Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance

Background Cystic fibrosis (CF) patients have reduced mucociliary clearance resulting in recurring and chronic bacterial lung infections. Pseudomonas aeruginosa is one of the most common pathogens to colonize the airways of CF patients and can persist in the lungs for decades. CF patients infected with P. aeruginosa are treated with macrolides to inhibit quorum sensing, mucoidity and has additional immunomodulatory effects. However, according to the EUCAST committee, P. aeruginosa is not susceptible to macrolides leaving resistance mechanisms largely overlooked. Methods Using a modified susceptibility testing protocol, P. aeruginosa isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics. Quorum sensing related properties, alterations in proteome composition and ribosome subunits distribution were further analysed to characterize the effect of the uL4 mutations on the physiology of the bacteria. Findings Several uL4 mutations were identified in isolates from P. aeruginosa collections from various sources and geographical locations. Most of them mapped to the conserved loop region of uL4 and resulted in increased survival upon macrolide exposure. uL4 mutations did not negatively impact the physiology of the bacteria and greater concentrations of antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity. Proteome analysis revealed that pathways involved in ribosome adaptation displayed altered expression levels possibly to compensate for the uL4 mutations, which changed the subunit distribution of the ribosome. Interpretation Macrolides used against P. aeruginosa cause selection of macrolide resistant mutants which is a widespread - but uncharacterized - phenomenon. Using a modified susceptibility test, revealed that macrolides are indeed effective bacteriostatic antibiotics against P. aeruginosa and that this effect - along with macrolide-induced quorum sensing modulation - are drastically reduced in uL4 mutants. Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated especially when patients are treated with prolonged courses of macrolides. Funding Cystic Fibrosis Foundation, Independent Research Fund Denmark, Novo Nordisk Foundation Research in context Evidence before this study Macrolide antibiotics are readily prescribed as anti-inflammatory therapy against Pseudomonas aeruginosa infections in cystic fibrosis patients. This treatment strategy has largely overlooked the direct antimicrobial effect of this drug class on this pathogen, which is considered non-susceptible according to the European Committee on Antimicrobial Susceptibility Testing. This is because standardized antimicrobial susceptibility testing is sub-optimal for the quantification of macrolide minimum inhibitory concentrations due to the interference of the growth medium. Mutations in ribosomal RNA and increased efflux has been shown to reduce the effect of macrolides on P. aeruginosa, however, the involvement of ribosomal proteins has not been investigated in P. aeruginosa. Work done in other bacterial species such as Escherichia coli, Streptococcus pneumoniae, Legionella pneumophila and Neisseria gonorrhoeae has identified the ribosomal proteins uL4 and uL22 as targets for evolved macrolide resistance in vitro and in vivo. Especially the extended loop region of uL4 has been identified as important for macrolide susceptibility. We found no other studies published addressing the emergence of macrolide resistance through uL4 or uL22 mutations in P. aeruginosa. Added value of this study We present evidence that macrolide antibiotics used as anti-inflammatory agents and bacterial modulators against P. aeruginosa infection in cystic fibrosis (CF) patients cause the emergence of resistant strains through mutations in the ribosomal protein uL4. The importance of this finding is underlined by the identification of uL4 mutations in multiple strains not only within our strain collection comprised of CF associated isolates but throughout all available sequences of clinical P. aeruginosa isolates spanning several continents and different infection types. uL4 mutations resulted in significantly reduced susceptibility towards macrolide antibiotics with respect to bacteriostatic and quorum sensing modulation effects. The generation time in presence of macrolide antibiotics was unaffected in strains harbouring uL4 mutations, while it was reduced in wild type strains indicating a fitness advantage of the mutation. Implications of all the available evidence The uL4 mutants identified in this study were significantly more resistant towards macrolide antibiotics than strains harbouring a wild type uL4 hereby revealing that not only are macrolides effective as antimicrobial agents against P. aeruginosa but also that the imposed selective pressure causes resistant mutants to arise through mutations in the ribosomal uL4 protein. These mutations are also present but uncharacterized in other collections of clinical P. aeruginosa isolates from patient groups who typically receive long courses of macrolide treatment. The effect of the uL4 and possibly other hitherto unknown mutations on macrolide susceptibility can be determined via modification of the standard susceptibility testing protocol. Along with evidence describing other types of macrolide resistance mechanisms in P. aeruginosa such as rRNA modification and increased efflux activity, our results demonstrate that macrolide resistance development is widespread in P. aeruginosa and macrolide antibiotics should be considered as antimicrobial agents against P. aeruginosa with the same precautions being taken to avoid resistance development as for any other antimicrobial agent.