Acquired resistance to macrolides inPseudomonas aeruginosafrom cystic fibrosis patients

Abstract

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that , considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth.We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP).Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain.Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

The microbiome in bronchiectasis

“…The impact of inhaled antibiotics on the microbiome has not been investigated. Ideally, the impact on the lung microbiome by all the commonly used antibiotics to treat respiratory infections in bronchiectasis needs to be investigated [60].…”

HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients

“…Consistent with this, all 2011 isolates carried mutations in gene PA4280.2, which encodes the 23S ribosomal subunit. In fact, isolate 2011_33 carried an A2044G substitution, whereas the remaining isolates from 2011 carried a C2597T mutation, both located in the secondary structure of domain V of the ribosomal RNA gene and previously reported to confer macrolide resistance (32, 75). Thus, macrolide resistance in coexisting CFD 2011 isolates was acquired by distinct mutations in the same gene.…”

“…In order to investigate the molecular bases of the antimicrobial resistance observed in CFD isolates, we explored the acquisition of mutations in a set of 168 chromosomal genes, here defined as the resistome, which have been described to be involved in P. aeruginosa antibiotic resistance mechanisms (27, 28, 32). Thus, using the 1991 genome as reference, we analyzed the distribution of a total of 5710 SNPs and 1078 indels accumulated in a period of 20 years of infection, which we have previously detected in the collection of isolates by WGS analysis (24).…”

“…From the beginning of 2004 to 2012, patient CFD received systematic long-term treatments with azithromycin combined with other antipseudomonal agents. Although macrolide resistance is frequent among CF isolates, only two reports describe the emergence of macrolide resistance in vivo (32, 75). As shown in Fig.…”

“…In order to correlate the documented resistance genotypes with the observed resistance phenotypes, single-nucleotide polymorphisms (SNPs) and indels (1- to 10-bp insertion/deletion mutations) for each isolate obtained from the previous study (24) were filtered based on an exhaustive literature review (27, 28). We also added PA0668.4, PA4280.2, PA4690.2 and PA5369.2 genes to the list, affecting macrolide resistance (32). Thus, we obtained a set of 168 genes known to be related to antibiotic resistance in P. aeruginosa .…”