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

Abstract

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. IMPORTANCE 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.

From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

“…Finally, several recent research studies on non-CF infections reported that P. aeruginosa adaptive mechanisms occur at the very beginning of the colonization, emphasizing the underappreciated role of genetic adaptation in acute infections [51, 58, 59]. Altogether, these results indicate that different modes of selection arise with time, according to infection stage and severity.…”

“…Indeed, it has been shown that antibiotic exposure promotes the emergence of hypermutability in P. aeruginosa, then favouring acquisition of antibiotic resistance [45,[48][49][50][51]. However, Mehta and colleagues also observed that some hypermutable lineages would spontaneously decline and disappear from the evolving population [49].…”

“…Compensation of the hypermutable phenotype by secondary mutations observed by Feliziani and colleagues [ 8 ] supports this notion, as it can rebalance the mutation rate to a regular level after a stage of rapid diversification and adaptation. Finally, several recent research studies on non-CF infections reported that P. aeruginosa adaptive mechanisms occur at the very beginning of the colonization, emphasizing the underappreciated role of genetic adaptation in acute infections [ 51, 58, 59 ]. Altogether, these results indicate that different modes of selection arise with time, according to infection stage and severity.…”

Mutators Enhance Adaptive Micro-Evolution in Pathogenic Microbes

“…This is evident in clinical populations in bacterial species such as Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus and Salmonella enterica, in which mutator strains are significantly more resistant to antibiotics than non-mutator strains [49,54,56,57,61,[85][86][87]. Serial whole genome sequencing of P. aeruginosa isolates from a patient, in whom clonal ceftazidime-avibactam (CZA) resistance emerged over the course of days, showed a lack of MMR which facilitated the rapid evolution of resistance during acute infection [87]. In vitro adaptive evolution experiments with a mutS strain also showed rapid acquisition of CZA resistance in vitro [87].…”

Mutators drive evolution of multi-resistance to antibiotics

“…Mutators are a potential concern for combination therapy, as multi-resistance is often found in mutator lineages, although this is believed to have arisen due to sequential application of monotherapy 22,[28][29][30][31] . Direct evidence for the efficacy of combinations against mutators is mixed [32][33][34] . We imposed selection for resistance by increasing the concentrations of antibiotics over a physiologically-relevant concentration gradient 35,36 .…”