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.

[1]  H. Schweizer,et al.  Loss of RND-Type Multidrug Efflux Pumps Triggers Iron Starvation and Lipid A Modifications in Pseudomonas aeruginosa , 2021, Antimicrobial agents and chemotherapy.

[2]  A. Oliver,et al.  Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Modification of the Pseudomonas aeruginosa MexCD-OprJ Efflux Pump , 2021, Antimicrobial agents and chemotherapy.

[3]  Adam M. Feist,et al.  Compensatory evolution of Pseudomonas aeruginosa’s slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis , 2021, Nature Communications.

[4]  Michael T. Wolfinger,et al.  Gene Expression Profiling of Pseudomonas aeruginosa Upon Exposure to Colistin and Tobramycin , 2021, Frontiers in Microbiology.

[5]  M. Adams,et al.  Emergence of Resistance to Ceftazidime-Avibactam in a Pseudomonas aeruginosa Isolate Producing Derepressed blaPDC in a Hollow-Fiber Infection Model , 2021, Antimicrobial Agents and Chemotherapy.

[6]  A. Carvalho-Assef,et al.  Carbapenem-Resistant Pseudomonas aeruginosa in Chronic Lung Infection: Current Resistance Profile and Hypermutability in Patients with Cystic Fibrosis , 2021, Current Microbiology.

[7]  S. Molin,et al.  Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis , 2020, Nature Reviews Microbiology.

[8]  Joseph K. Pickrell,et al.  Next Generation Genotyping (NGG) Using Riptide™. Performance Specifications when Best Practices are Applied. , 2020, Journal of biomolecular techniques : JBT.

[9]  A. Oliver,et al.  Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam , 2020, Antimicrobial Agents and Chemotherapy.

[10]  A. Oliver,et al.  In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa. , 2020, The Journal of antimicrobial chemotherapy.

[11]  S. Beatson,et al.  Antimicrobial Resistance in ESKAPE Pathogens , 2020, Clinical Microbiology Reviews.

[12]  P. Vivekanandan,et al.  Antibiotic Resistance and Epigenetics: More to It than Meets the Eye , 2019, Antimicrobial Agents and Chemotherapy.

[13]  T. Naas,et al.  A 2.5-Year Within-Patient Evolution of Pseudomonas aeruginosa Isolates with In Vivo Acquisition of Ceftolozane-Tazobactam and Ceftazidime-Avibactam Resistance upon Treatment , 2019, Antimicrobial Agents and Chemotherapy.

[14]  S. Molin,et al.  Hypermutator Pseudomonas aeruginosa Exploits Multiple Genetic Pathways To Develop Multidrug Resistance during Long-Term Infections in the Airways of Cystic Fibrosis Patients , 2019, Antimicrobial Agents and Chemotherapy.

[15]  R. Bonomo,et al.  Dynamic Emergence of Mismatch Repair Deficiency Facilitates Rapid Evolution of Ceftazidime-Avibactam Resistance in Pseudomonas aeruginosa Acute Infection , 2019, mBio.

[16]  M. Castanheira,et al.  Combination of MexAB-OprM overexpression and mutations in efflux regulators, PBPs and chaperone proteins is responsible for ceftazidime/avibactam resistance in Pseudomonas aeruginosa clinical isolates from US hospitals. , 2019, The Journal of antimicrobial chemotherapy.

[17]  A. Oliver,et al.  Epidemiology and Treatment of Multidrug-Resistant and Extensively Drug-Resistant Pseudomonas aeruginosa Infections , 2019, Clinical Microbiology Reviews.

[18]  R. Bonomo,et al.  Deciphering the Evolution of Cephalosporin Resistance to Ceftolozane-Tazobactam in Pseudomonas aeruginosa , 2018, mBio.

[19]  Y. Shamoo,et al.  The Essential Role of Hypermutation in Rapid Adaptation to Antibiotic Stress , 2018, Antimicrobial Agents and Chemotherapy.

[20]  J. Martínez,et al.  Mutation-Driven Evolution of Pseudomonas aeruginosa in the Presence of either Ceftazidime or Ceftazidime-Avibactam , 2018, Antimicrobial Agents and Chemotherapy.

[21]  Haixu Tang,et al.  The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli , 2018, Genetics.

[22]  A. Oliver,et al.  The Versatile Mutational Resistome of Pseudomonas aeruginosa , 2018, Front. Microbiol..

[23]  A. Oliver,et al.  Mechanisms leading to in vivo ceftolozane/tazobactam resistance development during the treatment of infections caused by MDR Pseudomonas aeruginosa , 2018, The Journal of antimicrobial chemotherapy.

[24]  James T. Robinson,et al.  Variant Review with the Integrative Genomics Viewer. , 2017, Cancer research.

[25]  S. Salipante,et al.  Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa , 2017, mBio.

[26]  B. Kreiswirth,et al.  Emergence of Ceftolozane-Tazobactam-Resistant Pseudomonas aeruginosa during Treatment Is Mediated by a Single AmpC Structural Mutation , 2017, Antimicrobial Agents and Chemotherapy.

[27]  S. Chevalier,et al.  Structure, function and regulation of Pseudomonas aeruginosa porins , 2017, FEMS microbiology reviews.

[28]  D. Bilton,et al.  Pseudomonas aeruginosa adaptation and diversification in the non-cystic fibrosis bronchiectasis lung , 2017, European Respiratory Journal.

[29]  R. Bonomo,et al.  Ceftazidime/Avibactam and Ceftolozane/Tazobactam: Second-generation β-Lactam/β-Lactamase Inhibitor Combinations. , 2016, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[30]  F. Klawonn,et al.  Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa , 2016, Antimicrobial Agents and Chemotherapy.

[31]  R. Kassen,et al.  The properties of spontaneous mutations in the opportunistic pathogen Pseudomonas aeruginosa , 2016, BMC Genomics.

[32]  Raymond Lo,et al.  Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database , 2015, Nucleic Acids Res..

[33]  Boo Shan Tseng,et al.  Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange , 2015, Nature Protocols.

[34]  I. Broutin,et al.  Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins , 2015, Antimicrobial Agents and Chemotherapy.

[35]  R. Alm,et al.  Selection and molecular characterization of ceftazidime/avibactam-resistant mutants in Pseudomonas aeruginosa strains containing derepressed AmpC. , 2015, The Journal of antimicrobial chemotherapy.

[36]  E. Picard,et al.  Is infection with hypermutable Pseudomonas aeruginosa clinically significant? , 2015, Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society.

[37]  Hiroshi Nikaido,et al.  The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria , 2015, Clinical Microbiology Reviews.

[38]  P. Cluzel,et al.  Adaptive Resistance in Bacteria Requires Epigenetic Inheritance, Genetic Noise, and Cost of Efflux Pumps , 2015, PloS one.

[39]  A. Oliver,et al.  Evolution of Pseudomonas aeruginosa Antimicrobial Resistance and Fitness under Low and High Mutation Rates , 2015, Antimicrobial Agents and Chemotherapy.

[40]  M. Lynch,et al.  Mutation Rate, Spectrum, Topology, and Context-Dependency in the DNA Mismatch Repair-Deficient Pseudomonas fluorescens ATCC948 , 2014, Genome biology and evolution.

[41]  Andrea M. Smania,et al.  Coexistence and Within-Host Evolution of Diversified Lineages of Hypermutable Pseudomonas aeruginosa in Long-term Cystic Fibrosis Infections , 2014, PLoS genetics.

[42]  Ronald N. Jones,et al.  Mutation-Driven β-Lactam Resistance Mechanisms among Contemporary Ceftazidime-Nonsusceptible Pseudomonas aeruginosa Isolates from U.S. Hospitals , 2014, Antimicrobial Agents and Chemotherapy.

[43]  A. Oliver,et al.  Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC , 2014, Antimicrobial Agents and Chemotherapy.

[44]  S. Feliziani,et al.  Simple Sequence Repeats Together with Mismatch Repair Deficiency Can Bias Mutagenic Pathways in Pseudomonas aeruginosa during Chronic Lung Infection , 2013, PloS one.

[45]  S. Molin,et al.  Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators , 2013, PLoS genetics.

[46]  J. Karlowsky,et al.  Ceftazidime-Avibactam: a Novel Cephalosporin/β-lactamase Inhibitor Combination , 2013, Drugs.

[47]  J. Blázquez,et al.  Mutational Spectrum Drives the Rise of Mutator Bacteria , 2013, PLoS genetics.

[48]  Haixu Tang,et al.  Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing , 2012, Proceedings of the National Academy of Sciences.

[49]  L. Gallagher,et al.  Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1 , 2012, Journal of bacteriology.

[50]  D. Ehmann,et al.  Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor , 2012, Proceedings of the National Academy of Sciences.

[51]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration , 2012, Briefings Bioinform..

[52]  H. Nagarajaram,et al.  A Study on Mutational Dynamics of Simple Sequence Repeats in Relation to Mismatch Repair System in Prokaryotic Genomes , 2012, Journal of Molecular Evolution.

[53]  Samuel I. Miller,et al.  PhoQ Mutations Promote Lipid A Modification and Polymyxin Resistance of Pseudomonas aeruginosa Found in Colistin-Treated Cystic Fibrosis Patients , 2011, Antimicrobial Agents and Chemotherapy.

[54]  R. Hancock,et al.  Genetic Determinants Involved in the Susceptibility of Pseudomonas aeruginosa to β-Lactam Antibiotics , 2010, Antimicrobial Agents and Chemotherapy.

[55]  A. Oliver,et al.  Bacterial hypermutation in cystic fibrosis, not only for antibiotic resistance. , 2010, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[56]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[57]  Nancy D. Hanson,et al.  Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms , 2009, Clinical Microbiology Reviews.

[58]  L. Christiansen,et al.  Antibiotic Resistance in Pseudomonas aeruginosa Strains with Increased Mutation Frequency Due to Inactivation of the DNA Oxidative Repair System , 2009, Antimicrobial Agents and Chemotherapy.

[59]  C. Pommerenke,et al.  Genomewide Identification of Genetic Determinants of Antimicrobial Drug Resistance in Pseudomonas aeruginosa , 2009, Antimicrobial Agents and Chemotherapy.

[60]  Mona Singh,et al.  Predicting functionally important residues from sequence conservation , 2007, Bioinform..

[61]  N. Høiby,et al.  Occurrence of Hypermutable Pseudomonas aeruginosa in Cystic Fibrosis Patients Is Associated with the Oxidative Stress Caused by Chronic Lung Inflammation , 2005, Antimicrobial Agents and Chemotherapy.

[62]  K. Poole,et al.  Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of Pseudomonas aeruginosa , 2004, Journal of bacteriology.

[63]  J. Blázquez,et al.  Hypermutation as a factor contributing to the acquisition of antimicrobial resistance. , 2003, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[64]  Y. Li,et al.  A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa. , 2003, The Journal of antimicrobial chemotherapy.

[65]  G. Bierbaum,et al.  An Elevated Mutation Frequency Favors Development of Vancomycin Resistance in Staphylococcus aureus , 2002, Antimicrobial Agents and Chemotherapy.

[66]  A. Oliver,et al.  High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. , 2000, Science.

[67]  R. Hancock,et al.  PhoP–PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer‐membrane protein OprH and polymyxin B resistance , 1999, Molecular microbiology.

[68]  T. Köhler,et al.  Carbapenem Activities against Pseudomonas aeruginosa: Respective Contributions of OprD and Efflux Systems , 1999, Antimicrobial Agents and Chemotherapy.