Fig 1.
Aztreonam resistance is associated with variation in NalD across independent clinical isolates from acute P. aeruginosa infection.
(A) Phylogenetic tree of isolates from acute infections of cancer patients reconstructed from core genes, including the type strains PA14, PAO1 and PA7. The minimal inhibitory concertation (MIC) of the aztreonam varies significantly across the phylogenetic tree, showing it is not a phylogenetically conserved trait. (B) NalD protein is the only protein in the mexAB-oprM efflux pathway that is strongly associated with aztreonam MIC in a rank sum test (***, p = 0.005). The table on the bottom shows the p-values for rank sum tests conducted on other proteins known from the mexAB-oprM efflux system and its regulatory pathway. (C) Expanding the analysis to all the transcriptional regulators encoded by the P. aeruginosa genome revealed 30 candidates whose protein sequences variation were associated with aztreonam MIC (see S2 Table), but NalD remained the strongest correlate.
Fig 2.
P. aeruginosa infection of a cancer patient hospitalized to receive hematopoietic cell transplantation evolved resistance to aztreonam in the course of therapy.
(A) Timeline shows clinical events: conditioning regimen (myeloablation), hematopoietic cell infusion, location in the hospital (bone marrow transplantation unit [BMT] or intensive care unit [ICU]), the period of neutropenia, antibiotics administered, the day (relative to the day of transplant, day 0) and body site of origin (rsw: rectal swab; spt: sputum; bld: blood) of the eight P. aeruginosa isolates analyzed here. (B) Antibiotic resistance profiles of blood isolates measured by the clinical microbiology laboratory as the infection progressed; the profiles informed clinicians that the infection was multi-drug-resistant but also that it was initially sensitive to aztreonam (isolates D+3bld, D+4bld and D+5bld). As the disease progressed to become life-threatening sepsis, the patient was transferred to the ICU and was given aztreonam; however, the isolate D+7bld demonstrated resistance to aztreonam. The patient died on day +8.
Fig 3.
Whole-genome sequencing of eight P. aeruginosa isolates pinpointed the mutation NalDF198L responsible for aztreonam resistance detected one day before the patient died.
(A) A genome-based phylogenetic tree shows that the eight sepsis isolates are highly related to each other compared to other clinical isolates and type strains. (B) Genomic analysis revealed that the aztreonam-resistant isolate (D+7bld) had only two unique variations compared to aztreonam-sensitive isolates. Vertical dashed lines highlight the common presence of a given variation across multiple isolates. (C) Aztreonam MIC confirmed the clinical laboratory results for the eight sepsis isolates. (D) Experimental validation in PA14 showed that mutation in NalD but not in peg.4653 (PA14_50010) confers aztreonam resistance. “-”and “+” denote absence or presence of mutation found in D+7bld; “Δ” denotes deletion of the 10th alpha helix in NalD protein. (**, p-value<0.01).
Fig 4.
NalD mutation contributes to a general signature of antibiotic resistance.
(A) A signature of aztreonam resistance obtained with LASSO regression shows only two transcriptional regulators, including NalD, and explains >60% of the variation in aztreonam MIC. The coefficients have units of fold-change. (B) Antibiotic inhibitions zones were measured using the disk assay for 8 antibiotics from several classes. The inhibition zone indices (shows as normalized areas of inhibition disk) show that the 8 sepsis isolates are resistant to multiple antibiotics. A resistance index computed from combining the negative values of the inhibition zone indices shows that the 8 sepsis isolates rank higher in multi-drug resistant than any other isolate tested. (C) The signature of multi-antibiotic resistance has six transcriptional regulators, including NalD, and explains >80% of the variation in the multi-drug resistance index. The coefficients have units of integrated fold-change across all the 8 antibiotics.
Fig 5.
Molecular details of mechanism of acquired aztreonam resistance in the sepsis patient infected with P. aeruginosa.
(A) 3D structure of NalD dimer (PDB id: 5daj) with residue 198F (phenylalanine) shown as stick model. One copy of NalD is rainbow colored, while the other is in gray. The 10th helix is shown in red. (B) A closer look of residue 198F and possible interaction with two nearby aromatic residues, 89Y (tyrosin) and 205W (tryptophan). Those three residues are close together and could have aromatic interactions or a possible hydrogen bond (3.2 Å) between 198F and 89Y. 198F also aligns well with 205W, both of which have ring structure and could form a displaced pi stacking that stabilizes the NalD structure (3). (C) Prediction of mutation effect based on NalD structure. The mutation F198L would widen the distance between carbon groups and lose the pi-pi interaction, which could ultimately destabilize NalD dimerization. (D) Wild-type NalD dimer represses transcription of mexAB-oprM operon. (E) The mutation NalDF198L could interfere with dimerization, and de-repress transcription. (F) MexAB and OprM form an anti-porter system that exports aztreonam, increasing resistance [29].
Fig 6.
RNA-seq analysis shows that the aztreonam-resistant isolate D+7bld up-regulates the mexAB-oprM efflux system and attenuates response to aztreonam stress.
(A) We compared the transcriptomes to reference isolate D-3rsw at 0μg/mL of aztreonam and found hundreds of differentially expressed genes. (B) The up-regulation of the mexAB-oprM efflux system in D+7bld supported that the NalD mutation released the transcriptional repression of mexAB-oprM. (C) The transcriptome of D+7bld at 4μg/mL aztreonam resembled the transcriptome of the aztreonam sensitive D-3rsw at half that dose (2μg/mL), confirming that the aztreonam resistance allows the strain to sustain higher levels of antibiotic challenge. azt, aztreonam. *, p-value<0.05.
Fig 7.
Validation of metabolic model using experimental growth data.
(A) Experimental growth curves of both sensitive (D-3rsw) and resistant (D+7bld) P. aeruginosa strains at various aztreonam concentrations (μg/mL). (B) Steady state distribution of biomass flux predicted from metabolic models for the same experimental conditions (except for D-3rsw at 4μg/mL aztreonam). PDF: probability density function. (C) Comparison of the measured growth rate (blue bars) with the model predictions (red bars). The measured growth rates were obtained by fitting an exponential growth model to the exponential phase of the growth curves shown in (A). The predicted growth rates were approximated from the mean of the biomass flux distributions shown in (B). The growth rates are relative to that of the sensitive strain in the absence of aztreonam (S0). Error bars: standard deviation.
Fig 8.
Evolved metabolic-level adaptations in the aztreonam-resistant strain.
(A) Metabolic flux changes relative to the reference condition S0 (D-3rsw, no aztreonam). (B) Venn diagram showing the overlap between reactions whose flux levels significantly altered by aztreonam alone, mutations alone, and their combination. Among all 48 reactions constitutively modulated by mutations (i.e., constitutive mutation effects), 30 are directly related to aztreonam resistance (because aztreonam can induce their responses) and 18 are indirectly related. (C) Grouping of the 48 constitutively modulated reactions by pathways they belong to. (D) Expression of eda serves as a bottleneck to the flux through the Entner-Doudoroff (ED) pathway (red arrows). The connected Embden-Meyerhof-Parnas (EMP) pathway (green arrows) and pentose phosphate (PP) pathway (blue arrows), as well as the relative expression changes of the major genes in the three pathways (heatmap) are also shown. Tpi: triose phosphate isomerase; fba: fructose-1,6-biphosphate aldolase; fbp: fructose-1,6-biphosphatase; pgi: glucose-6-phosphaate isomerase; zwf: glucose-6-phosphate dehydrogenase; pgl: 6-phosphogluconolactonase; edd: phosphogluconate dehydratase; eda: 2-dehydro-3-deoxy-phosphogluconate aldolase; DHAP: dihydroxyacetone phosphate: FDP: D-fructose-1,6-biphosphate; F6P: fructose-6-phosphate; G6P: glucose-6-phosphate; PGL: 6-phospho-D-glucono-1,5-lactone; 6PG: 6-phospho-D-gluconate; KPDG: 2-dehydro-3-deoxy-6-phospho-D-gluconate; G3P: glyceraldehyde 3-phosphate; PYR: pyruvate.