In Vitro Activities of Clinafloxacin against Contemporary Clinical Bacterial Isolates from 10 North American Centers

Abstract

Clinafloxacin was more active than ciprofloxacin against 4,213 aerobic and facultative anaerobic bacterial isolates from 10 medical centers, as tested by broth microdilution and disk diffusion methods. The percentage of 201 anaerobes susceptible to clinafloxacin by broth microdilution was comparable to cefoxitin. Our data support the proposed disk diffusion interpretive criteria for aerobic bacteria with 5-μg clinafloxacin disks.

Clinafloxacin (CI-960, PD 127391) is a new fluoroquinolone with a broad spectrum of antimicrobial activity that is superior to currently available fluoroquinolones, particularly against gram-positive bacteria (3–7) and anaerobes (3, 6, 13). The present study was a 10-laboratory survey designed to (i) determine the susceptibility of a wide range of current clinical isolates to clinafloxacin, compared to ciprofloxacin (or cefoxitin in the case of anaerobes), and (ii) assess the reliability of proposed disk diffusion interpretive zone diameter criteria for clinafloxacin (8).

The 10 participating centers are listed in the Acknowledgments. Fresh clinical isolates (first quarter 1997) from different patients were collected by each participating facility. Nonfastidious gram-negative bacterial isolates were tested by broth microdilution and disk diffusion methods at the collecting institution; all others were shipped to The Clinical Microbiology Institute for testing.

The bacterial species and numbers of isolates tested are listed in Tables 1 and 2. At least one quality control strain was tested on each test day; these strains included Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923 (disk diffusion only), Streptococcus pneumoniae ATCC 49619 (streptococcal tests only), Haemophilus influenzae ATCC 49247 (Haemophilus tests only), Neisseria gonorrhoeae ATCC 49226 (gonococcal tests only), Bacteroides fragilis ATCC 25285 (anaerobe tests only), Bacteroides thetaiotaomicron ATCC 29741 (anaerobes only), and Eubacterium lentum ATCC 43055 (anaerobes only). On alternating days, either E. coli ATCC 25922 or P. aeruginosa ATCC 27853 was tested by the 10 collaborating facilities; they tested only gram-negative species.

TABLE 1.

Susceptibilities of 4,213 aerobic and facultative clinical isolates to clinafloxacin (Clina) and ciprofloxacin (Cipro)

Microorganism (no. of isolates)a	MIC (μg/ml)b				% Susceptible to ≤1.0-μg/ml dose of:
	50%		90%
	Clina	Cipro	Clina	Cipro	Clina	Cipro
Acinetobacter baumannii (70)	0.06	0.5	2.0	>16	81.4	58.6
Acinetobacter spp.c (40)	0.03	0.25	2.0	>16	82.5	72.5
Burkholderia cepacia (13)	0.25	1.0	2.0	>16	84.6	69.2
Citrobacter freundii (20)	0.015	0.015	0.06	0.12	100	100
Enterobacter aerogenes (137)	0.015	0.03	0.25	0.12	96.4	89.1
Enterobacter agglomerans (12)	≤0.008	≤0.008	0.015	0.03	100	100
Enterobacter cloacae (152)	0.015	0.015	0.12	0.25	97.4	94.1
Enterobacter spp.d (21)	0.015	0.015	0.25	1.0	100	85.7
E. coli (375)	≤0.008	0.015	0.015	0.03	98.7	97.9
Klebsiella oxytoca (114)	≤0.008	0.015	0.03	0.06	100	99.1
Klebsiella pneumoniae (243)	0.015	0.03	0.25	0.5	96.3	94.2
Morganella morganii (80)	≤0.008	≤0.008	0.12	0.25	97.5	92.5
Pasteurella multocida (16)	≤0.008	≤0.008	≤0.008	≤0.008	100	100
Proteus mirabilis (111)	0.015	0.03	0.03	0.06	100	100
Proteus vulgaris (23)	0.015	0.015	0.03	0.03	100	100
Providencia rettgeri (13)	0.03	0.06	0.03	0.06	100	100
Providencia stuartii (31)	0.25	1.0	1.0	>16	96.8	54.8
P. aeruginosa (308)	0.12	0.25	1.0	2.0	95.5	85.7
Pseudomonas fluorescens group (24)	0.12	0.25	2.0	4.0	87.5	79.2
Pseudomonas spp. (13)	0.03	0.25	0.5	8.0	92.3	69.2
Salmonella spp. (128)	≤0.008	0.015	0.015	0.03	100	100
Serratia marcescens (154)	0.03	0.06	0.25	2.0	98.7	87.7
Serratia spp.e (11)	≤0.008	0.015	0.03	0.06	100	100
Shigella spp.f (81)	≤0.008	0.015	≤0.008	0.015	100	100
S. maltophilia (99)	0.12	2.0	0.5	8.0	94.9	43.4
Miscellaneousg (25)	≤0.008	0.015	0.06	0.25	100	100
E. faecalis (167)	0.12	1.0	4.0	>16	76.6	67.7
E. faecium (35)	2.0	>16	16	>16	34.3	20.0
Enterococcus spp.h (12)	0.06	0.5	2.0	>16	83.3	83.3
MRSA (86)	0.5	16	1.0	>16	96.5	16.3
S. aureus β-Lac− (20)	0.015	0.25	0.03	4.0	100	85.0
S. aureus β-Lac+ (186)	0.015	0.25	0.03	0.5	99.5	95.7
MR Coag− (200)	0.06	1.0	0.5	>16	99	50.5
Coag− β-Lac− (35)	0.03	0.25	0.03	0.5	100	97.1
Coag− β-Lac+ (69)	0.03	0.25	0.12	4.0	98.6	89.9
Stomatococcus sp. (26)	0.03	0.5	0.25	8.0	100	80.8
Streptococcus pyogenes (group A) (120)	0.03	0.25	0.06	0.5	100	96.7
Streptococcus agalactiae (group B) (75)	0.06	0.5	0.12	1.0	100	94.7
S. pneumoniae (PEN-S) (128)	0.03	0.5	0.06	1.0	100	99.2
S. pneumoniae (PEN-I) (32)	0.03	0.5	0.06	0.5	100	100
S. pneumoniae (PEN-R) (45)	0.03	0.5	0.06	1.0	100	100
Streptococcus viridans group (49)	0.06	1.0	0.12	2.0	100	77.6
Other streptococcii (37)	0.06	0.5	0.12	1.0	100	91.9
Miscellaneousj (25)	0.06	0.5	1.0	2.0	96	80
H. influenzae β-Lac− (126)	≤0.008	≤0.008	≤0.008	0.015	100	100
H. influenzae β-Lac+ (54)	≤0.008	≤0.008	≤0.008	0.015	100	100
Haemophilus spp.k (70)	≤0.008	≤0.008	≤0.008	0.015	100	100
Moraxella catarrhalisl (153)	≤0.008	0.03	0.015	0.03	100	100
N. gonorrhoeaem (99)	≤0.002	0.004	≤0.002	0.008	100	100
Neisseria meningitidis (50)	≤0.008	≤0.008	≤0.008	≤0.008	100	100

^aAbbreviations: β-Lac, β-lactamase; Coag⁻, coagulase-negative staphylococci; PEN, penicillin (S, susceptible; I, intermediate; R, resistant). 

^b50% and 90%, MICs at which 50 and 90% of the isolates are inhibited, respectively. 

^cIncludes 11 A. lwoffii, 17 A. anitratus, and 12 Acinetobacter sp. (no other specification [NOS]) strains. 

^dIncludes 5 E. amnigenus, 1 E. gergoviae, 1 E. sakazaki, and 14 Enterobacter sp. (NOS) strains. 

^eIncludes seven S. liquefaciens and four Serratia sp. (NOS) strains. 

^fIncludes 22 S. flexneri, 50 S. sonnei, and 9 Shigella sp. (NOS) strains. 

^gIncludes 14 Aeromonas sp., 1 Alcaligenes sp., 1 Flavobacterium sp., 2 Gemella sp., 1 Hafnia alvei, 2 Kluyvera sp., 1 Proteus penneri, and 3 Yersinia enterocolitica strains. 

^hIncludes two E. avium, three E. casseliflavus, one E. cecorum, five E. durans, and one E. raffinosus strain. 

ⁱIncludes seven Streptococcus bovis, one S. intermedius, five S. mitis, two S. milleri, one S. mutans, five Streptococcus sp. (NOS), one Streptococcus sp. (not pyogenes), and four S. sanguis strains and four group C streptococci, one group F streptococcus, and six group G streptococci. 

^jGram-positive bacteria, including one Aerococcus viridans, one Bacillus sp., one Brevibacterium casei, five C. jeikeium, eight Corynebacterium sp., one Lactobacillus casei, three Leuconostoc sp., two Listeria monocytogenes, one Micrococcus sp., and two Pediococcus pentosaceus strains. 

^kIncludes 2 H. aphrophilus, 2 H. haemolyticus, 2 H. parahaemolyticus, 21 H. parainfluenzae, and 43 Haemophilus sp. (NOS) strains. 

^lIncludes 5 β-lactamase-negative and 58 β-lactamase-positive strains. 

^mIncludes 90 β-lactamase-negative and 9 β-lactamase-positive strains.  

TABLE 2.

Susceptibilities of 201 anaerobes to clinafloxacin (Clina) and cefoxitin (Cfox)

Microorganism (no. of isolates)	MIC (μg/ml)a				% Susceptible
	50%		90%
	Clina	Cfox	Clina	Cfox	Clina	Cfox
B. fragilis (45)	0.25	8.0	2.0	32	80	80
B. thetaiotaomicron (15)	0.25	32	0.5	32	93.3	33.3
Bacteroides spp.b (24)	0.12	8.0	2.0	32	83.3	79.2
Clostridium perfringens (30)	0.12	1.0	0.12	2.0	100	100
Clostridium spp.c (22)	0.06	1.0	0.25	32	95.5	86.4
Fusobacterium spp.d (4)	≤0.03	≤0.03			100	100
Peptostreptococcus spp.e (20)	0.12	0.5	0.25	1.0	90	100
Prevotella spp.f (30)	0.06	2.0	2.0	8.0	86.7	100
Miscellaneous anaerobesg (11)	0.06	0.25	0.25	8.0	100	100

^a50% and 90%, MICs at which 50 and 90% of the isolates are inhibited. 

^bIncludes two B. caccae, three B. distasonis, three B. fragilis group, one B. ovatus, five Bacteroides sp. (NOS), one Bacteroides sp. (not fragilis), four B. uniformis, and five B. vulgatus strains. 

^cIncludes one C. bifermentans, one C. butyricum, one C. difficile, four C. septicum, one C. sporogenes, two C. tertium, and two Clostridium sp. (NOS) strains. 

^dIncludes two F. necrophorum, one F. nucleatum, and one Fusobacterium sp. (NOS) strain. 

^eIncludes 3 P. anaerobius, 5 P. asaccharolyticus, and 12 Peptostreptococcus sp. (NOS) strains. 

^fIncludes 7 P. bivia, 3 P. buccae, 1 P. intermedia, 1 P. loescheii, 7 P. melaninogenica, and 11 Prevotella sp. (NOS) strains. 

^gIncludes one E. lentum, nine Propionibacterium acnes, and one Veillonella sp. strain. 

Clinafloxacin was provided as standardized powder by Parke-Davis Pharmaceuticals; ciprofloxacin, cefoxitin, ampicillin, and penicillin G were procured from their respective manufacturers or from other commercial sources. The following commercially prepared disks were used for susceptibility testing: 5-μg clinafloxacin (BBL 604541) and 5-μg ciprofloxacin (BBL 606634).

Methods outlined by the National Committee for Clinical Laboratory Standards were used for all tests (9–11). Broth microdilution tests were performed with all organisms except N. gonorrhoeae. For the latter species, the agar dilution method was used with GC agar containing IsoVitaleX supplements (10). Cation-adjusted Mueller-Hinton broth was the test medium for all other aerobic isolates and was supplemented with 2 to 3% lysed horse blood for testing of streptococci and with Haemophilus Test Medium supplements for testing of Haemophilus species. Wilkins-Chalgren broth was used for anaerobes (9). Against anaerobes, clinafloxacin activity was compared with that of cefoxitin. Drug concentrations of both quinolones were twofold concentrations ranging from 0.008 to 16 μg/ml. During testing of streptococci, penicillin was tested at breakpoint concentrations, and ampicillin was included during testing of Haemophilus spp. β-Lactamase tests were also performed on all Haemophilus spp. isolates by using a nitrocefin reagent.

For disk diffusion tests, Mueller-Hinton agar was used and was supplemented with 5% defibrinated sheep blood for testing of streptococci, and Haemophilus Test Medium agar was used for testing of Haemophilus spp. (11). All tests yielding very major errors were repeated, and the results of the repeat test were recorded.

The susceptibilities of 4,213 aerobic and facultative gram-negative bacterial isolates to clinafloxacin and ciprofloxacin are summarized in Table 1. Although there were minor variations with different species, clinafloxacin was the most active of the two drugs against this group as a whole. All isolates susceptible to ciprofloxacin were susceptible to ≤1.0 μg of clinafloxacin per ml. Of 302 gram-positive bacterial isolates not susceptible to ciprofloxacin, 231 (76.5%) were susceptible to clinafloxacin, as were 163 (66.0%) of 247 nonfastidious gram-negative isolates resistant to ciprofloxacin. Nevertheless, clinafloxacin MICs for ciprofloxacin-resistant isolates tended to be higher than those for ciprofloxacin-susceptible isolates of the same species.

At ≤1.0 μg/ml, 96.2% of nonfastidious gram-negative bacteria were susceptible to clinafloxacin, whereas only 89.3% were susceptible to ciprofloxacin. Of 308 strains of P. aeruginosa, 95.5% were susceptible to clinafloxacin, compared to 86% for ciprofloxacin. Clinafloxacin was more than 10 times more active than ciprofloxacin against 99 strains of Stenotrophomonas maltophilia, resulting in 95% of strains being susceptible to clinafloxacin and 43% of strains being susceptible to ciprofloxacin. These results are comparable to those of previous studies (4, 12).

The geometric mean MICs of ciprofloxacin for gram-positive bacteria were 4- to 30-fold higher than those of clinafloxacin for the different species tested. This was particularly striking for methicillin-resistant S. aureus (MRSA) isolates, of which 96.5% were susceptible to clinafloxacin and 16.3% were susceptible to ciprofloxacin. Although most MRSA strains were susceptible to clinafloxacin at ≤1.0 μg/ml, the geometric mean MIC for this group was 15 times greater than for methicillin-susceptible strains. Enterococci were the least susceptible to both drugs, although clinafloxacin MICs were four to eight times lower than those of ciprofloxacin. The 76% of Enterococcus faecalis and 34% of Enterococcus faecium strains susceptible to clinafloxacin are much lower than the results reported in two studies (3, 4) and more consistent with the increasing resistance reported by others (5, 7). Of the 1,347 total gram-positive isolates, 94.7% were susceptible to clinafloxacin at ≤1.0 μg/ml, compared to 77.6% for ciprofloxacin.

The 552 fastidious gram-negative bacterial isolates were exquisitely susceptible to both drugs, with clinafloxacin being slightly more active than ciprofloxacin. All isolates were susceptible to both drugs. It should be noted, however, that N. gonorrhoeae isolates with decreased susceptibilities to ciprofloxacin have higher clinafloxacin MICs (2).

The susceptibilities of anaerobes to clinafloxacin were compared to cefoxitin (Table 2). Although the activity by drug concentration was greater for clinafloxacin for all species tested, the differences in susceptibility breakpoints for the two drugs (≤1.0 μg/ml for clinafloxacin and ≤16 μg/ml for cefoxitin) rendered the percent susceptible to the drugs nearly the same (90.5 and 87.6%, respectively). Although Wexler et al. reported all of 339 strains of anaerobic bacteria to be inhibited by 1.0 μg of clinafloxacin per ml (13), we encountered several strains with clinafloxacin MICs higher than this, including two isolates of Prevotella spp. with MICs of 16 μg/ml.

A scattergram showing the distribution of clinafloxacin MICs and disk diffusion zone diameters for all aerobic isolates (excluding N. gonorrhoeae) is provided in Fig. 1. MIC breakpoints for clinafloxacin have not yet been approved. MIC breakpoints of ≤1.0 μg/ml (susceptible) and ≥4.0 μg/ml (resistant) were tentatively selected in this study based on published pharmacokinetic data showing that a 200-μg dose in healthy volunteers yielded a mean C_max of 2.46 μg/ml and a 6.1-h half-life in one study (1) and a mean C_max of 1.34 μg/ml and a 5.65-h half-life in a second study (14). These breakpoints have been previously proposed (8). With corresponding zone diameter breakpoints of ≥21 and ≤17 mm, there were two very major (false-susceptible disk tests) errors, representing 0.05% of the total population and 3.2% of the total resistant isolates. There were 14 (0.3%) major (false-resistant disk tests) errors and 88 (2.2%) minor errors. For comparison, the error rates for ciprofloxacin were as follows: very major, 0.2% of all isolates and 2.1% of resistant isolates; major, 0.5%; and minor, 7.1%. These very low error rates with the clinafloxacin disk test support the proposed disk diffusion breakpoints (5).

FIG. 1.

Scattergram of clinafloxacin MICs (ordinate) and disk diffusion zone diameters (abscissa) for 4,057 aerobic clinical isolates. Horizontal lines represent proposed MIC breakpoints; vertical lines represent the corresponding zone diameter breakpoints.

Clinafloxacin disk diffusion susceptibility tests with viridans streptococci and pneumococci combined can be interpreted with the breakpoints that are proposed for other microorganisms. With these breakpoints for 284 strains, there was one major error and no very major or minor errors. Very major errors were not possible with this group of organisms, because all isolates were susceptible.

In summary, clinafloxacin exhibited greater activity against recent clinical isolates of aerobic bacteria than did ciprofloxacin, particularly against gram-positive isolates. The percentage of anaerobic clinical isolates susceptible to clinafloxacin was comparable to that of cefoxitin. The previously proposed clinafloxacin disk diffusion interpretive criteria performed satisfactorily with this large number of recent clinical isolates.