Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance. ABSTRACT Metabolic flexibility of Pseudomonas aeruginosa could lead to new strategies to combat bacterial infection. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate global metabolism in naturally and artificially evolved strains with cefoperazone-sulbactam (SCF) resistance (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). Inactivation of the pyruvate cycle and nitric oxide (NO) biosynthesis was identified as characteristic features of SCF resistance in both evolved strains. Nitrite-dependent NO biosynthesis instead of an arginine-dependent NO pathway is responsible for the reduced NO, which is attributed to lower nitrite and electrons from the oxidation of NADH to NAD+ provided by the pyruvate cycle. Exogenous fumarate, NADH, nitrate, and nitrite promoted the NO level and thereby potentiated SCF-mediated killing. Unexpectedly, fumarate caused the elevation of nitrite, while nitrite/nitrate resulted in the increase of Cyt bc1 complex (providing electrons). These interesting findings indicate that the nitrite-dependent NO biosynthesis and the pyruvate cycle are mutual to promote NO metabolism. In addition, the NO-potentiated sensitivity to SCF was validated by NO donor sodium nitroprusside. These results reveal an endogenous NO-mediated SCF resistance and develop its reversion by metabolites in P. aeruginosa. IMPORTANCE Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance. Since bacterial metabolic state impacts sensitivity and resistance to antibiotics, exploring and disclosing bacterial metabolic mechanisms can be used to develop a metabolome-reprogramming approach to elevate bacterial sensitivity to antibiotics. Therefore, GC-MS-based metabolomics is used to explore the similarities and differences of metabolomes between naturally and artificially evolved cefoperazone-sulbactam (SCF)-resistant P. aeruginosa (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). It identifies the depressed nitrite-dependent nitric oxide (NO) biosynthesis as the most overlapping characteristic feature between AP-RCLIN-EVO and AP-RLAB-EVO. This is because the pyruvate cycle fluctuates, thereby generating fewer NADH and providing fewer electrons for nitrite-dependent NO biosynthesis than the control. Interestingly, exogenous fumarate, NADH, nitrate, and nitrite as well as NO donor sodium nitroprusside promote NO generation to elevate sensitivity to SCF. These results highlight the way to understand metabolic mechanisms of antibiotic resistance and explore metabolic modulation to combat the bacterial pathogen.

[1]  Xuan-xian Peng,et al.  Enhanced Biosynthesis of Fatty Acids Is Associated with the Acquisition of Ciprofloxacin Resistance in Edwardsiella tarda , 2021, mSystems.

[2]  M. Schoenfisch,et al.  Exogenous Nitric Oxide Improves Antibiotic Susceptibility in Resistant Bacteria. , 2020, ACS infectious diseases.

[3]  S. Dolan.  Current Knowledge and Future Directions in Developing Strategies to Combat Pseudomonas aeruginosa Infection. , 2020, Journal of molecular biology.

[4]  Xuan-xian Peng,et al.  Reduced ROS-mediated antibiotic resistance and its reverting by glucose in Vibrio alginolyticus. , 2020, Environmental microbiology.

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

[6]  E. Schneider-Futschik,et al.  Metabolomic Description of Ivacaftor Elevating Polymyxin B Mediated Antibacterial Activity in Cystic Fibrosis Pseudomonas aeruginosa. , 2020, ACS pharmacology & translational science.

[7]  Xuan-xian Peng,et al.  Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus. , 2020, Environmental microbiology.

[8]  Fengqi Sun,et al.  Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake , 2020, mBio.

[9]  Jianming Chen,et al.  Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora pogona , 2020, Microbial Cell Factories.

[10]  Xuan-xian Peng,et al.  Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus. , 2019, Environmental microbiology.

[11]  Sławomir Jabłoński,et al.  Metabolomic studies of Pseudomonas aeruginosa , 2019, World Journal of Microbiology and Biotechnology.

[12]  J. Blair,et al.  TetR-family transcription factors in Gram-negative bacteria: conservation, variation and implications for efflux-mediated antimicrobial resistance , 2019, BMC Genomics.

[13]  Jian-ying Zhang,et al.  Glycine, serine and threonine metabolism confounds efficacy of complement-mediated killing , 2019, Nature Communications.

[14]  Xiaojun Yang,et al.  Comparative Extracellular Proteomics of Aeromonas hydrophila Reveals Iron-Regulated Secreted Proteins as Potential Vaccine Candidates , 2019, Front. Immunol..

[15]  Xuan-xian Peng,et al.  Metabolic mechanism of ceftazidime resistance in Vibrio alginolyticus , 2019, Infection and drug resistance.

[16]  P. Hertzog,et al.  Comparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa , 2019, mSystems.

[17]  B. Glick,et al.  Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. , 2019, Biotechnology advances.

[18]  Brian T. Tsuji,et al.  Alterations of Metabolic and Lipid Profiles in Polymyxin-Resistant Pseudomonas aeruginosa , 2018, Antimicrobial Agents and Chemotherapy.

[19]  D. Hoyer,et al.  Sputum Active Polymyxin Lipopeptides: Activity against Cystic Fibrosis Pseudomonas aeruginosa Isolates and Their Interactions with Sputum Biomolecules , 2018, ACS infectious diseases.

[20]  Jia-xin Zhu,et al.  Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria , 2018, Proceedings of the National Academy of Sciences.

[21]  Jason H. Yang,et al.  Carbon Sources Tune Antibiotic Susceptibility in Pseudomonas aeruginosa via Tricarboxylic Acid Cycle Control. , 2017, Cell chemical biology.

[22]  Junsong Wang,et al.  1H NMR-Based Global Metabolic Studies of Pseudomonas aeruginosa upon Exposure of the Quorum Sensing Inhibitor Resveratrol. , 2017, Journal of proteome research.

[23]  L. Högberg,et al.  ECDC publishes 2015 surveillance data on antimicrobial resistance and antimicrobial consumption in Europe , 2016, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[24]  Huajun Zheng,et al.  Tracking Cefoperazone/Sulbactam Resistance Development In vivo in A. baumannii Isolated from a Patient with Hospital-Acquired Pneumonia by Whole-Genome Sequencing , 2016, Front. Microbiol..

[25]  E. Gutiérrez-Cirlos,et al.  Cardiolipin deficiency causes a dissociation of the b6c:caa3 megacomplex in B. subtilis membranes , 2016, Journal of Bioenergetics and Biomembranes.

[26]  A. Bäumler,et al.  Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens. , 2016, Current opinion in microbiology.

[27]  Xuan-xian Peng,et al.  Functional metabolomics: from biomarker discovery to metabolome reprogramming , 2015, Protein & Cell.

[28]  C. Guo,et al.  Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria. , 2015, Cell metabolism.

[29]  E. Nudler,et al.  Endogenous Nitric Oxide Protects Bacteria Against a Wide Spectrum of Antibiotics , 2009, Science.

[30]  D. Hassett,et al.  Involvement of Nitric Oxide in Biofilm Dispersal of Pseudomonas aeruginosa , 2006, Journal of bacteriology.

[31]  L. Rice,et al.  Resistance to cefoperazone-sulbactam in Klebsiella pneumoniae: evidence for enhanced resistance resulting from the coexistence of two different resistance mechanisms , 1993, Antimicrobial Agents and Chemotherapy.