Several clinical studies have validated the beneficial effects of long-term treatment with azithromycin in CF patients chronically infected with Pseudomonas aeruginosa (P. aeruginosa) and its usage has progressively entered clinical guidelines (5-9). The efficiency of azithromycin has been attributed to its anti-inflammatory and anti-virulence properties including e.g. inhibition of motility, quorum sensing and protease activity (9, 10, 21, 22). Although P. aeruginosa is considered naturally resistant to macrolides, in vitro susceptibility was previously demonstrated upon testing in alternative media including eukaryotic cell medium RPMI 1640 (supplemented or not with FCS) or serum-supplemented CA-MHB, suggesting that macrolides might additionally exert direct antimicrobial activity on P. aeruginosa (10,11). The differences observed in phenotypic susceptibility to azithromycin depending on the test medium have been ascribed to increased outer-membrane permeability and decreased expression of efflux pumps in the presence of RPMI 1640 or serum, leading to enhanced azithromycin accumulation inside the bacteria (11). The authors therefore proposed that azithromycin MIC testing of P. aeruginosa CF isolates in RPMI 1640 could routinely be included in microbiological diagnostics (11).
Extending previous findings, we demonstrate here that in 17 out of 29 (59%) MDR P. aeruginosa CF isolates, MIC values were low when tested in RPMI supplemented with FCS, ranging from 0.25-8 µg/ml. In contrast, in vitro resistance with high MICs to azithromycin even in RPMI/FCS as found in 12 out of 29 MDR isolates in the present study could be explained by mutations in the 23S rRNA which are frequently detected in CF isolates. Indeed, Mustafa et al. observed mutations in domain V of 23S rRNA in 43% of CF P. aeruginosa isolates while mutations were absent in 48 tested strains derived from patients suffering from hospital acquired pneumonia (12). Thus, testing in RPMI/FCS might be an option to identify P. aeruginosa resistance caused by 23S rRNA mutation.
However, although RPMI and CA-MHB supplemented with FCS have been suggested to more closely resemble the eukaryotic environment and therefore to constitute the better test medium, these media do not necessarily reflect the particular milieu in the airways of CF patients. It was suggested that the physiological situation of CF airways might be better mimicked by SCFM which imitates the specific nutritional composition and ion concentrations of CF sputum and is therefore a less rich medium compared to RPMI (13). We therefore evaluated susceptibility of MDR P. aeruginosa in this medium. Of note, azithromycin MICs were consistently ≥256 µg/ml in SCFM in all P. aeruginosa clinical isolates as well as in reference strain PAO1, arguing against a direct antimicrobial effect of azithromycin in the airways of CF patients. Macrolides are protonated in acidic environments going along with reduced activity. SCFM was used with a pH of 6.8, which might interfere with activity, yet, a slightly acidic pH in airways of CF patients is well documented in the literature (13). As a conclusion, our data therefore do not support routine azithromycin MIC assessment in CF clinical isolates using RPMI/FCS, as proposed previously (12). This study shows that for CF isolates and macrolides in vitro testing is associated with a high level of uncertainty. SCFM, sputum adapted medium, might be more appropriate for antimicrobial susceptibility testing than conventional broth. This notion is also supported by a recent publication of Diaz Iglesias et al who investigated antibiotic susceptibility, biofilm formation and metabolic activity using different media (23).
Our results do not substantiate a direct antimicrobial effect of azithromycin on P. aeruginosa when tested in SCFM, a medium that represents the CF environment. Even though we could not detect an antibacterial effect of azithromycin on P. aeruginosa we could replicate the occurrence of a resistance mechanism, specifically the mutation of the 23S rRNA gene, in MDR isolates of CF patients. This might be interpreted as a hint that azithromycin has an effect on P. aeruginosa in vivo other than blocking bacterial growth. Indeed it has been demonstrated that azithromycin causes a modulation of protein expression instead of a complete block which would be needed for an antibacterial effect. This effect seems to be pronounced for genes involved in quorum sensing (22, 24, 25). Since this effect seems to take place at concentration far below the measured MIC and the quorum sensing system is involved in biofilm formation, virulence and immune modulation (26, 27) it is plausible that this drives the development of azithromycin resistance in P. aeruginosa in vivo. Since azithromycin does not seem to be effective in CF patients uninfected with P. aeruginosa (28) this indicates that azithromycin has indeed an effect on P. aeruginosa in vivo. Therefore it would be interesting to study the effect of azithromycin in patients with azithromycin resistant strains compared to patients with non-resistant strains. In this context testing for azithromycin resistance using RPMI might be a useful tool.
In conclusion our data do not support the implementation of azithromycin MIC assessment of P. aeruginosa CF isolates in routine microbiological diagnostics as suggested previously (12). The results warrant further assessment of the in vivo efficacy of azithromycin in the subgroup of MDR P. aeruginosa infected CF patients in prospective clinical trial.