Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins

Abstract

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections.

Fig. 1

Large chromosomal deletions in pyomelanin-producing P. aeruginosa clinical isolates. A. Location (in red) relative to sequenced P. aeruginosa strain PAO1 of chromosomal deletions in five brown P. aeruginosa mutants compared with wild-type precursors. B. Close-up 1A, indicating the deleted region for each pyomelanin-producing mutant in this study. The blue bars surround the genes deleted in all the pyomelanin-producing mutants. The yellow rectangle indicates hmgA gene. The length of the deletion and the deleted genes are indicated. The direct repeats putatively involved in the deletion are detailed (PCR-verified repeats are bold-typed). C. Name and function of the genes deleted in all the pyomelanin-producing mutants.

Fig. 2

Metabolic changes in pyomelanin-producing mutants of P. aeruginosa. A. Proportion of living bacteria after 24 h of incubation in minimal media M63 supplemented with 0.05% of tyrosine as the sole C-source as described before (see Supplementary Information). Ratio of means ± SEM from at least 3 independent experiments; two-sided Student’s t-tests; NS, P > 0.05; ***, P < 0.001. B. Bacterial growth on M63 plates supplemented with 0.1% geraniol as the only C-source and incubated for 48 h at 37°C followed by 10 days at 30°C.

Fig. 3

Survival of the pyomelanin-producing mutants of P. aeruginosa in harsh environments. A. The minimal inhibitory concentrations of tobramycin were determined by Etest. B and C. The resistance to osmotic shock was determined in Luria-Bertani broth supplemented with 2M NaCl. Ratio of means ± SEM from at least 3 independent experiments; two-sided Student’s t-tests; NS, P > 0.05; ***P < 0.001.

Fig. 4

Kinetics of the early phase of biofilm formation of pyomelanin-producing mutants of P. aeruginosa and their wild-type parents. Measures were done by the BioFilm Ring Test® (BioFilm Control). Means of BFI (BioFilm Indice) for four replicates from two independent experiments. Standard deviations were not displayed for ease of reading. Statistical differences at 4 h were calculated with two-sided Student’s t-tests; **P < 0.01; ***P < 0.001.

Fig. 5

Brown mutants can compete with their wild-type parents in coculture despite lower fitness in monoculture. Means 6 SEM from at least 3 independent experiments; two-sided Student’s t-tests; NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. A. Growth in Luria-Bertani broth under aerobic conditions at 37°C under shaking at 150 r.p.m. B. Growth in Luria-Bertani broth supplemented with 0.4 mM KNO3 under anaerobic conditions without shaking. C. Relative proportion of pyomelanin-producing mutants after 24 h of coculture with their isogenic parents in biofilm under anaerobic conditions as described elsewhere (Waite and Curtis, 2009). D. Inhibitory activity of 30 μl of the supernatant of an overnight culture of pyomelanin-producing mutants in MH broth containing 1 mg/l of the SOS response inducer mitomycin C against a culture on MH agar of their wild-type parent (up). Inhibitory activity of 30 μl of the supernatant of a 12-h culture of the wild-type parent in MH broth containing 1 mg/l of mitomycin C against a culture on MH agar of their pyomelanin-producing derivative (down). As a control, 30 μl of MH broth containing 1 mg/l of mitomycin C incubated overnight had no inhibitory activity against any of the strains and isolates shown on agar culture (data not shown). The 2 variant colonies growing in the inhibition spot of the C_wt were found – after an overnight subculture on MH agar – as susceptible to the C_pm supernatant as their parent (data not shown).