INTRODUCTION

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It is well known that the in vitro activity of the macrolide class of antimicrobial agents is influenced by the pH of the test medium (7, 8, 10). Susceptibility testing of bacterial isolates from lower respiratory tract infections is often carried out in a carbon dioxide-rich environment similar to that which exists in the lung. However, susceptibility testing of macrolides and azalides in the presence of carbon dioxide can lead to elevated MICs because the presence of carbon dioxide decreases the pH of the medium (5). This effect of pH and carbon dioxide has been well studied with obligate anaerobes (7, 10), but it is less well documented for facultative organisms. In 1986, Dibb et al. (3) tested the in vitro activity of erythromycin for clinical isolates of bacteria including Haemophilus influenzae and showed that the MICs obtained from susceptibility tests performed in air alone were much lower than those obtained after incubation in 8% carbon dioxide. The most striking difference was found for H. influenzae isolates, for which the MIC of erythromycin at which 50% of the isolates were inhibited (MIC₅₀) was 0.5 µg/ml in air, compared to 4 µg/ml in carbon dioxide. To complicate the issue further, when macrolides are tested against H. influenzae the resultant MICs have been shown to be extremely method dependent (2). In 1988, Barry et al. reported erythromycin MIC₉₀s of 2 to 16 µg/ml and MIC₅₀s of 1 to 8 µg/ml for H. influenzae isolates, depending on the method of susceptibility testing (2). These authors reported similar variabilities in clarithromycin MIC₅₀s and MIC₉₀s (2).

In the present study, the in vitro activities of erythromycin, clarithromycin, and azithromycin were determined for 178 clinical isolates, including Streptococcus pneumoniae, Moraxella catarrhalis, H. influenzae, and Haemophilus parainfluenzae, recovered from the sputum of patients with chronic obstructive pulmonary disease by an agar dilution method and incubation in either air or 5% carbon dioxide. A microbiological plate assay was used to quantify the reductions in the in vitro activities of these agents.

	MATERIALS AND METHODS

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Bacteria and growth conditions. Seventeen isolates of S. pneumoniae, 51 isolates of H. influenzae, 48 isolates of M. catarrhalis, and 62 isolates of H. parainfluenzae recovered from the sputum of patients with chronic obstructive pulmonary disease were used throughout. Two quality control strains were used, H. influenzae NCTC 8466 and S. pneumoniae NCTC 7466. The indicator strain Sarcina lutea was used in microbiological plate assay experiments. The liquid medium for all strains was brain heart infusion broth (Unipath, Basingstoke, United Kingdom) supplemented with NAD (Sigma), at a final concentration of 10 µg/ml, and hemin, at a final concentration of 10 µg/ml. The solid medium was Iso-sensitest agar (Unipath) supplemented with 5% lysed horse blood and 10 µg of NAD per ml.

Antimicrobial agents. The following antibiotics were obtained from their respective manufacturers and made up and used according to the manufacturers' instructions: erythromycin (Abbott), azithromycin (Pfizer), and clarithromycin (Abbott).

Growth conditions. All isolates were grown overnight in 5% carbon dioxide in liquid medium to give a viable count of ~10⁸ CFU/ml. Three strains of S. pneumoniae and three strains of H. influenzae were randomly selected and inoculated into duplicate brain heart infusion broths (supplemented with hemin and NAD for H. influenzae strains): one broth was incubated in 5% carbon dioxide and the other in ambient air. After overnight incubation, viable counts were obtained; there was no significant reduction in the numbers of viable cells after overnight incubation in air compared with 5% carbon dioxide, with similar numbers (5 × 10⁸ CFU/ml) for both incubation atmospheres for all strains. However, when grown in air, 26 clinical isolates of H. influenzae (n = 19) and S. pneumoniae (n = 7) grew poorly or did not grow at all, indicating a requirement for an atmosphere that is enriched with carbon dioxide.

Determinations of susceptibility. The MIC of each antibiotic for each strain was determined by the agar doubling dilution method on Iso-sensitest agar supplemented with 5% lysed horse blood and NAD. Two sets of plates containing doubling dilutions of antibiotic were inoculated by transferring 1 µl of undiluted overnight culture to the surface of the agar with a multipoint inoculator (Denley Tech, Billinghurst, United Kingdom) to give a final inoculum of approximately 10⁶ CFU. One set of plates was incubated overnight in air and the other in 5% carbon dioxide. All isolates were inoculated onto an antibiotic-free plate to confirm growth. After 24 h incubation, the lowest concentration of antibiotic that inhibited growth was defined as the MIC; single colonies or hazy growth were ignored. The MIC was expressed in micrograms per milliliter, and MIC₅₀s, MIC₉₀s, and geometric means were calculated for the four bacterial species against each antimicrobial agent and compared with the breakpoint concentrations recommended by National Committee for Clinical Laboratory Standards (NCCLS) and British Society of Antimicrobial Chemotherapy (BSAC) guidelines (9). These experiments were repeated on two further separate occasions. The MICs of the macrolides for 24 clinical isolates of H. influenzae and H. parainfluenzae were also determined in parallel with pH-adjusted medium (the pH was adjusted to 8.4 by the addition of filter-sterilized 1 M sodium hydroxide to the agar before the addition of antibiotic) and Iso-sensitest medium, pH 7.2. Both sets of plates were incubated in 5% carbon dioxide.

Microbiological assay of antimicrobial activity. To determine the stability of each agent in carbon dioxide and air, a bioassay was performed. Antibiotic agar no. 11 (150 ml) (Unipath) was prepared according to the manufacturer's instructions and autoclaved. When cooled to 50°C, 1.5 ml of a 48-h broth culture of the indicator organism, S. lutea NCTC 8340, was added and the seeded agar was poured into a square petri dish (243 cm by 243 cm by 18 mm) (Nunc, Life Technologies, Paisley, Scotland). After drying for 30 min in a drying cabinet, 25 wells were punched in the agar with a no. 4 cork borer. One set of standard concentrations of each of the three agents was prepared (10, 5, 2.5, 1.25, and 0.6 µg/ml) and aliquoted into the wells of two sets of agar plates. One set of plates was incubated in air, and an identical set was incubated overnight in 5% carbon dioxide. Bioassays were performed for each agent in parallel and in triplicate on 3 separate days. The diameters of the zones of inhibition (in millimeters) were measured after 18 h of incubation. Bioassays were also carried out in pH-adjusted medium to quantify any difference in the stability of the three agents caused by a change in pH. pH adjustment was achieved by aseptic addition of sterile 1 M sodium hydroxide until a pH of 8.4 was reached. These plates were then incubated in 5% carbon dioxide; an identical set was incubated in air overnight.

	RESULTS

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Antimicrobial activity. For S. pneumoniae isolates, all MICs were well below the NCCLS- and BSAC-recommended breakpoint concentrations (Table 1). For erythromycin, there was no difference between the MIC₅₀s and MIC₉₀s obtained in air or carbon dioxide. However, geometric mean data show that isolates tested in air had much lower mean values than those grown in carbon dioxide, indicating a real difference between the two incubation atmospheres. For azithromycin there was greater activity detected in air, reflected by a three-tube dilution difference between the MIC₅₀s for carbon dioxide and air; this difference is also reflected in geometric mean data. Clarithromycin was also more active in air, with a consistent one-tube dilution difference between the two incubation conditions, illustrated in both MIC₅₀s, MIC₉₀s, and geometric mean data.

Microbiological assay. After overnight incubation in air, the zone diameter of a 10-µg/ml standard of all three agents was unchanged (Fig. 1). However, overnight incubation in 5% carbon dioxide resulted in a substantial reduction in the potency of all three agents. The reduction in the in vitro activity was most marked for azithromycin; when measured after overnight incubation in carbon dioxide, the 10-µg/ml standard was found to contain <0.6 µg of active compound per ml. For erythromycin, overnight incubation in carbon dioxide reduced the 10-µg/ml standard to 1.25 µg/ml. For clarithromycin, the reduction was from 10 to 1.8 µg/ml after overnight incubation in carbon dioxide. For the 5- and 2.5-µg/ml concentrations, there were no measurable zones of inhibition after incubation in carbon dioxide.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Effects of incubation of bioassays in air (), 5% CO2 (), and pH-adjusted medium with CO2 () upon the microbiological activities of erythromycin, clarithromycin, and azithromycin.

	DISCUSSION

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These data show that the activities of azithromycin, clarithromycin, and erythromycin are dramatically affected by the incubation atmosphere, most likely due to an effect of carbon dioxide upon the agent. Some microbiology laboratories perform sensitivity testing of bacterial respiratory tract isolates in an atmosphere containing increased carbon dioxide, potentially leading to overestimation of the MICs of macrolides and an incorrect designation of resistance. The decrease in activity of these agents observed in carbon dioxide is therefore of major importance. Other studies have shown that incubation in carbon dioxide leads to a marked decrease in the pH of the medium (1, 5), with these agents becoming less active as the pH becomes more acidic. However, for testing of macrolides against organisms that require incubation in a carbon dioxide atmosphere for growth, the pH of the growth medium can be adjusted to 8.4 after autoclaving, thus compensating for (although not completely eliminating) the reduction in pH which occurs during incubation and allowing for the testing of bacterial species requiring carbon dioxide (1, 4). It is important to understand, however, that isolates recovered from the lower respiratory tract of patients with chronic obstructive pulmonary disease may be subjected in vivo to relatively high partial pressures of carbon dioxide in the lung. It has therefore been suggested that the activity of clarithromycin against isolates of H. influenzae may be significantly compromised in respiratory tract infections (6). In light of the data obtained in this study, it is recommended that susceptibility testing of macrolides for bacterial isolates from the lower respiratory tract, or those requiring carbon dioxide, be carried out wherever possible with pH-adjusted medium and incubation in carbon dioxide. Clinical trials of these agents with the emphasis on clinical outcome are critical for determining the significance of the in vitro findings of this study.