Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics.

[1]  R. Iino,et al.  A Microfluidic Device for Simple and Rapid Evaluation of Multidrug Efflux Pump Inhibitors , 2012, Front. Microbio..

[2]  Carla C. C. R. de Carvalho,et al.  Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli , 2009, Journal of biological engineering.

[3]  H. Sano,et al.  Korormicin, a novel antibiotic specifically active against marine gram-negative bacteria, produced by a marine bacterium. , 1997, The Journal of antibiotics.

[4]  J. Clardy,et al.  Discovery of 3-Formyl-Tyrosine Metabolites from Pseudoalteromonas tunicata through Heterologous Expression , 2009, Journal of the American Chemical Society.

[5]  H. Nikaido,et al.  Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli , 2013, Antimicrobial Agents and Chemotherapy.

[6]  K. Lewis,et al.  Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Elkins,et al.  Substrate Competition Studies Using Whole-Cell Accumulation Assays with the Major Tripartite Multidrug Efflux Pumps of Escherichia coli , 2007, Antimicrobial Agents and Chemotherapy.

[8]  S. Kjelleberg,et al.  Competitive Interactions in Mixed-Species Biofilms Containing the Marine Bacterium Pseudoalteromonas tunicata , 2005, Applied and Environmental Microbiology.

[9]  S. Matsunaga,et al.  Four new bioactive pyrrole-derived alkaloids from the marine sponge Axinella brevistyla. , 2001, Journal of natural products.

[10]  H. Nikaido,et al.  AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants , 1996, Journal of bacteriology.

[11]  Christopher M Thomas,et al.  Biosynthesis of thiomarinol A and related metabolites of Pseudoalteromonas sp SANK 73390 , 2014 .

[12]  F. Azam,et al.  Antagonistic Interactions among Marine Bacteria Impede the Proliferation of Vibrio cholerae , 2005, Applied and Environmental Microbiology.

[13]  L. Piddock,et al.  Bacterial efflux pump inhibitors from natural sources. , 2007, The Journal of antimicrobial chemotherapy.

[14]  L. Gram,et al.  Bioactivity, Chemical Profiling, and 16S rRNA-Based Phylogeny of Pseudoalteromonas Strains Collected on a Global Research Cruise , 2011, Marine Biotechnology.

[15]  Jiayuan Liu,et al.  Secondary Metabolites Produced by the Marine Bacterium Halobacillus salinus That Inhibit Quorum Sensing-Controlled Phenotypes in Gram-Negative Bacteria , 2008, Applied and Environmental Microbiology.

[16]  Xiao-ru Wang,et al.  Identification of norharman as the cytotoxic compound produced by the sponge (Hymeniacidon perleve)‐associated marine bacterium Pseudoalteromonas piscicida and its apoptotic effect on cancer cells , 2006, Biotechnology and applied biochemistry.

[17]  B. Jørgensen,et al.  Latitudinal patterns in the abundance of major marine bacterioplankton groups , 2010 .

[18]  J. Molnár,et al.  Review. Comparison of multidrug resistant efflux pumps of cancer and bacterial cells with respect to the same inhibitory agents. , 2007, In vivo.

[19]  Donald G Robertson,et al.  Metabonomics in pharmaceutical discovery and development. , 2007, Journal of proteome research.

[20]  N. Masuda,et al.  Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa , 2000, Antimicrobial Agents and Chemotherapy.

[21]  Paul D. Boudreau,et al.  Microbiota of Healthy Corals Are Active against Fungi in a Light-Dependent Manner , 2014, ACS chemical biology.

[22]  H. Nikaido,et al.  Mechanisms of RND multidrug efflux pumps. , 2009, Biochimica et biophysica acta.

[23]  J. Bowman.  Bioactive Compound Synthetic Capacity and Ecological Significance of Marine Bacterial Genus Pseudoalteromonas , 2007, Marine drugs.

[24]  L. Gram,et al.  Explorative solid-phase extraction (E-SPE) for accelerated microbial natural product discovery, dereplication, and purification. , 2010, Journal of natural products.

[25]  J. Kubanek,et al.  Do brominated natural products defend marine worms from consumers? Some do, most don’t , 2004 .

[26]  Ayush Kumar,et al.  Resistance-Nodulation-Division Multidrug Efflux Pumps in Gram-Negative Bacteria: Role in Virulence , 2013, Antibiotics.

[27]  V. Paul,et al.  The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals , 2014, Proceedings of the Royal Society B: Biological Sciences.

[28]  L. Amaral,et al.  Evaluation of efflux activity of bacteria by a semi-automated fluorometric system. , 2010, Methods in molecular biology.

[29]  Matej Oresic,et al.  MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data , 2010, BMC Bioinformatics.

[30]  L. Piddock.  Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria , 2006, Clinical Microbiology Reviews.

[31]  Michael R Hamblin,et al.  Microbial Efflux Systems and Inhibitors: Approaches to Drug Discovery and the Challenge of Clinical Implementation , 2013, The open microbiology journal.

[32]  K. Lewis.  Platforms for antibiotic discovery , 2013, Nature Reviews Drug Discovery.

[33]  Christina M. Jones,et al.  Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton , 2014, Proceedings of the National Academy of Sciences.

[34]  M. Schorn,et al.  Biosynthesis of polybrominated aromatic organic compounds by marine bacteria , 2014, Nature chemical biology.

[35]  M. Kimura.  A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences , 1980, Journal of Molecular Evolution.

[36]  J. Andrews,et al.  Determination of minimum inhibitory concentrations. , 2001, The Journal of antimicrobial chemotherapy.

[37]  F. Azam,et al.  2-n-Pentyl-4-Quinolinol Produced by a Marine Alteromonas sp. and Its Potential Ecological and Biogeochemical Roles , 2003, Applied and Environmental Microbiology.

[38]  A. Meyer.  Prospects and challenges of developing new agents for tough Gram-negatives. , 2005, Current opinion in pharmacology.

[39]  S. Kjelleberg,et al.  Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. , 1999, FEMS microbiology ecology.

[40]  Michael A Fischbach,et al.  New antibiotics from bacterial natural products , 2006, Nature Biotechnology.

[41]  Gregory L. Challis,et al.  Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. Simões,et al.  Plants as sources of new antimicrobials and resistance-modifying agents. , 2012, Natural product reports.

[43]  Angela Lee,et al.  Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy , 2001, Antimicrobial Agents and Chemotherapy.

[44]  Koichiro Tamura,et al.  MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. , 2013, Molecular biology and evolution.

[45]  M. Webber,et al.  A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli. , 2010, The Journal of antimicrobial chemotherapy.

[46]  K. Poole,et al.  Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. , 2001, Journal of molecular microbiology and biotechnology.

[47]  Michelle C. Swick,et al.  Expression of Multidrug Efflux Pump Genes acrAB-tolC, mdfA, and norE in Escherichia coli Clinical Isolates as a Function of Fluoroquinolone and Multidrug Resistance , 2010, Antimicrobial Agents and Chemotherapy.

[48]  Christopher M Thomas,et al.  A Natural Plasmid Uniquely Encodes Two Biosynthetic Pathways Creating a Potent Anti-MRSA Antibiotic , 2011, PloS one.

[49]  J. Pagés,et al.  Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria. , 2009, Biochimica et biophysica acta.

[50]  H. Goossens,et al.  Antibiotic resistance—the need for global solutions , 2013, BDJ.

[51]  R. Iino,et al.  Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels , 2011, PloS one.

[52]  S. Kjelleberg,et al.  Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples. , 2007, FEMS microbiology ecology.

[53]  J. Handzlik,et al.  Strategies for bypassing the membrane barrier in multidrug resistant Gram‐negative bacteria , 2011, FEBS letters.

[54]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[55]  U. Hentschel,et al.  Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts , 2014, Marine drugs.

[56]  H. Nikaido,et al.  Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. , 2012, FEMS microbiology reviews.

[57]  Tudor I. Oprea,et al.  Microbial efflux pump inhibition: tactics and strategies. , 2011, Current pharmaceutical design.

[58]  Mingchao Yu,et al.  Genome analysis of Pseudoalteromonas flavipulchra JG1 reveals various survival advantages in marine environment , 2013, BMC Genomics.

[59]  Tyler A. Johnson,et al.  Scrutinizing the scaffolds of marine biosynthetics from different source organisms: Gram-negative cultured bacterial products enter center stage. , 2014, Journal of natural products.

[60]  K. Bostian,et al.  Practical applications and feasibility of efflux pump inhibitors in the clinic--a vision for applied use. , 2006, Biochemical pharmacology.

[61]  F. M. Lovell.  The Structure of a Bromine-Rich Marine Antibiotic , 1966 .

[62]  R. A. van den Berg,et al.  Centering, scaling, and transformations: improving the biological information content of metabolomics data , 2006, BMC Genomics.

[63]  G. Aeby,et al.  Spatial and temporal patterns of scleractinian coral, soft coral, and zoanthid disease on a remote, near-pristine coral reef (Palmyra Atoll, central Pacific). , 2011, Diseases of Aquatic Organisms.

[64]  Anton Y Peleg,et al.  Hospital-acquired infections due to gram-negative bacteria. , 2010, The New England journal of medicine.

[65]  J. Felsenstein.  CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[66]  Christina Cramer,et al.  Antibiotic Susceptibility Profiles ofEscherichia coli Strains Lacking Multidrug Efflux Pump Genes , 2001, Antimicrobial Agents and Chemotherapy.

[67]  J. Pagés,et al.  Efflux Pumps Are Involved in the Defense of Gram-Negative Bacteria against the Natural Products Isobavachalcone and Diospyrone , 2010, Antimicrobial Agents and Chemotherapy.

[68]  M. Tutino,et al.  Exocellular Peptides from Antarctic PsychrophilePseudoalteromonas Haloplanktis , 2005, Marine Biotechnology.

[69]  L. Piddock.  Multidrug-resistance efflux pumps ? not just for resistance , 2006, Nature Reviews Microbiology.

[70]  Gerard D. Wright.  Something old, something new: revisiting natural products in antibiotic drug discovery. , 2014, Canadian journal of microbiology.

[71]  Otto X. Cordero,et al.  Ecological Populations of Bacteria Act as Socially Cohesive Units of Antibiotic Production and Resistance , 2012, Science.

[72]  James R. Baker,et al.  In Situ Maleimide Bridging of Disulfides and a New Approach to Protein PEGylation , 2011, Bioconjugate chemistry.

[73]  J. Molnár,et al.  Inducement and Reversal of Tetracycline Resistance in Escherichia coli K-12 and Expression of Proton Gradient-Dependent Multidrug Efflux Pump Genes , 2005, Antimicrobial Agents and Chemotherapy.

[74]  W. Fenical,et al.  Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. , 1989, Science.

[75]  R. Andersen,et al.  Antibiotic Metabolites from a Marine Pseudomonad , 1977, Antimicrobial Agents and Chemotherapy.

[76]  P. Burkholder,et al.  Production of a pyrrole antibiotic by a marine bacterium. , 1966, Applied microbiology.

[77]  Matej Oresic,et al.  Processing methods for differential analysis of LC/MS profile data , 2005, BMC Bioinformatics.

[78]  M. Totrov,et al.  Waltzing transporters and 'the dance macabre' between humans and bacteria , 2007, Nature Reviews Drug Discovery.

[79]  Christopher T. Walsh,et al.  Antibiotics for Emerging Pathogens , 2009, Science.

[80]  L. Amaral,et al.  Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. , 2008, International journal of antimicrobial agents.