None of the mutant strains showed collateral resistance or sensitivity toward other classes of antibiotic indicating that the uL4 loop region mutations are specific for macrolide resistance ( figure S3). PAO1 laboratory strain mirrored in all cases the behavior of the ancestral wt clinical isolates ( figure 2a). Both +T and +VT mutant strains, in contrast, did not have a detectible effect on susceptibility towards either macrolide relative to the respective ancestral wt isolates neither with respect to the MIC nor to the post-MIC effect ( figure 2a). For ΔKPW, ΔRA and +G mutant strains, the difference in post-MIC effect relative to the ancestral strains was greater than 20-fold increasing from 16 μg/ml for the ancestral strains up to 512 μg/ml for the mutants ( figure 2a). For that reason, the post-MIC effect was a much better predictor of azithromycin susceptibility. For azithromycin, the MIC measurements were somewhat confounded by low level growth, which however, did not result in viable colonies when the post-MIC effect was evaluated after the end of the MIC assay ( figure 2a). Since aggregation and cell lysis could confound the MIC readings, we also determined the viability of bacteria after the MIC assay. Isolates ΔKPW, ΔRA and +G, compared with their respective ancestral wt isolates, showed a larger than 10-fold difference in MIC for erythromycin increasing from 64 μg/ml to 1024 μg/ml ( figure 2a). All further analyses were, therefore, performed in 50% LB. Using 50% LB resulted in a better separation between mutants and ancestors, less variability between experiments and overall lower MICs compared with CA-MHB ( figure 2 and S1b).
The asterisk denotes strains in our collection of clinical strains of P. Only sequences with alterations compared to PAO1 in otherwise highly conserved regions of uL4 and uL22 are shown. d) Alignments of 2909 uL uL22 protein sequences from P. Numbering refers to Pseudomonas aeruginosa. The loop region (shaded green) is highly conserved across species. c) Alignment of protein sequence of uL4 in 4 Gram-negative ( Pseudomonas aeruginosa, Escherichia coli, Burkholderia cepacia and Haemophilus influenzae) and 2 Gram-positive species ( Staphylococcus aureus and Streptococcus pneumoniae). Strains 10 (ΔKPW and ΔRA mutants), 99 (+G mutant), K (+T mutant) and 280 (+VT mutant), are ancestor isolates containing a wild type copy of the uL4 protein. Isolate LRJ04 (DK06 clone type) contains a 57-KPW-59 (ΔKPW) deletion, isolate 249x14C (DK06 clone type) contains a 68-RA-69 (ΔRA) deletion, isolate 102 (DK12 clone type) contains a glycine 65 (+G) insertion, isolate C (DK17 clone type) contains a threonine 64 (+T) insertion, and isolate 278 (DK36 clone type), contains a 189-VT-190 (+VT) duplication. Outlined squares or triangles mark the uL4 mutant isolate used. Hexagons mark the strains containing a wt uL4 used as control for the respective uL4 mutants within the specific clone type. Whole genome sequences were analysed to determine if the isolate contained a wt uL4 (circles and hexagons) or a mutant uL4 (squares and triangles). b) Emergence of uL4 mutants over time aligned with patient macrolide treatment. Circles indicate the position of mutations in the uL4 protein in isolates sampled from cystic fibrosis patients subsequent to antibiotic treatment. a) Localization and structure of the ribosomal uL4 protein in P. Figure 1 uL4 and uL22 protein mutations in clinical strains of Pseudomonas aeruginosa.