In Vitro Activities of ABT-773, a New Ketolide, against Aerobic and Anaerobic Pathogens Isolated from Antral Sinus Puncture Specimens from Patients with Sinusitis

Abstract

The comparative in vitro activities of ABT-773 against 207 aerobic and 162 anaerobic antral sinus puncture isolates showed that erythromycin-resistant pneumococcal strains were susceptible to ABT-773 (≤0.125 μg/ml); the MIC at which 90% of the isolates tested were inhibited for Haemophilus influenzae and other Haemophilus spp. was 4 μg/ml; and all Moraxella spp. and beta-lactamase-producing Prevotella species strains were inhibited by ≤0.125 μg/ml. Among the anaerobes tested, only fusobacteria (45%) required ≥4 μg of ABT-773/ml for inhibition. ABT-773 may offer a therapeutic alternative for sinus infections.

Bacterial sinusitis is the fifth most common disorder requiring antimicrobial therapy and affects approximately 30 million Americans yearly (16, 19). Macrolides have been used to treat acute sinusitis, with targeted activity against pneumococci, Haemophilus spp., and Moraxella catarrhalis; however, resistance has emerged (2, 6, 8, 11, 18).

ABT-773 is a new ketolide agent with a broad spectrum of activity, including macrolide-resistant pathogens (1, 3). In order to evaluate the potential efficacy of ABT-773 in the therapy of sinusitis, we determined its comparative in vitro activities against 369 recent aerobic and anaerobic clinical isolates from patients with sinusitis.

Strains were isolated from antral puncture specimens obtained from adult patients with sinusitis between 1994 and 1999 and were identified by standard criteria (12, 17). Streptococcus pneumoniae ATCC 49619, Haemophilus influenzae ATCC 49247, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, and Bacteroides fragilis ATCC 25285 were tested simultaneously with the appropriate plates and environments. The numbers and species of clinical isolates tested are given in Table 1.

TABLE 1.

Comparative in vitro activities of ABT-773 against aerobic and anaerobic bacteria isolated from patients with sinusitis

Organism (no.)b and agent	MIC (μg/ml)
	Range	50%	90%
Haemophilus spp.c (33)
ABT-773	1–>4	4	4
Erythromycin	2–>8	4	8
Azithromycin	1–4	2	4
Clarithromycin	4–>8	>8	>8
Amoxicillin	≤0.06–>8	0.5	>8
Cefuroxime	≤0.06–1	0.5	1
Levofloxacin	≤0.03	≤0.03	≤0.03
Moraxella catarrhalis (11)
ABT-773	0.06–0.125	0.125	0.125
Erythromycin	0.125–0.25	0.25	0.25
Azithromycin	≤0.06	≤0.06	≤0.06
Clarithromycin	0.125–0.25	0.125	0.125
Amoxicillin	≤0.06–2	1	2
Cefuroxime	0.25–2	1	2
Levofloxacin	≤0.03–0.06	≤0.03	0.06
Staphylococcus aureus (39)
ABT-773	≤0.03–0.06	≤0.03	0.06
Erythromycin	0.125–>8	0.125	>8
Azithromycin	0.5–>8	1	>8
Clarithromycin	0.125–>8	0.25	>8
Amoxicillin	≤0.06–>8	8	>8
Cefuroxime	0.25–8	1	2
Levofloxacin	0.06–>4	0.125	0.25
Staphylococcus spp., coagulase  negatived (32)
ABT-773	≤0.03–>4	0.06	>4
Erythromycin	≤0.06–>8	>8	>8
Azithromycin	0.25–>8	>8	>8
Clarithromycin	≤0.06–>8	>8	>8
Amoxicillin	≤0.06–>8	1	8
Cefuroxime	0.125–4	0.5	4
Levofloxacin	0.124–>4	0.25	2
Streptococcus pneumoniae (27)
ABT-773	≤0.03–1	≤0.03	≤0.03
Erythromycin	≤0.06–8	≤0.06	≤0.06
Azithromycin	≤0.06–>8	0.125	0.5
Clarithromycin	≤0.06–8	≤0.06	≤0.06
Amoxicillin	≤0.06–1	≤0.06	0.5
Cefuroxime	≤0.06–8	≤0.06	1
Levofloxacin	0.125–1	0.5	1
Streptococcus spp.e (22)
ABT-773	≤0.03–0.06	≤0.03	≤0.03
Erythromycin	≤0.06–>8	≤0.06	4
Azithromycin	≤0.06–>8	≤0.06	>8
Clarithromycin	≤0.06–>8	≤0.06	4
Amoxicillin	≤0.06–2	≤0.06	1
Cefuroxime	≤0.06–>8	≤0.06	1
Levofloxacin	≤0.03–>4	1	2
Corynebacterium spp.f (6)
ABT-773	≤0.03	0.06	—n
Erythromycin	0.25–>8	4	—
Azithromycin	2–>8	>8	—
Clarithromycin	0.25–>8	1	—
Amoxicillin	≤0.06–0.5	0.125	—
Cefuroxime	≤0.06–0.25	0.125	—
Levofloxacin	0.06–>4	0.125	—
Eikenella corrodens (10)
ABT-773	0.125–2	1	1
Erythromycin	4–>8	8	>8
Azithromycin	1–>8	4	>8
Clarithromycin	4–>8	8	>8
Amoxicillin	0.5–1	0.5	1
Cefuroxime	1–>8	4	>8
Levofloxacin	≤0.03	≤0.03	≤0.03
Enterobacteriaceaeg (21)
ABT-773	4–>4	>4	>4
Erythromycin	>8	>8	>8
Azithromycin	4–>8	>8	>8
Clarithromycin	>8	>8	>8
Amoxicillin	2–>8	>8	>8
Cefuroxime	0.25–>8	8	>8
Levofloxacin	≤0.03–0.125	≤0.03	0.125
Miscellaneous gram-negative  bacteriah (6)
ABT-773	1–>4	>4	—
Erythromycin	4–>8	>8	—
Azithromycin	0.5–>8	>8	—
Clarithromycin	4–>8	>8	—
Amoxicillin	>8	>8	—
Cefuroxime	0.25–>8	>8	—
Levofloxacin	0.06–>4	0.5	—
Fusobacterium spp.i (20)
ABT-773	≤0.08–8	2	8
Erythromycin
pH 7	0.5–>32	8	32
pH 8	≤0.03–16	8	32
Azithromycin
pH 7	≤0.03–1	0.5	1
pH 8	≤0.03–2	0.5	1
Clarithromycin
pH 7	8–32	8	16
pH 8	≤0.03–16	0.25	8
Amoxicillin	≤0.03–2	≤0.03	0.25
Cefuroxime	≤0.03–1	≤0.03	0.5
Levofloxacin	≤0.03–2	1	1
Prevotella spp.j (10)
ABT-773	≤0.08–0.125	0.06	0.125
Erythromycin
pH 7	0.06–8	0.5	1
pH 8	≤0.03–0.5	0.125	0.25
Azithromycin
pH 7	0.06–16	0.125	0.25
pH 8	0.06–2	0.125	0.25
Clarithromycin
pH 7	≤0.03–4	0.06	0.25
pH 8	≤0.03–0.5	≤.03	0.125
Amoxicillin	≤0.03–>32	2	>32
Cefuroxime	0.06–>32	8	>32
Levofloxacin	0.5–4	1	1
Prevotella melaninogenica (13)
ABT-773	0.015–0.125	0.03	0.125
Erythromycin
pH 7	0.125–32	0.5	4
pH 8	≤0.03–2	0.125	0.25
Azithromycin
pH 7	0.06–>32	0.125	8
pH 8	0.06–1	0.125	0.5
Clarithromycin
pH 7	≤0.03–4	0.06	2
pH 8	≤0.03–0.25	≤0.03	0.06
Amoxicillin	0.125–>32	8	>32
Cefuroxime	0.25–>32	32	>32
Levofloxacin	0.5–>8	1	1
Propionibacterium spp.k (28)
ABT-773	≤0.08–0.015	≤0.08	0.015
Erythromycin
pH 7	≤0.03–0.06	0.06	0.06
pH 8	≤0.03	≤0.03	≤0.03
Azithromycin
pH 7	≤0.03–0.125	0.06	0.125
pH 8	≤0.03–0.06	≤0.03	≤0.03
Clarithromycin
pH 7	≤0.03–0.06	≤0.03	0.06
pH 8	≤0.03	≤0.03	≤0.03
Amoxicillin	≤0.03–0.5	0.125	0.25
Cefuroxime	0.06–1	0.125	1
Levofloxacin	0.125–0.5	0.25	0.5
Peptostreptococcus magnus (40)
ABT-773	≤0.08–0.5	0.03	0.03
Erythromycin
pH 7	2–>32	4	4
pH 8	0.5–>32	1	2
Azithromycin
pH 7	1–>32	2	4
pH 8	0.5–>32	0.5	2
Clarithromycin
pH 7	1–>32	2	2
pH 8	0.5–>32	1	1
Amoxicillin	0.06–0.25	0.25	0.25
Cefuroxime	0.5–32	4	16
Levofloxacin	0.125–>8	0.25	0.5
Peptostreptococcus micros (16)
ABT-773	≤0.08–>8	0.03	0.03
Erythromycin
pH 7	0.25–>32	1	2
pH 8	0.125–>32	0.25	0.5
Azithromycin
pH 7	0.5–>32	1	1
pH 8	0.125–>32	0.25	0.25
Clarithromycin
pH 7	0.25–>32	0.5	1
pH 8	0.125–>32	0.125	0.25
Amoxicillin	≤0.03–4	0.25	4
Cefuroxime	0.125–4	1	4
Levofloxacin	0.06–1	0.5	0.5
Peptostreptococcus spp.l (12)
ABT-773	≤0.08–0.5	0.015	0.03
Erythromycin
pH 7	≤0.03–>32	2	4
pH 8	≤0.03–>32	0.5	1
Azithromycin
pH 7	0.125–>32	2	2
pH 8	≤0.03–>32	0.5	2
Clarithromycin
pH 7	≤0.03–>32	0.5	1
pH 8	0.5–8	0.25	1
Amoxicillin	≤0.03–1	0.125	0.25
Cefuroxime	0.06–16	4	16
Levofloxacin	0.25–8	4	4
Veillonella spp. (13)
ABT-773	≤0.008–4	1	2
Erythromycin
pH 7	2–32	8	32
pH 8	0.25–8	2	8
Azithromycin
pH 7	1–16	2	16
pH 8	0.25–2	0.5	2
Clarithromycin
pH 7	1–32	4	32
pH 8	0.5–8	2	8
Amoxicillin	0.25–8	2	4
Cefuroxime	2–16	8	16
Levofloxacin	0.25–8	0.25	0.5
Other anaerobesm (10)
ABT-773	≤0.08–>8	1	2
Erythromycin
pH 7	0.125–>32	2	16
pH 8	≤0.03–>32	1	16
Azithromycin
pH 7	≤0.03–>32	4	16
pH 8	≤0.03–>32	1	16
Clarithromycin
pH 7	0.06–>32	1	8
pH 8	≤0.03–>32	0.5	8
Amoxicillin	≤0.03–>32	1	32
Cefuroxime	≤0.03–>32	8	>32
Levofloxacin	≤0.03–>8	1	8

^ano., number of isolates tested. 

^bMIC₅₀, MIC at which 50% of the isolates tested were inhibited. 

^cIncludes 25 H. influenzae, 6 H. parainfluenzae, and 2 H. paraphrophilus isolates. 

^dIncludes 24 Staphylococcus epidermidis, 1 S. hyicus, 1 S. lugdenensis, 1 S. saprophyticus, 1 S. warnerii, 1 S. auricularis, 1 S. capitis, and 2 other coagulase-negative species isolates. 

^eIncludes 1 Streptococcus agalactiae, 3 S. anginosus, 1 group G beta-hemolytic streptococcus, and 2 other alpha-hemolytic streptococcal isolates and 1 S. constellatus, 2 S. mitis, 7 S. sanguis I and II, 1 S. salivarius, 1 S. pyogenes, 2 Gemella morbillorum, and 1 Enterococcus faecalis isolates. 

^fIncludes 2 Corynebacterium amycolatum, 1 C. minutissimum, 1 C. pseudodiphtheriticum, and 2 other Corynebacterium species isolates. 

^gIncludes 6 E. coli, 2 Citrobacter koseri, 2 Enterobacter aerogenes, 3 Enterobacter cloacae, 1 Hafnia alvei, 2 Klebsiella oxytoca, 1 Klebsiella pneumoniae, 1 Pantoea agglomerans, 1 Serratia liquefaciens, and 2 Serratia marcescens isolates. 

^hIncludes 3 Pseudomonas aeruginosa, 1 Acinetobacter baumannii, 1 Acinetobacter lwoffi, and 1 Bordetella bronchiseptica isolates. 

ⁱIncludes 16 F. nucleatum, 1 F. necrophorum, 2 F. naviforme, and 1 F. varium isolates. 

^jIncludes 1 P. bivia, 6 P. buccae, 1 P. intermedia, and 2 P. oris isolates. 

^kIncludes 12 P. acnes, 8 P. avidum, and 8 P. granulosum isolates. 

^lIncludes 1 P. anaerobius, 5 P. asaccharolyticus, 2 P. prevotii, and 4 other Peptostreptococcus sp. isolates. 

^mIncludes 1 Actinomyces sp., 1 Bilophila wadsworthia, 1 Eubacterium lentum, 1 Bacteroides capillosus, 2 Bacteroides fragilis, 1 Bacteroides uniformis, 2 Campylobacter gracilis, and 1 Clostridium paraputrificum isolates. 

ⁿ—, not calculated. 

The standard laboratory powders and their suppliers were as follows: ABT-773 and clarithromycin, Abbott Laboratories, Abbott Park, Ill.; amoxicillin, SmithKline Beecham Laboratories, Philadelphia, Pa.; azithromycin, Pfizer Inc., New York, N.Y.; levofloxacin, R. W. Johnson Pharmaceutical Research Institute, Raritan, N.J.; cefuroxime, Glaxo-Wellcome, Research Triangle Park, N.C.

Methods used for identification and testing were done as previously published (9) using a broth microdilution method and an inoculum of ∼5 × 10⁴ CFU per well for aerobes and an agar dilution method with an inoculum of 10⁵ CFU per spot for anaerobes. Susceptibility testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) standards (13, 14). Brucella agar supplemented with hemin, vitamin K₁, and 5% laked sheep blood was the basal medium used for anaerobic species. A second set of plates was prepared for erythromycin, azithromycin, and clarithromycin and adjusted to pH 8 prior to autoclaving, to counteract the acidifying effects of incubation in a CO₂-containing atmosphere, and was run simultaneously with the regular pH 7 plates for anaerobic isolates to detect the effect of pH on susceptibility.

ABT-773 performed well in our study, with 340 of 369 (92%) isolates susceptible by current preliminary criteria (G. Stone, A. Nilius, D. Hensey-Rudloff, L. Almer, J. Beyer, R. Flamm, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2164, p. 181, 2000). Tentative breakpoints for ABT-773 were ≤1 μg/ml for susceptibility and ≥4 μg/ml for resistance for nonfastidious species, 4 μg/ml for susceptibility for H. influenzae, and ≤0.5 μg/ml for susceptibility for streptococci. Of the 29 resistant strains, 21 were aerobic gram negatives of the Enterobacteriaceae or Pseudomonas spp. and 4 were coagulase-negative staphylococci. Our study did not include any pediatric strains that may be more resistant than adult strains.

ABT-773 and clarithromycin each had a MIC at which 90% of the isolates tested were inhibited (MIC₉₀) of ≤0.03 μg/ml for pneumococci, which was similar to the activity of erythromycin but fourfold lower than the activity of azithromycin (MIC₉₀ of 0.5 μg/ml). By chance, none of our pneumococcal sinus isolates was resistant to amoxicillin when the newly proposed NCCLS breakpoints were used, while two isolates each were resistant to erythromycin, cefuroxime, and levofloxacin. Andrews et al. (1) noted an ABT-773 MIC₉₀ of 0.12 μg/ml for 49 British isolates of pneumococcus. Doern et al. (6) noted that 39% of sinus pneumococcal isolates were resistant to penicillin, compared to an overall resistance of 26% for general isolates. Thornsberry et al. (18) noted a 29% rate of resistance of pneumococci to clarithromycin, while in our study, 25 of 27 sinus isolates were susceptible to azithromycin and clarithromycin. Doern et al. (6) found that 10% of pneumococci were resistant to azithromycin, clarithromycin, and erythromycin.

For other Streptococcus spp., ABT-773 had a MIC₉₀ of ≤0.03 μg/ml. The drug was more active than erythromycin, azithromycin, and clarithromycin against S. aureus, with a MIC₉₀ of 0.06 μg/ml in comparison to >8 μg/ml for the other macrolides.

In our study, 33% of H. influenzae and other Haemophilus species were beta-lactamase producers and were resistant to amoxicillin, 90% were resistant to erythromycin and clarithromycin, all except one isolate were susceptible to azithromycin, and all isolates were susceptible to ABT-773 (MIC, ≤4 μg/ml). Thornsberry et al. (18) noted that 67 and 58% of H. influenzae isolates were amoxicillin and clarithromycin susceptible, respectively.

For M. catarrhalis, ABT-773 had a MIC₉₀ of 0.125 μg/ml, which was comparable to those of the other macrolides. Levofloxacin showed good activity against most aerobic sinus isolates, with the exception of one strain of Acinetobacter baumannii and some strains of streptococci, S. aureus, and coagulase-negative Staphylococcus spp. With the exception of some members of the family Enterobacteriaceae and 4 of 32 strains of coagulase-negative staphylococci, ABT-773 showed good activity against all aerobic sinus isolates.

Studies of the anaerobic activity of ABT-773 have focused on the B. fragilis group species, intra-abdominal or veterinary isolates, with scant information presented regarding respiratory anaerobic isolates (1, 5, 10, 15). In our study, ABT-773 was quite active against all the anaerobes tested, with the exception of one Actinomyces sp. strain, two strains of Fusobacterium nucleatum, and one strain of Peptostreptococcus micros; the MICs for all these strains were >4 μg/ml. Fusobacteria and Veillonella spp. were generally resistant to the macrolides tested. Citron and Appleman (5) noted an ABT-773 MIC₉₀ of 8 μg/ml for 13 strains of F. nucleatum, while Sillerstrom et al. (15) noted a MIC₉₀ of 1.0 μg/ml for 40 isolates. Methodological differences may account for this variability between the studies. Fifty-two percent (12 of 23) of our Prevotella isolates were resistant to amoxicillin (MIC = 2 μg/ml), as were 31% (4 of 13) of the Veillonella species (there is no amoxicillin breakpoint, but the ampicillin breakpoint is ≤1 μg/ml for intermediate or susceptible isolates).

The addition of CO₂ to the atmosphere of incubation has been shown to variably affect macrolide susceptibility results, depending on the species tested, primarily because of a decrease of pH on the agar surface, but the clinical relevance has not been determined (7, 8). In general, we found an inconsistent two- to fourfold decrease in the activity against anaerobic species with a shift of pH from 7 to 8. For anaerobic isolates for which the MICs were higher, the MICs were reduced to a greater degree at pH 8 than at pH 7. However, we noticed no pH effect at all on Propionibacterium species for any macrolide. In contrast to Canigia et al. (4), who found that fusobacteria were the anaerobic species most affected by pH variation, we found little effect on fusobacterium susceptibility to macrolides. Barry et al. (A. L. Barry, P. C. Fuchs, and S. D. Brown, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2144, p. 348, 1999) studied the effect of CO₂ on the in vitro activity of ABT-773 and concluded that incubation in increased CO₂ elevated MICs of ABT-773 by approximately 30% and that azithromycin and erythromycin were more profoundly affected by increased CO₂. Ramer et al. (N. C. Ramer, D. F. McDaniel, P. M. Johnson, V. D. Shortridge, Y. S. Or, Z. Ma, and R. K. Flamm, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2143, p. 348, 1999) noted a two- to fourfold MIC decrease with an increase of pH from 6.8 to 8.0 with ABT-773. These higher MICs lead to a lower estimate of the in vitro activity and more conservative data.

ABT-773 has an improved and broader spectrum of activity than currently available macrolides and consequently merits further evaluation as a therapeutic alternative for sinusitis.