Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway

Abstract

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues.

Figure 1. Mutation in lasR increases denitrification, and leads to the buildup of the toxic metabolite NO·.

(A) The reactions that comprise the dissimilatory denitrification pathway, or the serial reduction of NO₃ ⁻ to nitrogen gas, in bacteria. (B) Growth of the wild-type P. aeruginosa strain PA14 and its derived lasR mutant PA14-lasR::Gm in a chemically defined medium (PN minimal medium) with and without NO₃ ⁻ supplementation, under low oxygen mass-transfer conditions. Results representative of three separate experiments. (C) Nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) concentrations in shaken cultures of the indicated strains grown with LB and 5 mM added nitrate. Results shown are the averages of technical triplicates ±s.d. and are representative of two separate experiments. Slopes for lines fit to each dataset between 4 and 6 minutes were significantly different with p<0.001 for [NO₂ ⁻] and p<0.02 for [NO₃ ⁻]. Also shown for reference (inset) are concurrent growth curves for each strain; results representative of two separate experiments. (D) Rates of degradation of added NO· from a mixture of NO· donors (DEANO and ProliNO) by cells pre-grown in a sealed chamber in LB prior to donor addition. Results shown are average ±s.d. for technical duplicates and are representative of two separate experiments. Slopes for lines fit to each dataset between 25 and 100 seconds were significantly different (p<0.02). (E) NO· concentrations in sealed, stirred LB cultures of the indicated strains during growth with 50 mM added NO₃ ⁻. Results are average ±s.d. of three separate experiments. Slopes for lines fit to each dataset between 4 and 9 minutes were significantly different between wild-type and lasR mutant cultures (p<0.02). For all experiments shown, similar results were obtained in at least two separate experiments with the Patient 1 early isolate and its derived lasR mutant (described in Table 1), and complementation with the lasR gene on a plasmid restored wild-type phenotypes (not shown).

Figure 2. Mutation in lasR confers increased susceptibility to nitrosative stress, including acidified NO₂ ⁻.

(A) Growth rate of PA14 versus PA14-lasR::Gm in LB in the presence and absence of two amounts of added NO· donor SPER-NO, transiently generating the indicated concentrations of NO·. Each result representative of at least two separate experiments. (B) Disk diffusion diameters of the indicated strains and isolates on LB agar buffered to pH 6.5, with disks containing 100 µmol of NaNO₂, then incubated for 24 hours at 37°C under aerobic conditions. Average ±s.d. for triplicate experiments. Similar results were obtained with PA14ΔlasR, and complementation with a wild-type copy of lasR on a plasmid restored wild-type phenotypes to lasR mutants (not shown). (C) Spontaneous sectors displaying the lasR phenotype (metallic surface sheen and autolytic flattening, indicated by black arrows) arise during agar surface growth for 1 week of PA14-derived strains with transposon insertions in genes in the periplasmic NO₃ ⁻ reductase gene cluster (top, genes PA1173 and napA) but not from those with insertion in genes in the membrane-bound NO₃ ⁻ reductase gene cluster (bottom, genes narJ and narK2). Also visible around the lasR mutant sectors is the blue pigment pyocyanin, which is produced at higher levels by lasR mutant PA14 than by wild-type cells upon extended incubation . Results representative of four separate experiments. Complementation with a wild-type copy of lasR on a plasmid restored wild-type phenotypes to lasR mutants isolated from sectors (not shown).

Figure 3. lasR mutant P. aeruginosa growth is altered during co-culture with S. aureus, apparently due to detoxification of NO·.

(A) Cells in a lawn of lasR mutant PA14 growing near a disk containing hemoglobin, which stoichiometrically scavenges NO· (as opposed to the catalytic effect of S. aureus colonies in B–C), do not display the autolysis of cells more distant from the disk, as shown in a photograph from above with illumination from above (upper) and below (lower). (B) Clinical isolates of lasR mutant P. aeruginosa (black arrows) and S. aureus (white arrows) grown together on LB agar with 400 µM added KNO₃, with lasR colony autolysis and resulting translucency indicated through transillumination of the agar plate. Black arrows indicate areas of P. aeruginosa lysis and/or sheen, white arrows indicate colonies of S. aureus, and black arrowheads indicate where lasR colony autolysis is relieved in the presence of S. aureus. (C) Co-culture as in (B) except with laboratory strain PA14-lasR::Gm grown with wild-type S. aureus Newman strain (colored orange in silico for clarity) after inoculation at a cell ratio of 50∶1. Arrows as in (B). (D) Ratios of cell counts of P. aeruginosa lasR versus wild-type after inoculation of static cultures in liquid LB with 400 µM added KNO₃ with equal numbers of each P. aeruginosa strain followed by growth for 48 hours in the presence and absence of equal cell numbers of the indicated S. aureus strains. Results are averages ±s.d. for triplicate counts and are representative of three separate experiments. Total final cell count was similar in each experiment.

Figure 4. lasR mutant P. aeruginosa strains and isolates exhibit lower rates of oxygen utilization and resistance to paraquat, tobramycin and ciprofloxacin.

(A) Change in oxygen concentration during stirred incubation of washed cells of the indicated strains resuspended at equivalent cell densities in LB with 400 µM KNO₃ at 37°C. Average of 3 experiments ±s.d.; results representative of 3 separate experiments. Slopes for lines fit to each dataset between 1 and 5 minutes were significantly different (p<0.04). Complementation of lasR mutants with a wild-type copy of lasR on a plasmid restored wild-type phenotypes (not shown). The difference was no longer statistically significant in the absence of added KNO₃ (LB was shown previously to contain approximately 23 µM NO₃ ⁻ ; not shown). (B) Fluorescence yields generated by adding a saturated DMSO solution of hydroethidine (HE), a probe of superoxide concentration , for 5 minutes on lawns of the indicated strains (where plasR indicates complementation with a wild-type copy of lasR on a plasmid) grown on LB agar. Average ±s.d. of triplicates and representative of five separate experiments; similar results were obtained in liquid cultures and with clinical isolate pairs for Patient 1 (not shown). (C) Zone diameters of growth inhibition for the indicated clinical isolates and strains by disks containing 1 µmol of paraquat after 24 hours' incubation in air at 37°C on LB agar with 400 µM KNO₃. Results shown are average ±s.d. for triplicates and are representative of >10 separate experiments. Complementation with a copy of lasR on a plasmid restored wild-type phenotypes to lasR mutants (data not shown). (D) As in (C), except with disks containing 3.75 µg of ciprofloxacin or 3 µg of tobramycin on MH agar and 400 µM KNO₃ (the lasR mutant strain tested for tobramycin susceptibility was PA14-L1, which does not contain an engineered aminoglycoside resistance gene). Average ±s.d. for triplicates. *, p<0.001 compared both with wild-type and the complemented mutant. No decreases in susceptibility were noted with disks of control antibiotics: carbenicillin, tetracycline, aztreonam, and polymyxin. Results with the unmarked deletion strain PA14ΔlasR were similar to those with the lasR mutants shown for both (C) and (D). (E) Tobramycin disk diffusion diameters for experiments as in 5d except with the indicated strains. Experiment at right compares the oxyRkatA-lasR mutant carrying an empty plasmid vector with the same strain carrying the same plasmid but with a wild-type copy of lasR, and on agar media containing 300 µg/mL carbenicillin for plasmid maintenance. Similar results were observed for disks of ciprofloxacin (not shown). Results shown are averages ±s.d. for triplicates.

Figure 5. Resistance of lasR mutant P. aeruginosa to tobramycin and ciprofloxacin is oxygen-dependent.

(A) Colony counts from serial slices of an agar-suspended culture with LB+400 µM KNO₃ containing 5 mM paraquat inoculated with equal numbers of cells of PA14 and a derived lasR mutant (PA14-lasR::Gm) and incubated for 48 hours. (B) The same experiment as in (A), except with 1 µg/mL tobramycin instead of paraquat, and using PA14-L1 (because this lasR mutant lacks an aminoglycoside resistance cassette). (C) The same experiment as in (B), except with 0.25 µg/mL ciprofloxacin and with PA14-lasR::Gm. All results representative of at least 3 independent experiments. No differences in cell density were noted in the absence of antibiotics or paraquat under these conditions after 48 hours of growth (not shown), in agreement with liquid growth findings .

Figure 6. A model for metabolic changes in CF-adapted lasR mutant isolates of P. aeruginosa.

According to the model, patients are initially infected with environmental isolates carrying wild-type copies of the lasR gene (left). These isolates have relatively high utilization of oxygen (activities indicated by the sizes of the green arrows) and lower utilization of nitrogen oxides (NO_x). Selective pressures encountered in the host, including abundant host NO₃ ⁻ and amino acids (AA), low host NO·, the presence of other bacterial species that metabolize NO·, reduced O₂ concentrations, and treatment with β−lactams or antibiotics that generate ROS, favor the emergence of lasR mutant isolates with higher utilization of nitrates and lower utilization of oxygen. This metabolic shift confers a growth advantage in the nutrient conditions in the CF airway, including abundant NO₃ ⁻, and relative resistance to the antibiotics used most frequently to treat CF patients.