ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 306-313 0066-4804/90/020306-08$02.00/0 Copyright ©D 1990, American Society for Microbiology
Vol. 34, No. 2
In Vitro Evaluation of WIN 57273, a New Broad-Spectrum Fluoroquinolone RONALD N. JONESt* AND ARTHUR L. BARRY The Clinical Microbiology Institute, Tualatin, Oregon 97062 Received 14 August 1989/Accepted 9 November 1989
WIN 57273 is a new fluoroquinolone that has an expanded spectrum of activity against Staphylococcus spp. (MIC for 90% of isolates [MIC9J, 0.008 ,ug/ml), Enterococcus faecalis (MIC90, 0.06 ,Lg/ml), Bacillus spp. (MIC90, 0.03 ,Ig/ml), Listeria monocytogenes (MICso, 0.06 ,ug/ml), Streptococcus spp. (MIC,0, 0.03 ,ug/ml), and Bacteroidesfragilis group strains (MIC90, 0.5 ,ig/ml). Like other fluoroquinolone compounds, WIN 57273 was active against members of the family Enterobacteriaceae (97% of strains inhibited by 1.4 x 10-9. This new compound possesses a wide potential spectrum of use, and it should be evaluated further by in vitro and in vivo studies.
The halogenated 4-quinolones have generally shown expanded activity against members of the family Enterobacteriaceae and staphylococci compared with that of nalidixic acid, but they have been more variable in their activity against Streptococcus spp., enterococci, and Pseudomonas aeruginosa (7, 17). In addition, few of these compounds have exhibited a usable clinical potency for anaerobic organisms such as the Bacteroides spp. (3, 4, 15, 16). The newer fluoroquinolones that do possess significant activity against gram-positive cocci and/or anaerobic bacteria generally contain a pyrrolidinyl or pyrrolyl group replacing the most commonly used 7-piperazinyl side chain (3, 4, 6, 15, 16). WIN 57273 is an investigational fluoroquinolone with the structural formula 1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolonecarboxylic acid (Fig. 1). This structure resembles those of previously described compounds having greater in vitro utility against anaerobic bacteria and gram-positive species. Also, since WIN 57273 seems to possess antimicrobial activity similar to those of a number of recently studied 4-quinolones (data on file, Sterling-Winthrop Research Institute), we chose to compare this drug in vitro with other fluoroquinolones (1, 2, 5), including the most active clinically available agent, ciprofloxacin (2). The study of WIN 57273 included (i) an MIC comparison with five quinolones and three penicillins against over 700 stock culture strains; (ii) a series of tests to determine WIN 57273 activity against antimicrobial agentresistant bacteria; (iii) a study of WIN 57273 MBCs and effects of inoculum, pH, or medium on MICs; (iv) the quantitation of rates of mutation to WIN 57273 resistance; and (v) preliminary studies of WIN 57273 disk tests with five drug concentrations.
MATERIALS AND METHODS
Antimicrobial agents. WIN 57273 was obtained from Sterling-Winthrop Research Institute (Rensselaer, N.Y.). All other comparison drugs (ciprofloxacin, enoxacin, fleroxacin, norfloxacin, ofloxacin, ampicillin, oxacillin, penicillin, cefoxitin, clindamycin, chloramphenicol, and metronidazole) were provided by their principal U.S. manufacturers. The WIN 57273 disks containing 1, 2, 5, 10, and 20 ,ug were produced by the investigators. The comparison 10-jxg norfloxacin disks were prepared by BBL Microbiology Systems, Cockeysville, Md. Bacterial isolates. The stock culture organisms were typical clinical isolates previously collected from the microbiology laboratories at the Centers for Disease Control, Atlanta, Ga.; The Cleveland Clinic Foundation, Cleveland, Ohio; St. Francis Hospital, Wichita, Kans.; St. Vincent Hospital and Medical Center, Portland, Oreg.; Northwestern Memorial Hospital, Chicago, Ill.; and the Kaiser Permanente Health Care Program of the Northwest, Regional Laboratory, Clackamas, Oreg. Of these 702 isolates, 684 are listed in Tables 1 and 2. Seventeen organisms that possess known resistances to different antimicrobial agents were used for the MBC and inoculum effect studies. In addition, 26 gram-negative strains with reduced susceptibility to the clinically available fluoroquinolones (ciprofloxacin and/or norfloxacin; Table 3) were tested against WIN 57273 and three other similar comparison compounds. Organisms (28 strains) with well-characterized stably derepressed cephalosporinases were also processed (9). Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and S. aureus ATCC 29213 were the strains recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for quality assurance (11, 12). Susceptibility testing methods. The isolates were processed by the NCCLS broth microdilution method (12). A few
* Corresponding author. t Present address: Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, IA 52242.
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WIN 57273 IN VITRO ACTIVITY
VOL. 34, 1990 0
0 OH
H3C A
CH3 FIG. 1. Structure of WIN 57273.
strains were tested by the agar dilution method (12) to assess the effects of medium, supplements, and method on the WIN 57273 MICs. Anaerobic bacteria were tested by the NCCLS Mll-A procedure (13) on Wilkins-Chalgren agar, produced by Difco Laboratories (Detroit, Mich.). Quality control organisms were replicate processed against WIN 57273 disks containing 1, 2, 5, 10, and 20 jig of drug. The MBC studies followed the NCCLS M26-P guidelines (10), which use the rejection values published by Pearson et al. (14). Determination of mutational resistance. Spontaneous mutations to WIN 57273 resistance were determined by plating 1 ml of a steady-state culture (ca. 109 CFU/ml) onto MuellerHinton agar plates containing concentrations of four and eight times the previously determined WIN 57273 MIC (8). RESULTS Activity of WIN 57273 against gram-positive organisms. Table 1 summarizes the results of tests of 200 gram-positive strains against WIN 57273 and five other fluoroquinolones. WIN 57273 exhibited potent activity against gram-positive organisms; MICs for all strains were c2 jig/ml. For the tested species, the WIN 57273 activity could be ranked as follows: Staphylococcus spp. and JK group diphtheroids (MIC for 100% of isolates [MIC100], 0.015 jig/ml) > streptococci (MIC1o, 0.03 jig/ml) > Bacillus spp. and Listeria monocytogenes (MIC100, 0.06 jig/ml) > enterococci (MIC100, 2 jig/ml). When directly compared with the most active drug (ciprofloxacin), WIN 57273 was 16- to 256-fold more active, based on the MIC for 90% of isolates (MIC90) for each species. The only exceptions were for a few uncommonly isolated species (Enterococcus faecium and Bacillus spp.), for which WIN 57273 was only two- to fourfold more potent than ciprofloxacin. The ofloxacin activity against gram-positive microorganisms was most similar to that of ciprofloxacin, and the other tested fluoroquinolones were generally less potent against these species, especially in their failure to inhibit the enterococci and many Streptococcus spp. (see Streptococcus agalactiae, Streptococcus bovis, and Streptococcus pneumoniae data). Staphylococci were particularly susceptible to WIN 57273, the MIC90 being 0.008 jig/ml for all strains tested. Activity of WIN 57273 against gram-negative species. The results from MIC testing of 484 gram-negative strains against the six fluoroquinolones are found in Table 2. WIN 57273 was also very active against Branhamella catarrhalis (MIC10J, c0.004 jig/ml), Haemophilus influenzae (MIC10o, 0.03 jig/ml), and pathogenic Neisseria spp. (MIC100, c0.03 or s0.004 jig/ml). The P. aeruginosa strains were more resistant to all fluoroquinolones, with ciprofloxacin appearing most active (MICg, 0.5 jig/ml); ciprofloxacin was 4- and 16-fold more potent than norfloxacin and WIN 57273, respectively. Members of the family Enterobacteriaceae (254 strains) were most susceptible to ciprofloxacin and least inhibited by WIN 57273. However, for only 3.1% of isolates were WIN
307
57273 MICs >2 jig/ml, a concentration used as a nonsusceptible level for all fluoroquinolones except ciprofloxacin. Potential WIN 57273-resistant strains included minority populations found among Citrobacterfreundii, Proteus vulgaris, and Serratia marcescens. Bacteroides fragilis group strains (32 isolates) were quite susceptible to WIN 57273 with the agar dilution method on Wilkins-Chalgren medium (13). For all but one strain the MIC of WIN 57273 was either 0.25 or 0.5 ,ug/ml. Many of the test strains were resistant to cefoxitin (50%) or clindamycin (25%). All anaerobic bacteria were susceptible to metronidazole, chloramphenicol, and P-lactamase inhibitor combinations (data not shown). Several additional species were tested in more limited numbers (two strains each): all of the WIN 57273 MICs were c2 ,ig/ml. These species and MIC ranges included the following: Achromobacter xylosoxidans, 1 to 2 ,ug/ml; Aeromonas spp., 0.03 to 0.06 jig/ml; Cedecea lapagei, .0.004 to 0.5 ,ug/ml; Citrobacter amalonaticus, 0.25 to 0.5 jig/ml; Enterobacter sakazakii, 0.12 to 0.25 ,ug/ml; Flavobacterium spp., c0.004 to 0.008 ,ug/ml; Hafnia alvei, 0.5 jig/ml; Providencia alcalifaciens, 0.12 to 1 jig/ml; and Serratia liquefaciens, 0.06 to 0.5 jig/ml. WIN 57273 activity against drug-resistant organism populations. WIN 57273 was tested against several antimicrobial agent-resistant organism groups, including a fluoroquinolone-resistant gram-negative population (Table 4). WIN 57273 was comparable to fleroxacin and ofloxacin in its activity against these ciprofloxacin- and/or norfloxacin-resistant strains (11, 12). Enoxacin was least active (4% susceptible strains at c2 jig/ml), whereas ciprofloxacin (35% susceptible), WIN 57273 (35% susceptible), and fleroxacin (38% susceptible) showed the most inhibition at recommended or proposed susceptible breakpoints (1, 5, 11, 12). Tests were performed with 28 strains that were capable of producing stably derepressed type I cephalosporinases (9). WIN 57273 inhibited 79o of these 28 strains at c2 jig/ml with an MIC for 50% of isolates tested (MIC50) of 0.5 jig/ml; other fluoroquinolones were also active. P-Lactamase-producing strains of various types were also inhibited by WIN 57273 and other quinolones (data not shown). The elevated WIN 57273 MICs for P. aeruginosa strains were not significantly different from those for P-lactamase-deficient strains of the same species (Table 2). All quinolones were active against aminoglycoside-resistant gram-negative bacilli. Effect of inoculum on WIN 57273 MICs and MBCs. Seventeen organisms were tested against WIN 57273 with three inoculum concentrations, and MBCs were determined by a reference procedure (10, 14). WIN 57273 was clearly bactericidal, e.g., 16 of 17 (94%) MBC endpoints were 8 2-8 1->8 0.5->8
>8 8
WIN 57273
0.06
0.06
Ciprofloxacin
1
1
Enoxacin Fleroxacin Norfloxacin Ofloxacin
8 4 4 2
8 4 4 2
WIN 57273
WIN 57273
Ciprofloxacin
Staphylococcus spp., coagulase negative Oxacillin resistantd (15)
90%0
2
Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin Oxacillin susceptiblec (20)
50%
1
Ciprofloxacin
Listeria monocytogenes (10)
MIC (,ug/ml)
Fluoroquinolone
Enoxacin Fleroxacin Norfloxacin Ofloxacin WIN 57273
'0.004
'0.004
2 0.5 2
2 4
0.5
0.5
.0.004 0.5 1
_0.004
2
'0.004
Ciprofloxacin
0.25
Enoxacin Fleroxacin Norfloxacin Ofloxacin
0.5 0.5 1
WIN 57273 Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
.0.004 0.25 0.5
0.25
1 0.5 0.5
0.03-0.06
1-4
1
0.5
4-8 2-8 2-8 1-4
0.5->4 4->8 2-4 2->8
0.5
0.5
0.5-4
.0.004 0.5-1
1-2 0.25-1 1-4 0.25-0.5
1
1 2 1 4 0.5
.0.004
0.25-2 0.5-4 0.25-1 0.5-8 0.25-1
0.008 0.5 1 1 1 0.5
.0.004-0.008
0.0)15 0.5 2 4 4 1
.0.004-0.015 0.12-0.5
0.12-0.5 0.25-1 0.25-1 0.5-2 0.12-1
0.5-4 0.5-4 0.25-8 0.25-1
Continued on following page
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TABLE 1-Continued MIC (,ug/ml) Organism (n)
Fluoroquinolone
50%o
90o
Range
Streptococcus bovis (10)
WIN 57273 Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
0.015 2 >8 >8 >8 4
0.03 4 >8 >8 >8 4
0.008-0.03 2-4 >8 >8 >8 4
S. pneumoniaef (20)
WIN 57273 Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
0.015 1 8 8 8 2
0.03 2 >8 8 8 2
c0.004-0.03 0.5-4 1->8 2->8 2->8 0.5-4
S. pyogenes (20)
WIN 57273
Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
0.03 0.5 4 4 2 1
0.03 1 >8 8 4 2
0.008-0.03 0.25-2 2->8 2->8 2->8 0.5-4
WIN 57273 Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
0.015 1 >8 8 8 2
0.03 2 >8 >8 >8 2
0.008-0.03 1-2 8->8 8->8 4->8
0.03 0.5 8 4 4 1
0.03 0.5 8 8 4 1
0.015-0.03 0.5 4-8 4-8 2-4 1
0.015 0.5 8
0.03 1 >8 8 4 2
0.008-0.03 0.5-2 4->8 4-8 2-4 1-2
S. agalactiae (20)
Streptococcus spp. Serogroup C (10)
Serogroup G (10)
WIN 57273
Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin WIN 57273
Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
4 4 1
2-4
a All strains produced a 13-lactamase. Includes E. faecium (six strains), E. durans (two strains), and E. hirae (two strains). Ten strains produced a penicillinase. d Includes S. epidermidis (5 strains) and S. haemolyticus (10 strains). eIncludes S. auricularis (one strain), S. epidermidis (five strains), S. hominis (one strain), S. cohnii (one strain), S. capitis (one strain), S. similans (one strain), S. saprophyticus (two strains), S. warneri (two strains), and S. xylosus (one strain). f Ten isolates were resistant to penicillin (MICs, .0.12 ,ug/ml; 1-,ug oxacillin disk zones, '19 mm). b '
to a different medium base (DST) slightly increased the WIN 57273 MICs. The WIN 57273 MICs on Mueller-Hinton agar were essentially the same as those in cation-supplemented broth. The WIN 57273 MICs for the Neisseria gonorrhoeae isolates were determined on GC agar base with a cysteinefree IsoVitaleX (BBL) supplement known not to affect antimicrobial agent MIC endpoints. However, WIN 57273 MICs for gonococci remained the same (0.03 ,ug/ml) even when cysteine was included as a medium supplement (data not shown). Spontaneous rates of mutation to WIN 57273 resistance. The rates of spontaneously occurring mutants resistant to WIN 57273 concentrations four and eight times greater than the MIC for six organisms were determined by previously described methods (8). These enteric bacilli (Enterobacter cloacae, E. coli, Morganella morganii, Providencia rettgeri,
Klebsiella pneumoniae) and E. faecalis had low rates ranging from 5.6 x 108 to >1.4 x 10' at eightfold the WIN 57273 MIC. However, higher rates of resistance were observed for the gram-negative isolates at fourfold the WIN 57273 MIC. Preliminary disk test content studies. WIN 57273 was added to sterile paper disks at concentrations of 1, 2, 5, 10, and 20 p.g. Five replicate zones for each disk were then determined against NCCLS quality control organisms and also compared with their MICs. WIN 57273 zones for each strain did not significantly increase for disk contents of .2 ,ug. The control disk zone diameters for 10 ,ug of norfloxacin were significantly greater, especially for strains for which MICs of WIN 57273 were equal. Furthermore, WIN 57273 and norfloxacin 10-,ug disk inhibitory zones were equal (23 to 24 mm) for strains for which WIN 57273 was 32-fold more
310
JONES AND BARRY
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Activity of WIN 57273 compared with those of five other fluoroquinolones tested against 484 gram-negative organisms by NCCLS methods (12, 13) Organism (n)
MIC (,ug/ml)
Fluoroquinolone
Acinetobacter anitratus (10)
WIN 57273 Ciprofloxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin
Branhamella catarrhalisa (20)
WIN 57273
Ciprofioxacin Enoxacin Fleroxacin Norfloxacin Ofloxacin Haemophilus influenzae ,-Lactamase negative Ampicillin susceptible (25) Ampicillin resistant (19) ,B-Lactamase positive (21)
WIN 57273 WIN 57273 WIN 57273
Neisseria gonorrhoeaeb (59)
WIN 57273
509o
90%0
0.06 0.25 2 0.25 4 0.25
0.12 0.5 4 0.5 8 0.5
0.03-0.12 0.12-1 1-4 0.25-1 1->8 0.12-1
0.004