Telithromycin has been the first ketolide to be approved for clinical use, specifically developed for treatment of community acquired respiratory tract infections (CARTI) in order to overcome the spreading of resistance to macrolides among pneumococci 12. In comparison to macrolides, telithromycin shows notable improvements in antimicrobial and pharmacokinetic properties, even if it shares with macrolides the same bacterial target, represented by the ribosome 2. Particularly, telithromycin has significantly lower minimum inhibitory concentration (MIC) than the macrolide antibiotics for many gram-positive organisms, i.e Streptococcus. pneumoniae that shows, at the ribosome, a dual-site binding to the drug 3.

Telithromycin is a concentration-dependent antibiotic, thus the concentrations achieved at the infection site are recognized as an important determinant of efficacy 4. Due to the ability to penetrate into white blood cells and being characterized by high penetration rate, it may be delivered to sites of infections and reaches elevated concentrations in several tissues, particularly in the respiratory tract, comparable to those of macrolides and superior to β-lactams 56. H. influenzae, a major cause of CARTI 78910, demonstrates relatively good in vitro susceptibility to macrolides, azalides and ketolides, which show a unimodal MIC distribution and low prevalence of high level resistance when defined by current Clinical and Laboratory Standards Institute (CLSI) breakpoints 11.

Macrolides and azalides are currently recommended for treatment of community acquired pneumonia and acute exacerbations of chronic bronchitis, with the antipneumococcal fluoroquinolones, such as levofloxacin and moxifloxacin, as alternative agents in the most severe cases 1213141516.

Due to the fact that the bactericidal activity of macrolides and ketolides is related to the level of drug concentration in the infected tissue 1516, the evaluation of antibacterial activity of concentrations achievable in vivo, particularly in epithelial lining fluid (ELF), against H. influenzae, which is one of the most common pathogen of these anatomical districts, may provide further information on telithromycin activity. Carbon dioxide has been proved to affect antibacterial activity of macrolides and ketolides, when assessed by determination of MIC values 17181920. Influence of CO₂ on bactericidal activity of ketolides and macrolides as measured by time kill curves has been less investigated.

The present study aimed mainly to evaluate the antibacterial activity of telithromycin concentration achievable in ELF against H. influenzae clinical isolates with different pattern of resistance, in comparison with azithromycin and clarithromycin and the influence of CO₂ incubation on activity of telithromycin and macrolides.

Antibacterial activity of azithromycin, clarithromycin and telithromycin against H. influenzae expressed as MIC and MBC values and rate of susceptibility is depicted in Tables 1 and 2, where data obtained by incubating bacteria at ambient air or at 10% CO₂ are summarized. Telithromycin in ambient air showed activity against all the tested H. influenzae strains, with MIC values ranging from ≤ 0.03 to 4 mg/L, and susceptibility rates of 100%, similar to the azithromycin rates.

Generally, MICs and MBCs against β-lactamase negative H. influenzae strains were lower in absence of CO₂ for all the tested antibiotics, with a decrease from 2 to 4 fold in respect to the CO₂ incubation, being MIC₅₀/MIC₉₀ 2/8 and 1/2 mg/L for telithromycin, 8/16 and 2/8 mg/L for clarithromycin, 2/4 and 1/2 mg/L for azithromycin, after incubation with or without CO₂, respectively.

The MBCs of telithromycin were closer to the MIC values with respect to the two comparators. MBC₅₀/MBC₉₀ were 4/16 and 1/4 mg/L for telithromycin, 8/64 and 4/16 mg/L for clarithromycin, 4/8 and 1/4 mg/L for azithromycin after incubation with or without CO₂, respectively. In absence of CO₂, all the strains were fully susceptible to the study drugs (100%). After incubation in CO₂, the susceptibility rate was generally decreased (from 82% of telithromycin to 91% of clarithromycin), showing an interfering effect of this particular medium. The non-susceptible strains were included into the I (Intermediate) category, with the exclusion of 3 strains resistant to telithromycin. Similar results were observed for β-lactamase positive H. influenzae strains with MIC₅₀, MIC₉₀, MBC₅₀ and MBC₉₀ equal to those observed for β-lactamase negative strains. The microbiological activity of telithromycin was not significantly affected by β-lactamase production. Also in this case, the addition of CO₂ to the medium influenced the microbiological results, in terms of MIC and MBC values and susceptibility rates: 100% of susceptibility in open air for all the tested drugs, 87 and 89% in CO2 respectively for telithromycin and macrolides.

The 10 BLNAR strains were fully susceptible to the antibiotics in open air medium. In CO₂ atmosphere, MIC and MBC were higher in presence of CO₂ for all the tested antibiotics; the activity of telithromycin and clarithromycin was slightly superior to that of azithromycin being the susceptibility rate 90%, 90% and 80%, respectively.

Results obtained in time-kill curves for azithromycin, clarithromycin and telithromycin against β-lactamase-negative, β-lactamase-positive and BLNAR H. influenzae are shown in Figures 1, 2, 3 and in Tables 3, 4, 5. Bactericidal activities of telithromycin, clarithromycin and azithromycin against H. influenzae were similar, independently from the pattern of resistance. All these drugs were fully bactericidal after 12 hours at concentration of 4 × MIC and after 24 hours at the highest concentrations (2–4 × MIC, and ELF) against β-lactamase-negative strains (Figure 1, Table 3). Telithromycin and clarithromycin were bactericidal after 12 hours of incubation also at ELF concentration, while at the same time, azithromycin was bactericidal at 2 × MIC (Figure 1).

Similar trends in bactericidal activities were observed for β-lactamase producer and BLNAR strains (Figures 2 and 3, Tables 4 and 5).

Incubation of the bacterial culture in open air or in CO₂ seemed to slightly influence the killing curves, as, globally, no marked differences in bactericidal activity were observed between incubation in the presence or absence of CO₂.

The in vitro activity of telithromycin, the first ketolide developed for clinical use, has been widely evaluated in international and local studies, demonstrating a spectrum of activity that encompasses the key respiratory pathogens, including H. influenzae. As the bactericidal activity of macrolides and ketolides is related to the magnitude of drug concentration at the site of infection, it is instrumental the evaluation of antimicrobial activity of concentrations achievable in vivo at the bronchial tree. Thus the present study assessed the comparative in vitro bacteriological activity and the killing kinetics of telithromycin, azithromycin and clarithromycin against H. influenzae at concentrations multiple of the MIC and equal to ELF.

Telithromycin in ambient air showed activity against all the tested H. influenzae strains, with susceptibility rates of 100%, similar to azithromycin. The respective MBCs were from 2 to 4 fold higher than the MIC, generally lower than the comparators. Previous in vitro studies have already showed that the in vitro potency against H. influenzae of telithomycin is similar to azithromycin, considered the most active macrolide against this pathogen, and superior to clarithomycin 7112627282930313233.

The incubation in carbon dioxide affected the antibacterial activity of all the tested antibiotics, causing a notable increase in MICs and MBCs, resulting in a decreased rate of susceptibility among H. influenzae strains.

Susceptibility testing of respiratory tract pathogens is often performed in a CO₂ environment to ensure that the bacteria grow faster; however in this ambient the pH of the test medium decrease and macrolides and telithromycin activity seems adversely affected by this pH decrease. Thus the results of our study show that telithromycin susceptibility should be tested in ambient air, as well as that of macrolides. Notably, other recent in vitro studies have highlighted the adverse impact of CO₂ on susceptibility testing of telithromycin in key respiratory pathogens including H. influenzae 171819.

Few studies have evaluated the bactericidal activity of telithromycin alone or in comparison with macrolides against respiratory pathogens, and in particular against H. influenzae 83233. Our data confirm the results of these studies, indicating that the bactericidal activity of telithromycin is mainly evident at concentrations as high as twice and four times the MIC. For all the tested antibiotics the killing of H. influenzae was not affected by different resistance patterns of the strains included into the study. When MIC concentrations were considered, bactericidal activities of the studied drugs were quite similar, with azithromycin showing a more rapid killing at 2 × MIC. However, although not fully bactericidal, activity of telithromycin against H. influenzae seemed superior in comparison with that of clarithromycin and close to that of azithromycin. By contrast, when bactericidal activity of pulmonary concentrations was tested, telithromycin and clarithromycin showed a higher rate of killing than azithromycin on some strains, probably due to the inferior tissue distribution of this drug.

As both telithromycin and azithromycin are concentration dependent antibiotics, their penetration rate in site infection is an important determinant in predicting efficacy, thus MIC, breakpoints of macrolides, azalides and ketolides against this organism must be considered together with their levels in respiratory tissues and ELF. There is growing evidence that, even though MICs for macrolides against H. influenzae may be in the 'susceptible' range (as defined by current MIC breakpoints), in vivo bacteriological efficacy is poor against this pathogen, while PK/PD derived breakpoints seems to be more consistent with clinical outcomes 34. For both clarithromycin and azithromycin, the PK/PD breakpoint is five doubling dilutions lower than the CLSI breakpoint 35, while for telithromycin a breakpoint of 0.5 mg/L has been proposed 36. By considering these values, all the strains evaluated in the present study should be considered resistant to the two macrolides, while some of them should be classified as susceptible to telithromycin. However, determination of PK/PD breakpoints is usually based on serum concentrations chosen for optimal bacterial eradication and may not reflect the actual concentration at the site of infection, as occurs for the tested drugs which provide higher concentrations in the lungs than in other compartments, thus allowing higher susceptibility breakpoints when treating pulmonary infections.

In conclusion, telithromycin showed an in-vitro potency against H. influenzae comparable to azithromycin, with a superior in-vitro killing rate to clarithromycin at 2X-MIC against β-lactamase producers and BLNAR strains, and to azithromycin at ELF concentrations against β-lactamase negative strains. Against all strains, MICs and MBCs were lower in the absence of CO₂ for the tested antibiotics, showing an adverse effect of incubation in a CO₂ environment. The in-vitro potency together with the pharmacokinetic profile of the antimicrobial, should be considered in predicting its efficacy in the empirical therapy of CARTI.

LD received research funding from sanofi aventis.

LD participated in design and coordination of the study, interpretation of the data, and co-drafted the manuscript. EDV participated in study design, co-performed killing curves, analysis and interpretation of the data and co-drafted the manuscript. LN co-performed time kill curves and participated in analysis and interpretation of the data. ML participated in revising the manuscript.

All authors read and approved the final manuscript.