Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors.

Hypermutation due to DNA mismatch repair (MMR) deficiencies can accelerate the development of antibiotic resistance in Pseudomonas aeruginosa. Whether hypermutators generate resistance through predominantly similar molecular mechanisms to wild-type (WT) strains is not fully understood. Here, we show that MMR-deficient P. aeruginosa can evolve resistance to important broad-spectrum cephalosporin/beta-lactamase inhibitor combination antibiotics through novel mechanisms not commonly observed in WT lineages. Using whole-genome sequencing (WGS) and transcriptional profiling of isolates that underwent in vitro adaptation to ceftazidime/avibactam (CZA), we characterized the detailed sequence of mutational and transcriptional changes underlying the development of resistance. Surprisingly, MMR-deficient lineages rapidly developed high-level resistance (>256 μg/mL) largely without corresponding fixed mutations or transcriptional changes in well-established resistance genes. Further investigation revealed that these isolates had paradoxically generated an early inactivating mutation in the mexB gene of the MexAB-OprM efflux pump, a primary mediator of CZA resistance in P. aeruginosa, potentially driving an evolutionary search for alternative resistance mechanisms. In addition to alterations in a number of genes not known to be associated with resistance, 2 mutations were observed in the operon encoding the RND efflux pump MexVW. These mutations resulted in a 4- to 6-fold increase in resistance to ceftazidime, CZA, cefepime, and ceftolozane-tazobactam when engineered into a WT strain, demonstrating a potentially important and previously unappreciated mechanism of resistance to these antibiotics in P. aeruginosa. Our results suggest that MMR-deficient isolates may rapidly evolve novel resistance mechanisms, sometimes with complex dynamics that reflect gene inactivation that occurs with hypermutation. The apparent ease with which hypermutators may switch to alternative resistance mechanisms for which antibiotics have not been developed may carry important clinical implications

Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection

Antimicrobial resistance in bacteria represents one of the most consequential problems in modern medicine, and its emergence and spread threaten to compromise central advances in the treatment of infectious diseases. Ceftazidime-avibactam (CZA) belongs to a new class of broad-spectrum beta-lactam/beta-lactamase inhibitor combinations designed to treat infections caused by multidrug-resistant bacteria. Understanding the emergence of resistance to this important new drug class is of critical importance. In this work, we demonstrate that evolved mismatch repair deficiency in P. aeruginosa, an important pathogen responsible for significant morbidity and mortality among hospitalized patients, may facilitate rapid acquisition of resistance to CZA in the context of acute infection. These findings are relevant for both diagnosis and treatment of antimicrobial resistance emerging in acute infection in the hypermutator background and additionally have implications for the emergence of more virulent phenotypes.Strains of Pseudomonas aeruginosa with deficiencies in DNA mismatch repair have been studied in the context of chronic infection, where elevated mutational rates (“hypermutation”) may facilitate the acquisition of antimicrobial resistance. Whether P. aeruginosa hypermutation can also play an adaptive role in the more dynamic context of acute infection remains unclear. In this work, we demonstrate that evolved mismatch repair deficiencies may be exploited by P. aeruginosa to facilitate rapid acquisition of antimicrobial resistance in acute infection, and we directly document rapid clonal succession by such a hypermutating lineage in a patient. Whole-genome sequencing (WGS) was performed on nine serially cultured blood and respiratory isolates from a patient in whom ceftazidime-avibactam (CZA) resistance emerged in vivo over the course of days. The CZA-resistant clone was differentiated by 14 mutations, including a gain-of-function G183D substitution in the PDC-5 chromosomal AmpC cephalosporinase conferring CZA resistance. This lineage also contained a substitution (R656H) at a conserved position in the ATPase domain of the MutS mismatch repair (MMR) protein, and elevated mutational rates were confirmed by mutational accumulation experiments with WGS of evolved lineages in conjunction with rifampin resistance assays. To test whether MMR-deficient hypermutation could facilitate rapid acquisition of CZA resistance, in vitro adaptive evolution experiments were performed with a mutS-deficient strain. These experiments demonstrated rapid hypermutation-facilitated acquisition of CZA resistance compared with the isogenic wild-type strain. Our results suggest a possibly underappreciated role for evolved MMR deficiency in facilitating rapid adaptive evolution of P. aeruginosa in the context of acute infection

Colonies observed inside the zones of inhibition in disk diffusion susceptibility tests in MPAO1-<i>mutS</i>^Tn isolates.

Representative photos of Kirby–Bauer susceptibility testing with ATM, IPM, MEM, and piperacillin-tazobactam (TZP) in the MPAO1-WT ancestor (1X), MPAO1-mutSTn ancestor (2X), and 2 CZA-resistant MPAO1-mutSTn isolates (lineage 2A passage 12 and lineage 2B passage 7). Pictures display readings at both 16 h, as recommended per CLSI guidelines, as well as at 24 h of incubation at 37°C. Scattered colonies can be observed inside some zones of inhibition at 16 h, particularly in the MPAO1-mutSTn isolates and more markedly in the evolved CZA-resistant isolates. ATM, aztreonam; IPM, imipenem; MEM, meropenem; TZP, piperacillin-tazobactam. (PNG)</p

Consensus sequences and nucleotide and amino acid frequency tables in proximity to PA4373-mexV (4903384 T>C) and MexW E36K variants in 7,493 genomes from the NCBI pathogen database.

Orthologous regions neighboring 2 mutations in the MexVW operon were identified in a set of 7,493 P. aeruginosa genomes from the NCBI Pathogen Detection database. (a) Summary of the nucleotide sequence alignment in proximity to PA4373-mexV +/- 10 bases. (b) Amino acid sequence surrounding MexW E36K with the corresponding residue highlighted in orange background. (DOCX)</p

List of genomes (strain and accession number) used in <i>Pseudomonas</i> gene ortholog analysis.

(XLSX)</p

Bacterial strains and plasmids used in this work.

(DOCX)</p

Extended susceptibility testing results for the engineered mutants.

Five biological replicates (A–E) from each of the 3 mutants that underwent extended antibiotic testing by the Kirby–Bauer method as described. Results are given as zone diameters in mm and corresponding CLSI breakpoint susceptibility interpretations are given in parentheses. AMK, Amikacin; CI, Ciprofloxacin; GEN, Gentamycin; TOB, Tobramycin. (DOCX)</p

General metrics for RNA-seq samples and libraries used in this study.

Ribosomal bases (%) and Median 5′ to 3′ bias were obtained with Picard Tools. RIN = RNA Integrity Number; GT = Genotype. (XLSX)</p

Proportions and types of fixed variants acquired during CZA adaptation for each genotype and lineage.

The total number of fixed variants acquired is indicated in parentheses. The underlying data to generate this figure can be found in S2 Data. (PDF)</p

Variants in genes mutated in MPAO1-<i>mutS</i>^<i>Tn</i> lineages that were passaged with and without CZA.

Three lineages (A, B, and C; left) passaged without CZA, compared to the 3 lineages passaged with CZA (2A, 2B, 2D; right), and also described in Fig 2. The numbering following the CZA lineage indicates the passage number in which the variant was identified, followed by the isolate number (e.g., 2A4-2 indicates lineage 2A, passage 4, isolate 2). A plus sign indicates presence of the variant. Coordinate positions are given with respect to the PAO1 reference chromosome. The MPAO1-WT lineages did not acquire any variants in shared targets. FS, frameshift mutation. (DOCX)</p