In Vitro Activities of Gemifloxacin versus Five Quinolones and Two Macrolides against 271 Spanish Isolates of Legionella pneumophila: Influence of Charcoal on Susceptibility Test Results

Abstract

The MICs at which 90% of isolates are inhibited for gemifloxacin, trovafloxacin, and grepafloxacin were low (≤0.01 μg/ml) for 271 Legionella isolates when they were determined by the broth microdilution method but increased (≥6 dilutions) when they were determined by the agar dilution method. This was due to the charcoal in the agar dilution medium, as shown by the progressive decrease in the MICs when the charcoal concentrations decreased. As free drug is the active fraction, charcoal binding should be considered.

Extracellular susceptibility testing should be regarded as a screening method to determine drug inactivity (4) against Legionella isolates. The intracellular location of the infecting microorganism is responsible for the in vitro-in vivo activity-efficacy dichotomy seen with some antimicrobial agents (9).

Gemifloxacin is a broad-spectrum quinolone that has good intracellular penetration. This study investigated the antimicrobial susceptibilities of Legionella isolates to gemifloxacin compared with their susceptibilities to five quinolones and two macrolides. Two different test methods (the broth microdilution and agar dilution methods) were used, and the influence of the presence of charcoal in the test medium was also investigated.

The test panel included 251 clinical isolates and 20 environmental isolates of Legionella pneumophila, collected between 1983 and 1999 in 18 autonomous regions of Spain. The 251 clinical isolates of Legionella included 1 L. longbeachae isolate and 250 L. pneumophila isolates. L. pneumophila serogroups (SG) were determined by polyclonal antibody testing; 208 SG-1, 1 SG-2, 8 SG-3, 1 SG-4, 2 SG-5, 9 SG-6, 3 SG-8, 1 SG-9, 4 SG-10, 2 SG-12, 4 SG-4,8,10, 2 SG-3,6, 1 SG-3,5, 1 SG-5,10, 1 SG-5,8, and 2 SG-8,10 isolates were included. The 20 environmental isolates of L. pneumophila included 17 SG-1 strains, 2 SG-6 strains and 1 SG-4,8 strain. These isolates were epidemiologically related to outbreaks or single patients with Legionnaires' disease from whom the corresponding clinical isolate was available.

Overall, the test panels included 196 isolates of L. pneumophila SG-1 Pontiac, 18 isolates of L. pneumophila SG-1 Olda, 9 isolates of L. pneumophila SG-1 Bellingham, 2 isolates of L. pneumophila SG-1 nontypeable (as assessed with monoclonal antibodies), 45 isolates of L. pneumophila that belonged to other subgroups, and 1 L. longbeachae isolate. Fifty-four percent of the isolates came from the Mediterranean (east) coast of Spain, 10% came from the north of Spain (Cantabrian coast), 13% came from Madrid, and the remaining 23% came from other regions in the interior of the country. All strains were stored in skim milk at −70°C.

Levofloxacin, trovafloxacin, grepafloxacin, clarithromycin, and gemifloxacin (SmithKline Beecham Pharmaceuticals, Tonbridge, United Kingdom), erythromycin, ofloxacin (Sigma Chemical Co., St. Louis, Mo.), and ciprofloxacin (Bayer S.A., Barcelona, Spain) were kindly provided as powders of known potencies, diluted in distilled water, and frozen at −70°C until they were assayed.

Broth microdilution susceptibility testing was carried out with 96-well microtiter plates that contained 50 μl of buffered yeast extract supplemented with 0.1% α-ketoglutarate (BYEα) medium (3). The bacterial colonies were grown in buffered charcoal yeast extract agar supplemented with 0.1% α-ketoglutarate (BCYEα) at 35°C. After 3 days the colonies were suspended in distilled water and the turbidity was adjusted to that of a no. 2 McFarland standard (6 × 10⁸ CFU/ml). The test wells were then inoculated with 50 μl to give a final inoculum of 3 × 10⁵ CFU/ml. Each plate also included two antimicrobial agent-free control wells: the first one contained 100 μl of BYEα medium, and the second one contained 50 μl of the bacterial suspension plus 50 μl of BYEα medium. Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as control strains. The microtiter plates were incubated at 35°C in humidified air for 3 days.

Agar dilution susceptibility testing was performed as follows: a series of eight twofold dilutions in distilled water was prepared for each test antimicrobial agent, and 2 ml was added to plates that contained 18 ml of BCYEα (3). The test isolates were grown on BCYEα plates (1) at 35°C in a humidified environment. The colonies were suspended in distilled water, and the turbidity was adjusted to that of a no. 1 McFarland standard (10⁸ CFU/ml). A replicator device was used to inoculate 1 μl of the suspension onto antimicrobial agent-containing BCYEα plates, to give a final inoculum of 10⁴ to 10⁵ CFU/spot. Strain ATCC 33152 (Philadelphia) and a L. pneumophila SG-1 strain were used as controls. Up to 52 strains were inoculated onto each plate, which always included the control strains. In addition, two antimicrobial agent-free control plates, one with BCYEα and the other with BCYEα not supplemented with cysteine, were also included. All plates were incubated at 35°C in a humidified environment for 3 days.

In order to assess the effect of charcoal-containing medium, the MICs for 20% of the isolates (50 L. pneumophila isolates) were determined in BCYEα with decreasing charcoal concentrations (100, 75, 50, and 25% of the charcoal content in the original BCYEα medium [3]) by previously described methods (7).

From the point of view of resistance monitoring, no differences in susceptibility with respect to year of isolation, geographical origin, SGs, or subgroups were found. Despite the previously documented differences in susceptibility between clinical and environmental isolates (8), this study did not show differences, probably because the 20 environmental isolates tested were epidemiologically related to patients from whom the corresponding clinical isolate was also tested.

The antimicrobial susceptibility testing results for the total test panel of Legionella spp. are shown in Table 1. By the broth microdilution method, the most active agents were trovafloxacin (MIC at which 90% of isolates are inhibited [MIC₉₀], 0.0018 μg/ml), gemifloxacin (MIC₉₀, 0.003 μg/ml), and grepafloxacin (MIC₉₀, 0.007 μg/ml). Levofloxacin and clarithromycin showed similar activities (MIC₉₀s, 0.015 μg/ml) and were more active than ofloxacin and ciprofloxacin (MIC₉₀s, 0.03 μg/ml) and than erythromycin (MIC₉₀, 0.12 μg/ml). A completely different picture is seen when one considers the results obtained by the agar dilution method. Clarithromycin and levofloxacin appeared to be the most active compounds (MIC₉₀s, 0.12 μg/ml), followed by erythromycin and trovafloxacin (MIC₉₀s, 0.25 μg/ml), with the MIC₉₀s of all other quinolones tested being high (≥0.5 μg/ml).

TABLE 1.

MIC₅₀s, MIC₉₀s, and MIC ranges for 271 Legionella isolates determined by the microdilution method (BYEα medium) and the agar dilution method (BCYEα)

Antimicrobial agent	MIC (μg/ml)
	BYEα medium			BCYEα
	50%	90%	Range	50%	90%	Range
Gemifloxacin	0.003	0.003	0.0009–0.03	1	1	0.5–4
Trovafloxacin	0.0018	0.0018	0.0004–0.015	0.25	0.25	0.12–1
Grepafloxacin	0.007	0.007	0.0018–0.06	0.5	0.5	0.25–2
Levofloxacin	0.007	0.015	0.007–0.03	0.12	0.12	0.06–0.25
Ofloxacin	0.015	0.03	0.007–0.06	0.5	0.5	0.25–1
Ciprofloxacin	0.015	0.03	0.0018–0.03	0.5	0.5	0.12–1
Clarithromycin	0.007	0.015	0.0004–0.03	0.06	0.12	0.03–0.12
Erythromycin	0.06	0.12	0.03–1	0.12	0.25	0.03–0.5

It has been reported that the use of BCYEα results in 4- to 10-fold increases in the MICs compared with those obtained by tests that use BYEα medium (4). The activities of most antimicrobial agents are inhibited by BCYEα to a greater extent than by BYEα (5, 6). The degree of inhibition caused by BCYEα compared with that caused by BYEα was not the same for all the quinolones tested. While the ratio of the MIC₉₀ obtained with BCYEα to the MIC₉₀ obtained with BYEα medium for gemifloxacin, trovafloxacin, and grepafloxacin was more than 70, ratios for ciprofloxacin and ofloxacin were 16, and the ratio for levofloxacin was 8, which was similar to that for clarithromycin. Erythromycin was not affected by the use of BCYEα.

Table 2 shows the effect of decreasing the concentrations of charcoal in BCYEα on the MICs determined by the agar dilution method. The presence of charcoal in BCYEα has been shown to adversely affect the in vitro activities of some quinolones (2, 7). In this study, decreasing the charcoal concentration in the test media by up to 75% had no effect on the MIC₅₀s and MIC₉₀s of the two macrolides or levofloxacin. In contrast, for all the other quinolones tested, a decrease in the charcoal concentration of 75% resulted in decrease in the MIC₅₀s and MIC₉₀s of four- to eightfold. When the MICs of the macrolides and levofloxacin for each strain tested are compared, an increase in the charcoal concentration from 25 to 100% resulted in no effect or a twofold increase in the MIC. With ofloxacin and ciprofloxacin there was at least an eightfold reduction in the MIC for more than 70% of the strains tested when 25% charcoal instead of 100% charcoal was used, with the difference for <10% of strains being 16-fold. For the newer quinolones, gemifloxacin, trovafloxacin, and grepafloxacin, at least an eightfold decrease in MIC was detected for a similar proportion of the strains (≥70% of the strains). However, the percentage of strains for which there was at least a 16-fold difference was much higher (47% for gemifloxacin, 73% for trovafloxacin, and 38% for grepafloxacin) compared with percentage obtained in tests with the older agents.

TABLE 2.

Effects of various charcoal concentrations in BCYEα on the MIC₅₀s and MIC₉₀s for 50 isolates of L. pneumophila as determined by the agar dilution method

Antimicrobial agent	MIC (μg/ml) with the following % charcoal in BCYEα
	100%		75%		50%		25%
	50%	90%	50%	90%	50%	90%	50%	90%
Gemifloxacin	1	1	1	1	0.25	0.5	0.12	0.25
Trovafloxacin	0.25	0.25	0.12	0.12	0.06	0.06	0.015	0.03
Grepafloxacin	0.5	0.5	0.25	0.5	0.12	0.25	0.06	0.06
Levofloxacin	0.12	0.12	0.12	0.12	0.12	0.12	0.12	0.12
Ofloxacin	0.5	0.5	0.25	0.25	0.12	0.12	0.06	0.06
Ciprofloxacin	0.5	0.5	0.5	0.5	0.25	0.25	0.06	0.12
Clarithromycin	0.06	0.12	0.06	0.12	0.06	0.12	0.06	0.12
Erythromycin	0.12	0.25	0.12	0.25	0.12	0.25	0.12	0.25

If the MIC₉₀ obtained in tests with BYEα medium (0% charcoal) is compared with the MIC₉₀ obtained in tests with BCYEα (25% charcoal), it can be deduced that even low concentrations of charcoal have a marked effect on the activities of the newer quinolones (16-fold for trovafloxacin and 64-fold for gemifloxacin) but not on that of erythromycin, indicating that differences between the MICs of quinolones are due to charcoal binding and not to other differences between methods.

Laboratories should be aware of the fact that only the unbound fraction of an antibiotic has antibacterial activity and that new quinolones are profoundly affected by the binding to charcoal so that they can use test methods which reflect the true activities of new quinolones.