The Resistome of Pseudomonas aeruginosa in Relationship to Phenotypic Susceptibility

ABSTRACT Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa, obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. β-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa. This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.

[1]  M. Pfaller,et al.  Rapid detection of antibiotic-resistant organism carriage for infection prevention. , 2013, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[2]  Garth D Ehrlich,et al.  Deletion and acquisition of genomic content during early stage adaptation of Pseudomonas aeruginosa to a human host environment. , 2012, Environmental microbiology.

[3]  C. Koh,et al.  Pseudomonas aeruginosa AmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors , 2005, Antimicrobial Agents and Chemotherapy.

[4]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[5]  G. Smith,et al.  Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology. , 2013, JAMA internal medicine.

[6]  M. Radice,et al.  Resistencia a carbapenemes en aislamientos de Pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos , 2011 .

[7]  R. Hall,et al.  The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons. , 1987, Nucleic acids research.

[8]  Julian Parkhill,et al.  Rapid whole-genome sequencing for investigation of a neonatal MRSA outbreak. , 2012, The New England journal of medicine.

[9]  R. Barrangou,et al.  CRISPR/Cas, the Immune System of Bacteria and Archaea , 2010, Science.

[10]  Ronald N. Jones,et al.  Antimicrobial susceptibility of Gram-negative organisms isolated from patients hospitalised with pneumonia in US and European hospitals: results from the SENTRY Antimicrobial Surveillance Program, 2009-2012. , 2014, International journal of antimicrobial agents.

[11]  D. Lipman,et al.  Improved tools for biological sequence comparison. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Paterson,et al.  Coproduction of Novel 16S rRNA Methylase RmtD and Metallo-β-Lactamase SPM-1 in a Panresistant Pseudomonas aeruginosa Isolate from Brazil , 2006, Antimicrobial Agents and Chemotherapy.

[13]  L. Marraffini,et al.  CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea , 2010, Nature Reviews Genetics.

[14]  Malika Kumarasiri,et al.  Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance , 2013, Antimicrobial Agents and Chemotherapy.

[15]  R. Hancock,et al.  Adaptive Resistance to the “Last Hope” Antibiotics Polymyxin B and Colistin in Pseudomonas aeruginosa Is Mediated by the Novel Two-Component Regulatory System ParR-ParS , 2010, Antimicrobial Agents and Chemotherapy.

[16]  Annalisa Ballarini,et al.  Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray , 2012, BMC Microbiology.

[17]  J. Burke,et al.  Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial stewardship. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[18]  M. McConnell,et al.  Progress on the development of rapid methods for antimicrobial susceptibility testing. , 2013, The Journal of antimicrobial chemotherapy.

[19]  K. Poole.  Efflux-mediated antimicrobial resistance. , 2005, The Journal of antimicrobial chemotherapy.

[20]  Alan R. Davidson,et al.  Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system , 2012, Nature.

[21]  François Laviolette,et al.  Ray: Simultaneous Assembly of Reads from a Mix of High-Throughput Sequencing Technologies , 2010, J. Comput. Biol..

[22]  Andreas Dötsch,et al.  Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance , 2012, Antimicrobial Agents and Chemotherapy.

[23]  Nicola K. Petty,et al.  Global dissemination of a multidrug resistant Escherichia coli clone , 2014, Proceedings of the National Academy of Sciences.

[24]  U. Pennsylvania,et al.  Clinical and Laboratory Standards Institute , 2019, Springer Reference Medizin.

[25]  J. Jeukens,et al.  Comparative Genomics of Isolates of a Pseudomonas aeruginosa Epidemic Strain Associated with Chronic Lung Infections of Cystic Fibrosis Patients , 2014, PloS one.

[26]  Evan S Snitkin,et al.  Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae with Whole-Genome Sequencing , 2012, Science Translational Medicine.

[27]  Kelli L. Palmer,et al.  Multidrug-Resistant Enterococci Lack CRISPR-cas , 2010, mBio.

[28]  Alimuddin Zumla,et al.  Rapid nucleic acid diagnostics for the detection of antimicrobial resistance in Gram-negative bacteria: is it time for a paradigm shift? , 2014, The Journal of antimicrobial chemotherapy.

[29]  N. Woodford,et al.  Evaluation of an Expanded Microarray for Detecting Antibiotic Resistance Genes in a Broad Range of Gram-Negative Bacterial Pathogens , 2012, Antimicrobial Agents and Chemotherapy.

[30]  P. H. Roy,et al.  Potential Role of Group IIC-attC Introns in Integron Cassette Formation , 2009, Journal of bacteriology.

[31]  J. R. Johnson,et al.  Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data , 2013, The Journal of antimicrobial chemotherapy.

[32]  Scott R. Miller,et al.  Genotypic and Phenotypic Variation in Pseudomonas aeruginosa Reveals Signatures of Secondary Infection and Mutator Activity in Certain Cystic Fibrosis Patients with Chronic Lung Infections , 2011, Infection and Immunity.

[33]  Robert E W Hancock,et al.  Function of pseudomonas porins in uptake and efflux. , 2002, Annual review of microbiology.

[34]  R. Hancock,et al.  Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa , 1999, Journal of bacteriology.

[35]  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.

[36]  J. Rothberg,et al.  Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology , 2011, PloS one.

[37]  H. Grundmann,et al.  Development of a Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Pseudomonas aeruginosa , 2004, Journal of Clinical Microbiology.

[38]  Ole Lund,et al.  Genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing. , 2013, The Journal of antimicrobial chemotherapy.

[39]  T. Pirzadeh,et al.  Role of efflux pumps: MexAB-OprM and MexXY(-OprA), AmpC cephalosporinase and OprD porin in non-metallo-β-lactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients. , 2014, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[40]  Y. Carmeli,et al.  Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting , 2005, Current opinion in infectious diseases.

[41]  M. Ferraro.  Performance standards for antimicrobial susceptibility testing , 2001 .

[42]  A. Fraise,et al.  Genome sequencing and characterization of an XDR ST 111 serotype O 12 hospital outbreak strain of Pseudomonas aeruginosa , 2014 .

[43]  Anthony Maxwell,et al.  Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser83and Asp87 , 2001, Antimicrobial Agents and Chemotherapy.

[44]  R. Olsen,et al.  Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae , 2014, Proceedings of the National Academy of Sciences.

[45]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[46]  F. Rojo,et al.  Population Structure of Pseudomonas aeruginosa from Five Mediterranean Countries: Evidence for Frequent Recombination and Epidemic Occurrence of CC235 , 2011, PloS one.

[47]  A. Gales,et al.  SPM-1-producing Pseudomonas aeruginosa: analysis of the ancestor relationship using multilocus sequence typing, pulsed-field gel electrophoresis, and automated ribotyping. , 2011, Microbial drug resistance.

[48]  P. Woo,et al.  Rapid Identification and Validation of Specific Molecular Targets for Detection of Escherichia coli O104:H4 Outbreak Strain by Use of High-Throughput Sequencing Data from Nine Genomes , 2011, Journal of Clinical Microbiology.

[49]  J. Deane,et al.  Recent trends in resistance to cell envelope–active antibacterial agents among key bacterial pathogens , 2013, Annals of the New York Academy of Sciences.

[50]  Alan R. Davidson,et al.  A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa , 2014, mBio.

[51]  Alexandros Stamatakis,et al.  RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models , 2006, Bioinform..

[52]  P. H. Roy,et al.  Complete Genome Sequence of the Multiresistant Taxonomic Outlier Pseudomonas aeruginosa PA7 , 2010, PloS one.

[53]  J. Waitz.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically , 1990 .

[54]  D. Hocquet,et al.  Tracking Down Antibiotic-Resistant Pseudomonas aeruginosa Isolates in a Wastewater Network , 2012, PloS one.

[55]  N. Woodford,et al.  Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. , 2011, FEMS microbiology reviews.

[56]  Daniel Yordanov,et al.  Pseudomonas aeruginosa - a phenomenon of bacterial resistance. , 2009, Journal of medical microbiology.

[57]  D. Livermore.  Of Pseudomonas, porins, pumps and carbapenems. , 2001, The Journal of antimicrobial chemotherapy.

[58]  T. Nishino,et al.  Alterations of susceptibility of Pseudomonas aeruginosa by overproduction of multidrug efflux systems, MexAB-OprM, MexCD-OprJ, and MexXY/OprM to carbapenems: substrate specificities of the efflux systems , 2002, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy.

[59]  A. Oliver,et al.  VIM-2–producing Multidrug-Resistant Pseudomonas aeruginosa ST175 Clone, Spain , 2012, Emerging infectious diseases.

[60]  George A. O'Toole,et al.  The CRISPR/Cas Adaptive Immune System of Pseudomonas aeruginosa Mediates Resistance to Naturally Occurring and Engineered Phages , 2012, Journal of bacteriology.

[61]  G. Jacoby.  Mechanisms of resistance to quinolones. , 2005, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[62]  Gerald B. Pier,et al.  Lung Infections Associated with Cystic Fibrosis , 2002, Clinical Microbiology Reviews.

[63]  H. Yoneyama,et al.  Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa , 1993, Antimicrobial Agents and Chemotherapy.

[64]  V. Jarlier,et al.  Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa , 1999, Antimicrobial Agents and Chemotherapy.

[65]  K. Poole.  Pseudomonas Aeruginosa: Resistance to the Max , 2011, Front. Microbio..

[66]  W. Pearson.  Effective protein sequence comparison. , 1996, Methods in enzymology.

[67]  H. Nikaido,et al.  Contributions of MexAB-OprM and an EmrE Homolog to Intrinsic Resistance of Pseudomonas aeruginosa to Aminoglycosides and Dyes , 2003, Antimicrobial Agents and Chemotherapy.

[68]  K. Ko,et al.  Genomic variations between colistin-susceptible and -resistant Pseudomonas aeruginosa clinical isolates and their effects on colistin resistance. , 2014, The Journal of antimicrobial chemotherapy.

[69]  R. Hancock,et al.  Pseudomonas aeruginosa: all roads lead to resistance. , 2011, Trends in microbiology.

[70]  P. H. Roy,et al.  Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[71]  A. Leal,et al.  Frequency of microorganisms isolated in patients with bacteremia in intensive care units in Colombia and their resistance profiles , 2013, The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases.

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

[73]  K. Katoh,et al.  MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability , 2013, Molecular biology and evolution.

[74]  K. Poole,et al.  Contribution of the MexXY Multidrug Transporter to Aminoglycoside Resistance in Pseudomonas aeruginosa Clinical Isolates , 2003, Antimicrobial Agents and Chemotherapy.

[75]  P. Nordmann,et al.  Molecular Epidemiology and Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa , 2009, Antimicrobial Agents and Chemotherapy.

[76]  M. J. Lynch,et al.  Emergence of resistance to imipenem in Pseudomonas aeruginosa , 1987, Antimicrobial Agents and Chemotherapy.

[77]  A. Robicsek,et al.  Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase , 2006, Nature Medicine.

[78]  P. Plésiat,et al.  A Two-Component Regulatory System Interconnects Resistance to Polymyxins, Aminoglycosides, Fluoroquinolones, and β-Lactams in Pseudomonas aeruginosa , 2010, Antimicrobial Agents and Chemotherapy.

[79]  James H. Bullard,et al.  Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. , 2011, The New England journal of medicine.

[80]  M. Souli,et al.  Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. , 2008, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[81]  M. Nogueira,et al.  Detection of P. aeruginosa harboring bla CTX-M-2, bla GES-1 and bla GES-5, bla IMP-1 and bla SPM-1 causing infections in Brazilian tertiary-care hospital , 2012, BMC Infectious Diseases.

[82]  Martin C. J. Maiden,et al.  BIGSdb: Scalable analysis of bacterial genome variation at the population level , 2010, BMC Bioinformatics.

[83]  S. Nakamura,et al.  Proportion of DNA gyrase mutants among quinolone-resistant strains of Pseudomonas aeruginosa , 1990, Antimicrobial Agents and Chemotherapy.

[84]  Julian Parkhill,et al.  Whole-genome sequencing for analysis of an outbreak of meticillin-resistant Staphylococcus aureus: a descriptive study , 2013, The Lancet. Infectious Diseases.