 961 yet susceptible to 128 mg/L of MF 922. A less pronounced difference between the poor activity of MF 922 and the other quinolones was seen for other strains of P. stuartii. There was considerable variation amongst susceptibilities of the Enterobacteriaceae to the quinolones, for example Citrobacter. Salmonella and Shigella spp. were highly susceptible to rufloxacin (MIC*, 4 mg/L). Acinetobacter spp. were markedly less susceptible to the metabolite MF 922 than to the parent compound, rufloxacin. All four quinolones displayed decreased activity against strains with known or unknown mechanisms of resistance to other quinolones. For example a known gyr A resistant mutant E. coli isolated from a clinical trial was susceptible to 8 mg/L rufloxacin and 2 mg/L MF 961 and 0-25 mg/L norfloxacin whereas the pre-treatment isolate (wild-type) was susceptible to 0-25, 0-25 and 006 mg/L respectively. An outer membrane protein (OMP) F deficient variant of E. coli was susceptible to 16 mg/L rufloxacin. Strains of H. wfluauae were highly susceptible to the three new compounds, the rufloxacin MIC*, being 0-5 mg/L. The 12 /J-lactamase producing strains were as
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
The protein binding of rufloxacin and MF 922 was assessed by comparing samples at a concentration of 1 and 10 mg/L prepared in either pooled human serum (Flow Labs, McLean, VA, USA) or distilled water, after separation of bound from free drug by centrifugation at 2000 rpm for 10 min using 'Centriflo' units (Amicon Corp., Danvers, MA, USA). A microbiological assay method was used to measure drug concentrations. Standards were prepared in phosphate buffer at pH 7, and the indicator organism was E. coli 4004 (obtained from Bayer AG, Germany, being highly sensitive to quinolones). The activities of rufloxacin and ciprofloxacin against 4 isolates of C. trachomatis and one of C. pnewnoniae were determined by the method of Webberley et al. (1987). Briefly, McCoy cell cover slip cultures were infected with approximately 1000 inclusionforming units of chlamydia, exposed to the various dilutions of the antimicrobials for 48 h and then stained with 'Imagen Chlamydia test' (NovoBiolabs, Cambridge, England). The MIC was the lowest concentration that inhibited all inclusion development The minimum lethal concentration (MLC) was defined as the lowest concentration that inhibited inclusion development in cell sheets exposed to antimicrobial for 48 h, then reincubated in drug-free medium for an additional 48 h. Again a 99-9% loll was used as the endpoint.
652
R. W b e r t o l
T«bk I. The in-vitro activity of rufloxacin, MF 961 and MF 922 compared with other antimicrobial agents (mg/L)
Antimicrobial
MIC*
MIC*
Range
E. coli (59)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxklav
05 025 05 012 1 2 >128 4
2 1 2 025 4 8 >128 8
025-> 128 O12->128 O12->128 003-> 128 O5->128 05-32 O5->128 05-64
Klebsiella sp. (39)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 4 05 1 2 128 2
32 8 32 8 4 16 >128 16
O5->128 O25->128 O5-128 006-64 0 5 - > 128 0 5 - > 128 2->128 1-32
Enterobacter sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
64 16 64 8 >128 >128 >128 64
1-128 05-32 1-64 012-16 2->128 2->128 16->128 4-64
Citrobacter sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
05 025 025 006 4 4 128 8
1 1 1 012 >128 32 >128 64
025-1 012-1 025-2 003-012 O5->128 O5->128 64->128 1-128
Morganella morganii (23)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
1 05 05 003 128 16 64 64
2 1 1 012 >128 64 >128 64
012-4 O06-1 025-2 003-025 2->128 O12-M28 O5->128 05-128
Shlgella sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
05 025 025 006 2 4 4 4
05 025 05 006 64 16 >128 32
025-O5 012-025 025-05 003-006 1-64 1-16 2->128 2-32
4 2 2 1 >128 16 >128 32
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Bacterium (no. tested)
653
In-vitro activity ofrafloxadnand MF 961 Table L—continued Bacterium (no. tested)
Antimicrobial
MIC»
Providencia sp. (30)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoiidav
2 2 4 0-12 16 1 128 64
Salmonella sp. (10)
rufloxatin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav
1 0-5 05 O06 0-5 4 1 0-5
1 05 1 012 1 8 1 1
05-1 025-05 05-2 006-012 05-16 2-8 0 5 - > 128 05-16
Proteus mdgaris (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav
1 0-5 0-5 0O6 32 16 64 2
1 05 05 006 >128 >128 >128 8
1-2 025-05 025-O5 003-006 8->128 2->128 16->128 1-64
Providencia rettgeri (5)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav
2 0-5 0-5 006 0-5 025 05 05
2 1 1 006 2 4 1 1
Proteus mirabilis (39)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoddav
2 05 05 006 1 1 05 05
Serratia sp. (31)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 2 025 >128 >128 >128 64
MIC* 8 4 64 1 >128 16 >128 128
32 8 16 2 >128 >128 >128 128
1-32 05-32 05-128 006-8 O5->128 012-64 1->128 1->128
1-2 05-1 05-1 006 05-2 006-4 025-1 025-1
.
05-64 025-8 025-8 006-05 05-64 025-16 O5->128 025-16 1->128 05-64 1->128 006-64 >128 32->128 64->128 16->128
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
4 1 4 05 2 2 >128 8
Range
654
ILWbertfli Tabk I.—continued
Antimicrobial
MIC a
MIC,,
Range
Acinetobacter spp. (28)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav erythromycin rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav penicillin erythromycin rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 4 4 32 32 16 4 4 4 4 05 >128 >128 >128 128 O5 012 05 006 8 1 8 1 4 012 003 012 0015 025 003 025 025 006 025
4 2 64 8 128 64 >128 16 8 8 8 05 >128 >128 >128 >128 05 012 05 006 16 2 >128 2 8 012 006 025 003 05 012 16 5 4 5 012 006 025 003 025 006 012 012
O25-128 025-> 128 OO6->128 025-> 128 O25->128 003-> 128 O5->128 4-16 2-8 2-8 025-1 >128 128->128 128->128 64->128 025-1 003-025 025-1 003-006 05-128 012-32 025-> 128 025-8 1-8
P. aerugtnosa (30)
H. inftuenzae (44) (including 12 0-lactamase producers)
N. gonorrhoeae (30) (inrlnrling 1 /?-lfirtfirTM"f^
producers)
JV. meningitidis (9)
S. pneumoniae (28)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin
012 006 025 003 025 003 012 006 16 4 32 8 012 0015 0015
32 8 32 16 2 1 025
006-025 O015-O12 006-025 0015-003 006-1 0008-1 006-64 0015-1 0015-64 0015-1 012 006 012-025 003 012-025 0015-006 006-012 006-012 8-32 2-8 8-32 2-16 025-2 0008-4 0015-2
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Bacterium (no. tested)
655
In-vttro activity of rnfloxadn and MF 961 Table L—continued
Bacterium (no. tested)
MIC,,
Antimicrobial
MIC*
Range
0015 0O15 025
025 05 32
0015-2 0008-1 006-32
Enterococcus spp. (19)
rufloxacin MF961 MF922 norfloxacin cefador cefuroximc amoxycillin co-amoxiclav penicillin erythromycin
Lancefield group A streptococcci (16)
rufloxacin MF961 MF922 norfloxacin cefaclor ccfuroxime amoxycillin co-amoxiclav penicillin erythromycin
32 4 16 8 1 012 012 012 006 006 8 2 4 2 012 0008 0015 O015 0015 012
32 8 32 8 64 4 4 4 2 2 16 2 16 2 012 0015 0015 0015 0015 8
16-32 4-8 4-32 4-8 1-64 O008-4 O015-4 0015-4 008-2 O06-4 2-16 2 4-16 1-4 006-012 0008-0015 O015 0015 0015 006-8
Lancefield group B streptococci (19)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin cc-amoxidav penicillin erthromycin
2 8 4 05 O03 006 006 003 006
32 4 8 8 05 003 006 006 003 012
8-32 2-8 8 4-8 05 O03 006 006 003 006-012
M. catanhalis (49)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav penicillin erythromycin
1 05 2 025 05 1 05 006 2 012
1 05 2 025 1 2 2 025 8 025
012-1 O12-O5 1-2 012-025 025-1 025-2 O015-4 0015-1 0015-16 003-2
S. saprophyticus (30)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime
4 2 4 4 2 1
4 2 8 4 2 2
•
8
05-4 05-4 2-8 025-4 006-4 O06-2
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
co-amoxiclav penicillin erythromycin
656
R. Wbertoi Table L—continued
Bacterium (no. tested)
S. aureus (30)
B.fragilis (27)
C. perfringens (7)
C. difficile (5)
Clostridhon spp. (5)
MIC,,
MIC*
Range
amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxiclav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav
0-25 0-25 2 05 8 2 1 1 025 012 1 05 4 1 05 012 012 012 16 8 4 8 >128 16 16 1 4 1 4 8 8 2 025 012 32 16 4 32 64 64 025 025 8 05 2 8 8 1 012 012
05 05 2 1 16 4 2 2 025 025 2 1 4 1 2 1 05 025 32 8 8 32 >128 >128 >128 4
O03-O5 O03-O5 05-2 05-2 2-32 025-4 025-4 006-1 003-1 O03-O5 05-2 05-1 1-4 025-1 006-4 006-1 003-1 O03-O5 16-32 4-8 4-8 8-32 >128 8->128 2->128 025-32 4 1 4 8 8 2-4 012-1 012-O25 32 4-32 4-8 8-32 8-128 1-128 012-05 012-025 4-32 05-32 2-8 8-64 8-128 1-16 012-025 012-025
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
S. epidermidis (19)
Antimicrobial
In-rhro actirfty of rnfloxadn and MF 961
657
Discussion
A limited number of fluoroquinolones having a 1, 8 bridge structure have been described (Wentland, 1990). Ofloxacin is of this general structure as are S 25932 and
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
susceptible as the non-producers. Amoxycillin and (to a lesser extent) cefaclor showed a bimodal distribution of susceptibilities depending on the /Mactamase producing status of the strains. An amoxycillin resistant non-/?-lactamase producing strain (MIC 128mg/L) was less susceptible to all /Mactams but susceptible to the quinolones. Similarly strains of M. catarrhalis were highly susceptible to all four quinolones, but their susceptibility to amoxycillin and, to a lesser extent, the cephalosporins was determined by their ability to produce /J-lactamase (as determined by the nitrocefin test, data not shown). Neisseria gonorrhoeae and Neisseria meningitidis are highly susceptible to all four quinolones. The /Mactamase producing strains being as susceptible as the non-producers. Streptococcus pneumoniae was relatively insusceptible to rufloxacin and its metabolite. MF 961 was apparently two-fold more active than norfloxacin against these strains. Other streptococci (including enterococci) were relatively insusceptible to all the quinolones tested, although MF 961 was four to eight-fold more active than rufloxacin. Strains of 5. aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus have similar susceptibilities to rufloxacin, the M I Q Q being 2-4 mg/L. MF 961 was consistently two-fold more active than rufloxacin. The three methicillin resistant S. aureus were all susceptible to rufloxacin (MIC < 1 mg/L) and the other quinolones. Norfloxacin was as active or marginally less active than the rufloxacin against the staphylococci tested. All the quinolones tested had little useful activity against Bacteroides fragUis, although the MIC*, of MF 961 was four-fold lower than rufloxacin or norfloxacin. It was of interest to note that the MIC*, of the metabolite MF 922 was similarily four-fold lower than the parent compound, rufloxacin. Against Clostridia spp. there was variable activity with Clostridium difficile generally being resistant to all agents (except amoxycillin and co-amoxiclav), but Clostridium perfringens was more susceptible to quinolones. Of note was the high activity of MF 961 against C. perfringens (MIC* 1 mg/L). In Tables II and III the MBCs of rufloxacin and MF 922 are shown for eight selected bacterial strains, together with the effect of human serum and urine on the antimicrobial activity of these comounds. In general the MBC was within one dilutional step of the MIC, the exception being at pH 6 where the bactericidal activity of both compounds tended to be significantly less than that required to inhibit growth. Increasing amounts of human serum tended to decrease the activity of both compounds by about four-fold and an acid pH decreased the activity considerably. The protein binding of rufloxacin at 1 and 10 mg/L were 55-0% and 63-8%respectively.The protein binding of MF 922 was 30-3% and 32-6% respectively. The MIC of rufloxacin for the four strains of C. trachomatis was 4-8 mg/L (for ciprofloxacin the MICs were 1-2 mg/L). The minimum lethal concentrations, MLC, were 8-16 and 2-4 mg/L respectively for the two agents. The MIC of rufloxacin for the C. pneumoniae was 4 mg/L (ciprofloxacin 8 mg/L) and the MLC 8 mg/L (ciprofloxacin 16 mg/L).
1 8
F361 F283
S. aureus
2 4
4 16
4 8 16 32
8 4
>32 4
16 8
>32 32
32 32
32 32
>32 32
32 32
2 4
>32 2
1 2 >32 >32
32 8
>32 4
8 16
>32 2
>32 >32 4 012
4 8
1 1
8 8
4 2
4 2
2 1
pH8 MIC MBC
05 012 16 025
1265 1322 H284 H200 R50 R44 F361 F283
E.coli
Klebsiella spp.
M. catarrhalis
S. aureus O5 16
05 025
MIC
No.
Bacterium
32 05 05 05 1 2
1 1 1 16
1 05
8 8
2 2
>32 1
1 2
MIC MBC
16 025
05 025
MBC
1 4
1 1
>32 1
4 1
MIC
4 8
2 1
>32 1
4 2
MBC
4 4
>32 16
16 8
4 8
>32 16
16 8
05 2
>32 1
1 05
2 4
>32 2
2 1
Urine pH pH6 MIC MBC
05 2
>32 05
4 4
>32 4
4 2
pH7 MIC MBC to to
pH5 MIC MBC
1 1
>32 1
2 1
>32 4
8 1
pH8 MIC MBC
00 OO
Human serum concentration
Table III. The effect of human serum and urine (of different pH) on the inhibitory and bactericidal activity of MF 922. MIC and MBC values are given in mg/L.
4 32
4 4
4 2
1 05
R50 R44
M. catarrhalis
>32 4
>32 2
>32 2
>32 >32 05 05
H284 H200
Klebsiella spp.
8 2
4 4
05 05
2 1
1265 1322
E. coli
1 05
No.
Bacterium
to to
to to
OO OO
Urine pH pH6 pH7 MIC MBC MIC MBC
OO OO
pH5 MIC MBC
to to
Human serum concentration 0% 20% 70% MIC MBC MIC MBC MIC MBC
Table II. The effect of human serum and urine (of different pH) on the inhibitory and bactericidal activity of rufloxacin. MIC and MBC values are given in mg/L
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
In-Tttro activity of mfloxidn and MF 961
659
Acknowledgement We thank B. Imbimbo of Mcdiolanum Farmaccutici for his financial support and advice. References Cecchetti, V., Fravolini, A., FringueJli, R., Mascellani, G., Pagella, P., Palmioli, M. et al. (1987). Quinolonecarboxylic acids. 2. Synthesis and antibacterial evaluation of 7-oxo-2, 3-dihydro7-H-pyrido{l ,2,3-de] [1,4] bcnzothiazine-6-carboxylic acids. Journal of Medicinal Chemistry 30, 465-73. Hooper, D. C , Wolfson, J. S., Souza, K. S., Tung, C , McHugh, G. L. & Swartz, M. N. (1986). Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 29, 639-44.
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
S 25930 (Piddock et al., 1986). The in-vitro activities of rufloxacin and MF 961 are1 closer to those of S 25932/S 25930 than that of ofloxacin. In this study we confirm the preliminary data of Cecchetti et al., (1987) with the exception that we find rufloxacin less active against Klebsiella spp. Similarly we confirm that MF 961 is twice as active as rufloxacin (Pagella et al., 1989). Both rufloxacin and its desmethylated metabolite MF 922 and the more recently synthesized compound MF 961 have a remarkably similar degree of in-vitro activity, MF 961 being usually slightly more active. Norfloxacin was the most active quinolone investigated in this study, with the interesting exception of Acinetobacter spp. where MF 961 and rufloxacin were four- and two-fold more active respectively. Similarly against staphylococci, MF 961 and rufloxacin were as active or slightly more active than norfloxacin. As would be expected, strains known to be resistant to other quinolones whether by a gyr A mutation or by an alteration in the OMP F porin (Hooper et al., 1986), show cross resistance to the three agents we have studied. It was of interest to note that whereas rufloxacin was less active than ciprofloxacin against C. trachomatis it was twice as active against the single strain of C. pneumoniae. This should be verified with the study of further strains of these organisms. The considerable in-vitro activity of the metabolite of rufloxacin probably has little significance other than possibly interfering with the microbiological assay of the parent compound. Data from this laboratory (not shown) suggest that this will not be a significant problem with the assay of serum, but may be if urine samples are being studied. Preliminary information on the pharmacokinetics of rufloxacin indicate that the maximum serum level following a 400 mg oral dose is 3-6 mg/L and the elimination half-life is greater than 28 h (Wise, unpublished data; Imbimbo et al., 1991). This suggests that rufloxacin may well find clinical use in the treatment of urinary tract infections, bacterial gastroenteritis and gonococcal disease. Although the activity against H. influenzae and M. catarrhalis is high, the poor activity against streptococci including S. pneumoniae (MIC,,, 32 mg/L) make it unlikely to be useful in chest infections. However, many quinolones are concentrated within many components of the respiratory tract (Wise et at., 1990). The greater activity of MF 961 (especially against S. pneumoniae), if it were combined with good pharmacokinetic characteristics (which are as yet unknown) might suggest a greater clinical role for this agent
660
R.Wbe rtaZ.
(Received 24 July 1991; revised version accepted 25 January 1992)
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Imbimbo, B. P., Broccati, G., Cesana, M., Crema, F. & Attardo-Parrinello, G. (1991). Inter- and Intrasubject variabilities in the pharmacokinetics of rufloxacin after single oral administration to healthy volunteers. Antimicrobial Agents and Chemotherapy 35, 390-3. PageQa, P. G., Terai, P., Rugarli, P. L., Papagni, A., Maiorana, S. & Martina, R. (1989). MF-961, a quinolone of the new difluorinated benzothiazine series, endowed with high antibacterial activity. In Program and Abstracts of the Twenty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, 1989. Abstract 1251, p. 313. American Society for Microbiology, Washington, DC. 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. Piddock, L. J. V., Andrews, J. M., Diver, J. M. & Wise, R. (1986). In vitro studies of S-25930 and S-25932, 2 new 4-quinolones. European Journal of Clinical Microbiology 5, 303-10. Webberley, J. M., Matthews, R. S., Andrews, J. M. & Wise, R. (1987). Commercially available fluorescein-conjugated monoclonal antibody for determining the in vitro activity of antimicrobial agents against Chlamydia trachomatis. European Journal of Clinical Microbiology 6, 587-9. Wentland, M. P. (1990). Structure-activity relationships of fluroquinolones. In The New Generation of Quinolones (Siporin, C , Hcifetz, C. L. & Domagala, J. M., Eds), pp. 1-43. Marcel Dekker, New York. Wise, R., Ashby, J. P. & Andrews, J. M. (1988). In vitro activity of PD 127,391 on enhanced spectrum quinolones. Antimicrobial Agents and Chemotherapy 32, 1251-6. Wise, R., Baldwin, D. R., Honeybourne, D. & Andrews, J. M. (1990). Penetration of antibiotics into the bronchial mucosa—a review. Research Clinical Forums 12 (4), 95-102.
The in-vitro activity of two new qmnolones: rufloxacin and MF 961 R. Wise, J. M. Andrews, R. Matthews and M. Wobtenbohne
The in-vitro activity of two new quinolone antimicrobials, rufloxacin and MF 961, together with the dcsmethylated metabolite of rufloxacin (MF 922) were compared with other orally administered agents against 622 bacterial strains. Against Enterobacteriaceae and Pseudomonas aentginosa rufloxacin was generally active (MIQo 1-8 mg/L) with the exception of Klebsiella and Serratia spp. (MIC*, 32 mg/L and Enterobacter spp. (MlQo) 64 mg/L. The respiratory pathogens Haemophilus influenzae and Moraxella catarrhalis were susceptible to rufloxacin (MIC^ 05 and 1 mg/L respectively) but Streptococcus pneumoniae was less susceptible (MIC*, 32 mg/L). Staphylococcus attreus were susceptible to rufloxacin (MIC,, 2 mg/L). The rufloxacin metabolite MF 922 was generally as active as its parent. MF 961 was usually two-fold more active than rufloxacin. AU three compounds were four to 16 times less active than norfloxacin, but rufloxacin was as active or somewhat more active than norfloxacin against Staphylococcus spp. Any strains showing decreased susceptibility to other quinolones exhibited cross resistance to these new agents. The MBC of rufloxacin and MF 922 was within one dilution of the MIC and human serum had little effect upon the activity of both agents. The protein binding of rufloxacin and MF 922 at 1 and 10 mg/L were 55% and 63-8% and 30-3% and 32-6% respectively. The activity of rufloxacin against four strains of Chlamydia trachomatis and one strain of Chlamydia pneumoniae was determined. The MICs for C. trachomatis were 4-8 mg/L and 4 mg/L for C. pneumoniae. Introduction Rufloxacin (MF 934) is a new broad spectrum quinolone (Cecchetti et al., 1987) and has the formula 9-fluoro-10-(-methyl) piperazinyl-7-oxo-2, 3-dihydro-7H-pyrido-(l,2,3, de) (1,4) benzothiazine-6-carboxylic acid, the structure is shown in Figure 1. Rufloxacin undergoes a modest amount of metabolism in man and an ^-desmethylated derivative is found in urine (J. Kisicki, personal communication), which has been designated MF 922. In addition a molecular modification of rufloxacin has been produced (MF 961) which differs in that there is a fluoromethyl substituent in the benzothiazine ring (Figure 2).
COOH
Figure 1. The structure of rufloxacin (MF 934). 649 0305-7453/92/060649+12 $02.00/0 © 1992 The British Society for Antnnkrobud Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Department of Medical Microbiology, Dudley Road Hospital, Birmingham B18 7QH, UK
650
R.Wbetf aL
COOH
sv Figure 2. The itructure of MF 961.
Materials and methods
A total of 622 strains were studied, of which 589 were recent clinical isolates, eight were well characterized /Mactamase producers, 15 aminoglycoside resistant, and ten had been previously shown to have reduced sensitivity to the quinolones. The following antimicrobials of known potency were studied: rufloxacin, MF 961 and MF 922, obtained from Mediolanum Farmaceutici, Milan, Italy; cefuroxime, Glaxo Laboratories, Greenford, Middlesex, UK; norfloxacin, Merck Sharpe and Dohme, Hoddesdon, Herts., UK; penicillin, amoxycillin, davulanic acid and methicillin, Beechams, Brentford, UK; cefaclor and erythromycin, Eh" Lilly, Basingstoke, Hampshire, UK; ciprofloxacin, Bayer Pharmaceuticals, Newbury, UK. Minimum inhibitory concentrations (MICs) were determined by an agar plate dilution method (Wise, Ashby & Andrews, 1988). The final inoculum used was 104 cfu for all strains. For selected strains a comparison was made between results obtained with a final inocula of 104 and 10* cfu. IsoSensitest agar (pH 7-2, Oxoid, Basingstoke, UK) was used, supplemented with a 5% horse blood and 20 mg/L NAD (B.D.H. Ltd, Poole, Dorset) for fastidious organisms. For anaerobes Wilkins and Chalgren agar (Oxoid) with 5% horse blood was used. All plates were incubated in air for 18 h except for the following: Haemophilia inftuenzae and Neisseria spp. were incubated in air enriched with 6% CO2 for 18 h; anaerobes were incubated in an atmosphere containing 10% COj, 80% Nj, 10% H2 in an anaerobic cabinet (Don Whitley Ltd, Shipley, West Yorkshire, UK) for 48 h. The amoxycillin/clavulanate was tested in a ratio of 2: 1 and the susceptibilities were expressed in terms of the MIC to amoxycillin. The effect of human serum and urine on the MIC and MBC values of rufloxacin and MF 922 were studied with 8 strains (2 each of Staphylococcus aureus, Escherichia coli, Klebsiella sp., and Moraxella catarrhalis) by a method based on that of Pearson et al. (1980). In a microdilution system, Iso-Sensitest broth alone or supplemented with 20% or 70% human serum and pooled antibiotic free human urine at pH 5, 6, 7 and 8 were used as media, and broth dilution MICs were performed with an inoculum of 10*cfu/mL. All dilutions showing no visible growth after overnight incubation were subcultured on to antibiotic free agar, and the MBC was defined as a 99-9% kill as the endpoint
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
In this study we have compared the in-vitro activity of these two new agents and the desmethylated derivative of rufloxacin with those of norfloxacin, cefaclor, cefuroxime, amoxycillin and a 2: 1 mixture of amoxycillin/clavulanic acid. The antibacterial activity of rufloxacin against Chlamydia trachomatis and Chlamydia pneumoniae was also determined and compared to that of ciprofloxacin.
In-rttro activity of rafloudn and MF 961
651
Results Table I shows the activity of the different agents against the recent clinical isolates and the known /Mactamase producers. The activity of rufloxacin and its metabolite (MF 922) against the frequently encountered species of Enterobacteriaceae were remarkably similar and the activity of MF 961 was generally two-fold greater than that of rufloxacin. All three compounds were 4-16-times less active than norfloxacin, but were generally more active than the /Mactam antibiotics studied. If an individual strain was less susceptible to one of the quinolones it tended to be less susceptible to the others: an exception to this was an isolate of Providencia stuartii which was susceptible to 4 mg/L of rufloxacin, norfloxacin and MF 961 yet susceptible to 128 mg/L of MF 922. A less pronounced difference between the poor activity of MF 922 and the other quinolones was seen for other strains of P. stuartii. There was considerable variation amongst susceptibilities of the Enterobacteriaceae to the quinolones, for example Citrobacter. Salmonella and Shigella spp. were highly susceptible to rufloxacin (MIC*, 4 mg/L). Acinetobacter spp. were markedly less susceptible to the metabolite MF 922 than to the parent compound, rufloxacin. All four quinolones displayed decreased activity against strains with known or unknown mechanisms of resistance to other quinolones. For example a known gyr A resistant mutant E. coli isolated from a clinical trial was susceptible to 8 mg/L rufloxacin and 2 mg/L MF 961 and 0-25 mg/L norfloxacin whereas the pre-treatment isolate (wild-type) was susceptible to 0-25, 0-25 and 006 mg/L respectively. An outer membrane protein (OMP) F deficient variant of E. coli was susceptible to 16 mg/L rufloxacin. Strains of H. wfluauae were highly susceptible to the three new compounds, the rufloxacin MIC*, being 0-5 mg/L. The 12 /J-lactamase producing strains were as
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
The protein binding of rufloxacin and MF 922 was assessed by comparing samples at a concentration of 1 and 10 mg/L prepared in either pooled human serum (Flow Labs, McLean, VA, USA) or distilled water, after separation of bound from free drug by centrifugation at 2000 rpm for 10 min using 'Centriflo' units (Amicon Corp., Danvers, MA, USA). A microbiological assay method was used to measure drug concentrations. Standards were prepared in phosphate buffer at pH 7, and the indicator organism was E. coli 4004 (obtained from Bayer AG, Germany, being highly sensitive to quinolones). The activities of rufloxacin and ciprofloxacin against 4 isolates of C. trachomatis and one of C. pnewnoniae were determined by the method of Webberley et al. (1987). Briefly, McCoy cell cover slip cultures were infected with approximately 1000 inclusionforming units of chlamydia, exposed to the various dilutions of the antimicrobials for 48 h and then stained with 'Imagen Chlamydia test' (NovoBiolabs, Cambridge, England). The MIC was the lowest concentration that inhibited all inclusion development The minimum lethal concentration (MLC) was defined as the lowest concentration that inhibited inclusion development in cell sheets exposed to antimicrobial for 48 h, then reincubated in drug-free medium for an additional 48 h. Again a 99-9% loll was used as the endpoint.
652
R. W b e r t o l
T«bk I. The in-vitro activity of rufloxacin, MF 961 and MF 922 compared with other antimicrobial agents (mg/L)
Antimicrobial
MIC*
MIC*
Range
E. coli (59)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxklav
05 025 05 012 1 2 >128 4
2 1 2 025 4 8 >128 8
025-> 128 O12->128 O12->128 003-> 128 O5->128 05-32 O5->128 05-64
Klebsiella sp. (39)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 4 05 1 2 128 2
32 8 32 8 4 16 >128 16
O5->128 O25->128 O5-128 006-64 0 5 - > 128 0 5 - > 128 2->128 1-32
Enterobacter sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
64 16 64 8 >128 >128 >128 64
1-128 05-32 1-64 012-16 2->128 2->128 16->128 4-64
Citrobacter sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
05 025 025 006 4 4 128 8
1 1 1 012 >128 32 >128 64
025-1 012-1 025-2 003-012 O5->128 O5->128 64->128 1-128
Morganella morganii (23)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
1 05 05 003 128 16 64 64
2 1 1 012 >128 64 >128 64
012-4 O06-1 025-2 003-025 2->128 O12-M28 O5->128 05-128
Shlgella sp. (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
05 025 025 006 2 4 4 4
05 025 05 006 64 16 >128 32
025-O5 012-025 025-05 003-006 1-64 1-16 2->128 2-32
4 2 2 1 >128 16 >128 32
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Bacterium (no. tested)
653
In-vitro activity ofrafloxadnand MF 961 Table L—continued Bacterium (no. tested)
Antimicrobial
MIC»
Providencia sp. (30)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoiidav
2 2 4 0-12 16 1 128 64
Salmonella sp. (10)
rufloxatin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav
1 0-5 05 O06 0-5 4 1 0-5
1 05 1 012 1 8 1 1
05-1 025-05 05-2 006-012 05-16 2-8 0 5 - > 128 05-16
Proteus mdgaris (10)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav
1 0-5 0-5 0O6 32 16 64 2
1 05 05 006 >128 >128 >128 8
1-2 025-05 025-O5 003-006 8->128 2->128 16->128 1-64
Providencia rettgeri (5)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav
2 0-5 0-5 006 0-5 025 05 05
2 1 1 006 2 4 1 1
Proteus mirabilis (39)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoddav
2 05 05 006 1 1 05 05
Serratia sp. (31)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 2 025 >128 >128 >128 64
MIC* 8 4 64 1 >128 16 >128 128
32 8 16 2 >128 >128 >128 128
1-32 05-32 05-128 006-8 O5->128 012-64 1->128 1->128
1-2 05-1 05-1 006 05-2 006-4 025-1 025-1
.
05-64 025-8 025-8 006-05 05-64 025-16 O5->128 025-16 1->128 05-64 1->128 006-64 >128 32->128 64->128 16->128
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
4 1 4 05 2 2 >128 8
Range
654
ILWbertfli Tabk I.—continued
Antimicrobial
MIC a
MIC,,
Range
Acinetobacter spp. (28)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav erythromycin rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav penicillin erythromycin rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxidav
2 1 4 4 32 32 16 4 4 4 4 05 >128 >128 >128 128 O5 012 05 006 8 1 8 1 4 012 003 012 0015 025 003 025 025 006 025
4 2 64 8 128 64 >128 16 8 8 8 05 >128 >128 >128 >128 05 012 05 006 16 2 >128 2 8 012 006 025 003 05 012 16 5 4 5 012 006 025 003 025 006 012 012
O25-128 025-> 128 OO6->128 025-> 128 O25->128 003-> 128 O5->128 4-16 2-8 2-8 025-1 >128 128->128 128->128 64->128 025-1 003-025 025-1 003-006 05-128 012-32 025-> 128 025-8 1-8
P. aerugtnosa (30)
H. inftuenzae (44) (including 12 0-lactamase producers)
N. gonorrhoeae (30) (inrlnrling 1 /?-lfirtfirTM"f^
producers)
JV. meningitidis (9)
S. pneumoniae (28)
rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin
012 006 025 003 025 003 012 006 16 4 32 8 012 0015 0015
32 8 32 16 2 1 025
006-025 O015-O12 006-025 0015-003 006-1 0008-1 006-64 0015-1 0015-64 0015-1 012 006 012-025 003 012-025 0015-006 006-012 006-012 8-32 2-8 8-32 2-16 025-2 0008-4 0015-2
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Bacterium (no. tested)
655
In-vttro activity of rnfloxadn and MF 961 Table L—continued
Bacterium (no. tested)
MIC,,
Antimicrobial
MIC*
Range
0015 0O15 025
025 05 32
0015-2 0008-1 006-32
Enterococcus spp. (19)
rufloxacin MF961 MF922 norfloxacin cefador cefuroximc amoxycillin co-amoxiclav penicillin erythromycin
Lancefield group A streptococcci (16)
rufloxacin MF961 MF922 norfloxacin cefaclor ccfuroxime amoxycillin co-amoxiclav penicillin erythromycin
32 4 16 8 1 012 012 012 006 006 8 2 4 2 012 0008 0015 O015 0015 012
32 8 32 8 64 4 4 4 2 2 16 2 16 2 012 0015 0015 0015 0015 8
16-32 4-8 4-32 4-8 1-64 O008-4 O015-4 0015-4 008-2 O06-4 2-16 2 4-16 1-4 006-012 0008-0015 O015 0015 0015 006-8
Lancefield group B streptococci (19)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin cc-amoxidav penicillin erthromycin
2 8 4 05 O03 006 006 003 006
32 4 8 8 05 003 006 006 003 012
8-32 2-8 8 4-8 05 O03 006 006 003 006-012
M. catanhalis (49)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav penicillin erythromycin
1 05 2 025 05 1 05 006 2 012
1 05 2 025 1 2 2 025 8 025
012-1 O12-O5 1-2 012-025 025-1 025-2 O015-4 0015-1 0015-16 003-2
S. saprophyticus (30)
rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime
4 2 4 4 2 1
4 2 8 4 2 2
•
8
05-4 05-4 2-8 025-4 006-4 O06-2
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
co-amoxiclav penicillin erythromycin
656
R. Wbertoi Table L—continued
Bacterium (no. tested)
S. aureus (30)
B.fragilis (27)
C. perfringens (7)
C. difficile (5)
Clostridhon spp. (5)
MIC,,
MIC*
Range
amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefador cefuroxime amoxycillin co-amoxiclav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxiclav rufloxacin MF961 MF922 norfloxacin cefaclor cefuroxime amoxycillin co-amoxidav
0-25 0-25 2 05 8 2 1 1 025 012 1 05 4 1 05 012 012 012 16 8 4 8 >128 16 16 1 4 1 4 8 8 2 025 012 32 16 4 32 64 64 025 025 8 05 2 8 8 1 012 012
05 05 2 1 16 4 2 2 025 025 2 1 4 1 2 1 05 025 32 8 8 32 >128 >128 >128 4
O03-O5 O03-O5 05-2 05-2 2-32 025-4 025-4 006-1 003-1 O03-O5 05-2 05-1 1-4 025-1 006-4 006-1 003-1 O03-O5 16-32 4-8 4-8 8-32 >128 8->128 2->128 025-32 4 1 4 8 8 2-4 012-1 012-O25 32 4-32 4-8 8-32 8-128 1-128 012-05 012-025 4-32 05-32 2-8 8-64 8-128 1-16 012-025 012-025
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
S. epidermidis (19)
Antimicrobial
In-rhro actirfty of rnfloxadn and MF 961
657
Discussion
A limited number of fluoroquinolones having a 1, 8 bridge structure have been described (Wentland, 1990). Ofloxacin is of this general structure as are S 25932 and
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
susceptible as the non-producers. Amoxycillin and (to a lesser extent) cefaclor showed a bimodal distribution of susceptibilities depending on the /Mactamase producing status of the strains. An amoxycillin resistant non-/?-lactamase producing strain (MIC 128mg/L) was less susceptible to all /Mactams but susceptible to the quinolones. Similarly strains of M. catarrhalis were highly susceptible to all four quinolones, but their susceptibility to amoxycillin and, to a lesser extent, the cephalosporins was determined by their ability to produce /J-lactamase (as determined by the nitrocefin test, data not shown). Neisseria gonorrhoeae and Neisseria meningitidis are highly susceptible to all four quinolones. The /Mactamase producing strains being as susceptible as the non-producers. Streptococcus pneumoniae was relatively insusceptible to rufloxacin and its metabolite. MF 961 was apparently two-fold more active than norfloxacin against these strains. Other streptococci (including enterococci) were relatively insusceptible to all the quinolones tested, although MF 961 was four to eight-fold more active than rufloxacin. Strains of 5. aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus have similar susceptibilities to rufloxacin, the M I Q Q being 2-4 mg/L. MF 961 was consistently two-fold more active than rufloxacin. The three methicillin resistant S. aureus were all susceptible to rufloxacin (MIC < 1 mg/L) and the other quinolones. Norfloxacin was as active or marginally less active than the rufloxacin against the staphylococci tested. All the quinolones tested had little useful activity against Bacteroides fragUis, although the MIC*, of MF 961 was four-fold lower than rufloxacin or norfloxacin. It was of interest to note that the MIC*, of the metabolite MF 922 was similarily four-fold lower than the parent compound, rufloxacin. Against Clostridia spp. there was variable activity with Clostridium difficile generally being resistant to all agents (except amoxycillin and co-amoxiclav), but Clostridium perfringens was more susceptible to quinolones. Of note was the high activity of MF 961 against C. perfringens (MIC* 1 mg/L). In Tables II and III the MBCs of rufloxacin and MF 922 are shown for eight selected bacterial strains, together with the effect of human serum and urine on the antimicrobial activity of these comounds. In general the MBC was within one dilutional step of the MIC, the exception being at pH 6 where the bactericidal activity of both compounds tended to be significantly less than that required to inhibit growth. Increasing amounts of human serum tended to decrease the activity of both compounds by about four-fold and an acid pH decreased the activity considerably. The protein binding of rufloxacin at 1 and 10 mg/L were 55-0% and 63-8%respectively.The protein binding of MF 922 was 30-3% and 32-6% respectively. The MIC of rufloxacin for the four strains of C. trachomatis was 4-8 mg/L (for ciprofloxacin the MICs were 1-2 mg/L). The minimum lethal concentrations, MLC, were 8-16 and 2-4 mg/L respectively for the two agents. The MIC of rufloxacin for the C. pneumoniae was 4 mg/L (ciprofloxacin 8 mg/L) and the MLC 8 mg/L (ciprofloxacin 16 mg/L).
1 8
F361 F283
S. aureus
2 4
4 16
4 8 16 32
8 4
>32 4
16 8
>32 32
32 32
32 32
>32 32
32 32
2 4
>32 2
1 2 >32 >32
32 8
>32 4
8 16
>32 2
>32 >32 4 012
4 8
1 1
8 8
4 2
4 2
2 1
pH8 MIC MBC
05 012 16 025
1265 1322 H284 H200 R50 R44 F361 F283
E.coli
Klebsiella spp.
M. catarrhalis
S. aureus O5 16
05 025
MIC
No.
Bacterium
32 05 05 05 1 2
1 1 1 16
1 05
8 8
2 2
>32 1
1 2
MIC MBC
16 025
05 025
MBC
1 4
1 1
>32 1
4 1
MIC
4 8
2 1
>32 1
4 2
MBC
4 4
>32 16
16 8
4 8
>32 16
16 8
05 2
>32 1
1 05
2 4
>32 2
2 1
Urine pH pH6 MIC MBC
05 2
>32 05
4 4
>32 4
4 2
pH7 MIC MBC to to
pH5 MIC MBC
1 1
>32 1
2 1
>32 4
8 1
pH8 MIC MBC
00 OO
Human serum concentration
Table III. The effect of human serum and urine (of different pH) on the inhibitory and bactericidal activity of MF 922. MIC and MBC values are given in mg/L.
4 32
4 4
4 2
1 05
R50 R44
M. catarrhalis
>32 4
>32 2
>32 2
>32 >32 05 05
H284 H200
Klebsiella spp.
8 2
4 4
05 05
2 1
1265 1322
E. coli
1 05
No.
Bacterium
to to
to to
OO OO
Urine pH pH6 pH7 MIC MBC MIC MBC
OO OO
pH5 MIC MBC
to to
Human serum concentration 0% 20% 70% MIC MBC MIC MBC MIC MBC
Table II. The effect of human serum and urine (of different pH) on the inhibitory and bactericidal activity of rufloxacin. MIC and MBC values are given in mg/L
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
In-Tttro activity of mfloxidn and MF 961
659
Acknowledgement We thank B. Imbimbo of Mcdiolanum Farmaccutici for his financial support and advice. References Cecchetti, V., Fravolini, A., FringueJli, R., Mascellani, G., Pagella, P., Palmioli, M. et al. (1987). Quinolonecarboxylic acids. 2. Synthesis and antibacterial evaluation of 7-oxo-2, 3-dihydro7-H-pyrido{l ,2,3-de] [1,4] bcnzothiazine-6-carboxylic acids. Journal of Medicinal Chemistry 30, 465-73. Hooper, D. C , Wolfson, J. S., Souza, K. S., Tung, C , McHugh, G. L. & Swartz, M. N. (1986). Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy 29, 639-44.
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
S 25930 (Piddock et al., 1986). The in-vitro activities of rufloxacin and MF 961 are1 closer to those of S 25932/S 25930 than that of ofloxacin. In this study we confirm the preliminary data of Cecchetti et al., (1987) with the exception that we find rufloxacin less active against Klebsiella spp. Similarly we confirm that MF 961 is twice as active as rufloxacin (Pagella et al., 1989). Both rufloxacin and its desmethylated metabolite MF 922 and the more recently synthesized compound MF 961 have a remarkably similar degree of in-vitro activity, MF 961 being usually slightly more active. Norfloxacin was the most active quinolone investigated in this study, with the interesting exception of Acinetobacter spp. where MF 961 and rufloxacin were four- and two-fold more active respectively. Similarly against staphylococci, MF 961 and rufloxacin were as active or slightly more active than norfloxacin. As would be expected, strains known to be resistant to other quinolones whether by a gyr A mutation or by an alteration in the OMP F porin (Hooper et al., 1986), show cross resistance to the three agents we have studied. It was of interest to note that whereas rufloxacin was less active than ciprofloxacin against C. trachomatis it was twice as active against the single strain of C. pneumoniae. This should be verified with the study of further strains of these organisms. The considerable in-vitro activity of the metabolite of rufloxacin probably has little significance other than possibly interfering with the microbiological assay of the parent compound. Data from this laboratory (not shown) suggest that this will not be a significant problem with the assay of serum, but may be if urine samples are being studied. Preliminary information on the pharmacokinetics of rufloxacin indicate that the maximum serum level following a 400 mg oral dose is 3-6 mg/L and the elimination half-life is greater than 28 h (Wise, unpublished data; Imbimbo et al., 1991). This suggests that rufloxacin may well find clinical use in the treatment of urinary tract infections, bacterial gastroenteritis and gonococcal disease. Although the activity against H. influenzae and M. catarrhalis is high, the poor activity against streptococci including S. pneumoniae (MIC,,, 32 mg/L) make it unlikely to be useful in chest infections. However, many quinolones are concentrated within many components of the respiratory tract (Wise et at., 1990). The greater activity of MF 961 (especially against S. pneumoniae), if it were combined with good pharmacokinetic characteristics (which are as yet unknown) might suggest a greater clinical role for this agent
660
R.Wbe rtaZ.
(Received 24 July 1991; revised version accepted 25 January 1992)
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 11, 2015
Imbimbo, B. P., Broccati, G., Cesana, M., Crema, F. & Attardo-Parrinello, G. (1991). Inter- and Intrasubject variabilities in the pharmacokinetics of rufloxacin after single oral administration to healthy volunteers. Antimicrobial Agents and Chemotherapy 35, 390-3. PageQa, P. G., Terai, P., Rugarli, P. L., Papagni, A., Maiorana, S. & Martina, R. (1989). MF-961, a quinolone of the new difluorinated benzothiazine series, endowed with high antibacterial activity. In Program and Abstracts of the Twenty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, 1989. Abstract 1251, p. 313. American Society for Microbiology, Washington, DC. 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. Piddock, L. J. V., Andrews, J. M., Diver, J. M. & Wise, R. (1986). In vitro studies of S-25930 and S-25932, 2 new 4-quinolones. European Journal of Clinical Microbiology 5, 303-10. Webberley, J. M., Matthews, R. S., Andrews, J. M. & Wise, R. (1987). Commercially available fluorescein-conjugated monoclonal antibody for determining the in vitro activity of antimicrobial agents against Chlamydia trachomatis. European Journal of Clinical Microbiology 6, 587-9. Wentland, M. P. (1990). Structure-activity relationships of fluroquinolones. In The New Generation of Quinolones (Siporin, C , Hcifetz, C. L. & Domagala, J. M., Eds), pp. 1-43. Marcel Dekker, New York. Wise, R., Ashby, J. P. & Andrews, J. M. (1988). In vitro activity of PD 127,391 on enhanced spectrum quinolones. Antimicrobial Agents and Chemotherapy 32, 1251-6. Wise, R., Baldwin, D. R., Honeybourne, D. & Andrews, J. M. (1990). Penetration of antibiotics into the bronchial mucosa—a review. Research Clinical Forums 12 (4), 95-102.
