Exploring novel amides as efflux pump inhibitors for overcoming antibiotic resistance in multidrug-resistant Pseudomonas aeruginosa
-
Fahad Ullah
,
Maqsood Ali
,
Falak Niaz
,
Israr Ali Khan
,
Sher Wali Khan
,
Momin Khan
,
Rafaqat Ishaq
,
Abdul Manan
,
Ying Yu
,
Muhammad Ilyas
Abstract
Background and Objectives: Pseudomonas aeruginosa (P. aeruginosa), a multidrug-resistant bacterium, represents a considerable risk in healthcare environments owing to its capacity to induce various infections. The resistance of P. aeruginosa is frequently linked to efflux pumps that actively remove antibiotics from the bacterial cell. This study investigates novel amide compounds as potential alternatives to address P. aeruginosa isolates exhibiting multidrug resistance mediated by efflux pumps.
Materials and Methods: Gram staining and biochemical assays revealed thirty-three multi-drug-resistant P. aeruginosa isolates from a tertiary care hospital Peshawar. After antibiotic susceptibility testing, efflux pumps were detected using Ethidium Bromide (EtBr) agar cartwheel technique and UV transilluminator. Novel amides were tested for efflux pump and anti-pseudomonal action against efflux pump-positive isolates utilizing agar well diffusion and micro broth dilution, including synergy with ciprofloxacin and gentamicin.
Results: Three high efflux pump activity P. aeruginosa isolates were chosen using ETBr agar cartwheel technique. Novel amides (ITC, ITD, ITE, DEP) block efflux pump, although TEM-cu is very antimicrobial. TEM-cu, DEP, ITC, and ITE have 0.19, 0.78, and 0.78 mg/ml MICs. Effectiveness against efflux pump-expressing P. aeruginosa is lowest with ITE (1.56 mg/ml). Together with ciprofloxacin and gentamicin, TEM-cu and DEP improved antimicrobial effectiveness.
Conclusion: TEM-cu is highly effective against efflux pump-positive P. aeruginosa, while amides like ITC, ITD, ITE, and DEP block these pumps. With significant reductions, DEP and TEM-cu improve ciprofloxacin and gentamicin efficacy. This method may help overcome P. aeruginosa efflux pump-mediated resistance.
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7. Blair JM, Piddock LJ. How to measure export via bacterial multidrug resistance efflux pumps. mBio 2016; 7(4): e00840-16.
8. Hajiagha MN, Kafil HS. Efflux pumps and microbial biofilm formation. Infect Genet Evol 2023; 112: 105459.
9. Abdi SN, Ghotaslou R, Ganbarov K, Mobed A, Tanomand A, Yousefi M, et al. Acinetobacter baumannii efflux pumps and antibiotic resistance. Infect Drug Resist 2020; 13: 423-434.
10. Huang L, Wu C, Gao H, Xu C, Dai M, Huang L, et al. Bacterial multidrug efflux pumps at the frontline of antimicrobial resistance: an overview. Antibiotics (Basel) 2022; 11: 520.
11. Puzari M, Chetia P. RND efflux pump mediated antibiotic resistance in Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a major issue worldwide. World J Microbiol Biotechnol 2017; 33: 24.
12. Lorusso AB, Carrara JA, Barroso CDN, Tuon FF, Faoro H. Role of efflux pumps on antimicrobial resistance in Pseudomonas aeruginosa. Int J Mol Sci 2022; 23: 15779.
13. Verma P, Tiwari M, Tiwari V. Efflux pumps in multidrug-resistant Acinetobacter baumannii: Current status and challenges in the discovery of efflux pumps inhibitors. Microb Pathog 2021; 152: 104766.
14. Avakh A, Grant GD, Cheesman MJ, Kalkundri T, Hall S. The art of war with Pseudomonas aeruginosa: targeting Mex efflux pumps directly to strategically enhance antipseudomonal drug efficacy. Antibiotics (Basel) 2023; 12: 1304.
15. Kumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, et al. Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 2023; 181: 106182.
16. Gaurav A, Bakht P, Saini M, Pandey S, Pathania R. Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors. Microbiology (Reading) 2023; 169: 001333.
17. Threlfall T. The infrared spectra of amides. Part 1. The stretching vibrations of primary carboxamides. Vib Spectrosc 2022; 121: 103386.
18. Martin N, Cirujano FG. Heterogeneous catalytic direct amide bond formation. Catal Commun 2022; 164: 106420.
19. Puri S, Negi D. Simple to complex Amide Derivatives as potent Anti‐Tuberculosis Agents: A Literature Survey of the past Decade. ChemistrySelect 2022; 7(43): e202202584.
20. Pal R, Singh K, Paul J, Khan SA, Naim MJ, Akhtar MJ. Overview of chemistry and therapeutic potential of non-nitrogen heterocyclics as anticonvulsant agents. Curr Neuropharmacol 2022; 20: 1519-1553.
21. Alam A, Ali M, Rehman NU, Latif A, Shah AJ, Wazir NU, et al. Synthesis and characterization of biologically active flurbiprofen amide derivatives as selective prostaglandin-endoperoxide synthase II inhibitors: In vivo anti-inflammatory activity and molecular docking. Int J Biol Macromol 2023; 228: 659-670.
22. Galewicz-Walesa K, Pachuta-Stec A. The synthesis and properties of N-substituted amides of 1-(5-methylthio-1, 2, 4-triazol-3-yl)-cyclohexane-2-carboxylic acid. Ann Univ Mariae Curie Skłodowska Sect AA Chem 2003; 58: 118-121.
23. Graybill TL, Ross MJ, Gauvin BR, Gregory JS, Harris AL, Ator MA, et al. Synthesis and evaluation of azapeptide-derived inhibitors of serine and cysteine proteases. Bioorg Med Chem Lett 1992; 2: 1375-1380.
24. Moise M, Sunel V, Profire L, Popa M, Lionte C. Synthesis and antimicrobial activity of some new (sulfon-amidophenyl)-amide derivatives of N-(4-nitrobenzoyl)-phenylglycine and N-(4-nitrobenzoyl)-phenylalanine. FARMACIA 2008; 56: 283-289.
25. Warnecke A, Fichtner I, Sass G, Kratz F. Synthesis, cleavage profile, and antitumor efficacy of an albumin‐binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B. Arch Pharm (Weinheim) 2007; 340: 389-395.
26. Suravaram S, Hada V, Siddiqui IA. Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints. Indian J Med Microbiol 2021; 39: 315-319.
27. Sepehr A, Fereshteh S, Shahrokhi N. Detection of efflux pump using ethidium bromide-agar cartwheel method in Acinetobacter baumannii clinical isolates. J Med Microbiol Infect Dis 2022; 10: 36-41.
28. Palladini G, Garbarino C, Luppi A, Russo S, Filippi A, Arrigoni N, et al. Comparison between broth microdilution and agar disk diffusion methods for antimicrobial susceptibility testing of bovine mastitis pathogens. J Microbiol Methods 2023; 212: 106796.
29. Kamble PA, Phadke M. Use of checkerboard assay to determine the synergy between essential oils extracted from leaves of Aegle marmelos (L.) Correa and nystatin against Candida albicans. Ayu 2023; 44: 38-43.
30. Black C, Al Mahmud H, Howle V, Wilson S, Smith AC, Wakeman CA. Development of a Polymicrobial Checkerboard Assay as a tool for Determining Combinatorial Antibiotic Effectiveness in Polymicrobial Communities. Antibiotics (Basel) 2023; 12: 1207.
31. Süer D (2021). Molecular detection of some Virulence Genes In Pseudomonas aeruginosa isolated from clinical Source: Doctoral dissertation, Near East University. https://docs.neu.edu.tr/library/9299808495.pdf
32. Adilabdulhady D, Kadhim HM. Molecular detection of Pseudomonas aeruginosa isolated from different clinical cases and test antibiotics Sensitivity on the Bacterial Growth. Pak J Med Health Sci 2022; 16: 587-588.
33. Rashid A, Akram M, Kayode OT, Kayode A. Clinical features and epidemiological patterns of infections by multidrug resistance Staphylococcus aureus and Pseudomonas aeruginosa in patients with burns. Biomed J Sci Tech Res 2020; 25: 19272-19279.
34. Rehman A, Patrick WM, Lamont IL. Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem. J Med Microbiol 2019; 68: 1-10.
35. Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, Gómez-Zorrilla S, et al. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev 2019; 32(4): e00031-19.
36. Tilahun M, Shibabaw A, Adane M. Prevalence and multidrug resistance patterns of bacterial pathogens in wastewater and drinking water systems from hospital and non-hospital environments in Ethiopia: a systematic review and meta-analysis. BMC Infect Dis 2025; 25: 250.
37. Yuan Y, Rosado-Lugo JD, Zhang Y, Datta P, Sun Y, Cao Y, et al. Evaluation of heterocyclic carboxamides as potential efflux pump inhibitors in Pseudomonas aeruginosa. Antibiotics (Basel) 2021; 11: 30.
38. Lamut A, Peterlin Mašič L, Kikelj D, Tomašič T. Efflux pump inhibitors of clinically relevant multidrug resistant bacteria. Med Res Rev 2019; 39: 2460-2504.
39. Mech P (2024). Modulation of antibacterial resistance with special reference to inhibition of efflux pump in Staphylococcus aureus: Indian Veterinary Research Institute.
40. Srichaiyapol O, Thammawithan S, Siritongsuk P, Nasompag S, Daduang S, Klaynongsruang S, et al. Tannic acid-stabilized silver nanoparticles used in biomedical application as an effective antimelioidosis and prolonged efflux pump inhibitor against melioidosis causative pathogen. Molecules 2021; 26: 1004.
41. Khameneh B, Iranshahy M, Vahdati-Mashhadian N, Sahebkar A, Fazly Bazzaz BS. Non-antibiotic adjunctive therapy: a promising approach to fight tuberculosis. Pharmacol Res 2019; 146: 104289.
42. Talebi-Taher M, Gholami A, Rasouli-Kouhi S, Adabi M. Role of efflux pump inhibitor in decreasing antibiotic cross-resistance of Pseudomonas aeruginosa in a burn hospital in Iran. J Infect Dev Ctries 2016; 10: 600-604.
43. Kuok C-F, Hoi S-O, Hoi C-F, Chan C-H, Fong I-H, Ngok C-K, et al. Synergistic antibacterial effects of herbal extracts and antibiotics on methicillin-resistant Staphylococcus aureus: A computational and experimental study. Exp Biol Med (Maywood) 2017; 242: 731-743.
44. Kalishwaralal K, BarathManiKanth S, Pandian SRK, Deepak V, Gurunathan S. Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis. Colloids Surf B Biointerfaces 2010; 79: 340-344.
45. Ilyas M, Niaz F, Ishaq R, Khanum A. Detection of KatG Mutation in MDR Mycobacterium tuberculosis isolates by PCR-RFLP and DNA Sequencing. Bangladesh J Med Sci 2023; 22: 804-808.
2. Rossi E, La Rosa R, Bartell JA, Marvig RL, Haagensen JAJ, Sommer LM, et al. Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis. Nat Rev Microbiol 2021; 19: 331-342.
3. Morin CD, Déziel E, Gauthier J, Levesque RC, Lau GW. An organ system-based synopsis of Pseudomonas aeruginosa virulence. Virulence 2021; 12: 1469-1507.
4. Joseph MPS, Gautam MS, Verma MT, Lal MMN, Madhale DMD (2022). Infection control & safety: Xoffencerpublication. https://www.amazon.in/INFECTION-CONTROL-SAFETY-PAMELA-SHALINI/dp/B0BTJ92ZMT
5. Salam MA, Al-Amin MY, Salam MT, Pawar JS, Akhter N, Rabaan AA, et al. Antimicrobial resistance: a growing serious threat for global public health. Healthcare (Basel) 2023; 11: 1946.
6. Mohanty H, Pachpute S, Yadav RP. Mechanism of drug resistance in bacteria: efflux pump modulation for designing of new antibiotic enhancers. Folia Microbiol (Praha) 2021; 66: 727-739.
7. Blair JM, Piddock LJ. How to measure export via bacterial multidrug resistance efflux pumps. mBio 2016; 7(4): e00840-16.
8. Hajiagha MN, Kafil HS. Efflux pumps and microbial biofilm formation. Infect Genet Evol 2023; 112: 105459.
9. Abdi SN, Ghotaslou R, Ganbarov K, Mobed A, Tanomand A, Yousefi M, et al. Acinetobacter baumannii efflux pumps and antibiotic resistance. Infect Drug Resist 2020; 13: 423-434.
10. Huang L, Wu C, Gao H, Xu C, Dai M, Huang L, et al. Bacterial multidrug efflux pumps at the frontline of antimicrobial resistance: an overview. Antibiotics (Basel) 2022; 11: 520.
11. Puzari M, Chetia P. RND efflux pump mediated antibiotic resistance in Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a major issue worldwide. World J Microbiol Biotechnol 2017; 33: 24.
12. Lorusso AB, Carrara JA, Barroso CDN, Tuon FF, Faoro H. Role of efflux pumps on antimicrobial resistance in Pseudomonas aeruginosa. Int J Mol Sci 2022; 23: 15779.
13. Verma P, Tiwari M, Tiwari V. Efflux pumps in multidrug-resistant Acinetobacter baumannii: Current status and challenges in the discovery of efflux pumps inhibitors. Microb Pathog 2021; 152: 104766.
14. Avakh A, Grant GD, Cheesman MJ, Kalkundri T, Hall S. The art of war with Pseudomonas aeruginosa: targeting Mex efflux pumps directly to strategically enhance antipseudomonal drug efficacy. Antibiotics (Basel) 2023; 12: 1304.
15. Kumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, et al. Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 2023; 181: 106182.
16. Gaurav A, Bakht P, Saini M, Pandey S, Pathania R. Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors. Microbiology (Reading) 2023; 169: 001333.
17. Threlfall T. The infrared spectra of amides. Part 1. The stretching vibrations of primary carboxamides. Vib Spectrosc 2022; 121: 103386.
18. Martin N, Cirujano FG. Heterogeneous catalytic direct amide bond formation. Catal Commun 2022; 164: 106420.
19. Puri S, Negi D. Simple to complex Amide Derivatives as potent Anti‐Tuberculosis Agents: A Literature Survey of the past Decade. ChemistrySelect 2022; 7(43): e202202584.
20. Pal R, Singh K, Paul J, Khan SA, Naim MJ, Akhtar MJ. Overview of chemistry and therapeutic potential of non-nitrogen heterocyclics as anticonvulsant agents. Curr Neuropharmacol 2022; 20: 1519-1553.
21. Alam A, Ali M, Rehman NU, Latif A, Shah AJ, Wazir NU, et al. Synthesis and characterization of biologically active flurbiprofen amide derivatives as selective prostaglandin-endoperoxide synthase II inhibitors: In vivo anti-inflammatory activity and molecular docking. Int J Biol Macromol 2023; 228: 659-670.
22. Galewicz-Walesa K, Pachuta-Stec A. The synthesis and properties of N-substituted amides of 1-(5-methylthio-1, 2, 4-triazol-3-yl)-cyclohexane-2-carboxylic acid. Ann Univ Mariae Curie Skłodowska Sect AA Chem 2003; 58: 118-121.
23. Graybill TL, Ross MJ, Gauvin BR, Gregory JS, Harris AL, Ator MA, et al. Synthesis and evaluation of azapeptide-derived inhibitors of serine and cysteine proteases. Bioorg Med Chem Lett 1992; 2: 1375-1380.
24. Moise M, Sunel V, Profire L, Popa M, Lionte C. Synthesis and antimicrobial activity of some new (sulfon-amidophenyl)-amide derivatives of N-(4-nitrobenzoyl)-phenylglycine and N-(4-nitrobenzoyl)-phenylalanine. FARMACIA 2008; 56: 283-289.
25. Warnecke A, Fichtner I, Sass G, Kratz F. Synthesis, cleavage profile, and antitumor efficacy of an albumin‐binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B. Arch Pharm (Weinheim) 2007; 340: 389-395.
26. Suravaram S, Hada V, Siddiqui IA. Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints. Indian J Med Microbiol 2021; 39: 315-319.
27. Sepehr A, Fereshteh S, Shahrokhi N. Detection of efflux pump using ethidium bromide-agar cartwheel method in Acinetobacter baumannii clinical isolates. J Med Microbiol Infect Dis 2022; 10: 36-41.
28. Palladini G, Garbarino C, Luppi A, Russo S, Filippi A, Arrigoni N, et al. Comparison between broth microdilution and agar disk diffusion methods for antimicrobial susceptibility testing of bovine mastitis pathogens. J Microbiol Methods 2023; 212: 106796.
29. Kamble PA, Phadke M. Use of checkerboard assay to determine the synergy between essential oils extracted from leaves of Aegle marmelos (L.) Correa and nystatin against Candida albicans. Ayu 2023; 44: 38-43.
30. Black C, Al Mahmud H, Howle V, Wilson S, Smith AC, Wakeman CA. Development of a Polymicrobial Checkerboard Assay as a tool for Determining Combinatorial Antibiotic Effectiveness in Polymicrobial Communities. Antibiotics (Basel) 2023; 12: 1207.
31. Süer D (2021). Molecular detection of some Virulence Genes In Pseudomonas aeruginosa isolated from clinical Source: Doctoral dissertation, Near East University. https://docs.neu.edu.tr/library/9299808495.pdf
32. Adilabdulhady D, Kadhim HM. Molecular detection of Pseudomonas aeruginosa isolated from different clinical cases and test antibiotics Sensitivity on the Bacterial Growth. Pak J Med Health Sci 2022; 16: 587-588.
33. Rashid A, Akram M, Kayode OT, Kayode A. Clinical features and epidemiological patterns of infections by multidrug resistance Staphylococcus aureus and Pseudomonas aeruginosa in patients with burns. Biomed J Sci Tech Res 2020; 25: 19272-19279.
34. Rehman A, Patrick WM, Lamont IL. Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem. J Med Microbiol 2019; 68: 1-10.
35. Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, Gómez-Zorrilla S, et al. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev 2019; 32(4): e00031-19.
36. Tilahun M, Shibabaw A, Adane M. Prevalence and multidrug resistance patterns of bacterial pathogens in wastewater and drinking water systems from hospital and non-hospital environments in Ethiopia: a systematic review and meta-analysis. BMC Infect Dis 2025; 25: 250.
37. Yuan Y, Rosado-Lugo JD, Zhang Y, Datta P, Sun Y, Cao Y, et al. Evaluation of heterocyclic carboxamides as potential efflux pump inhibitors in Pseudomonas aeruginosa. Antibiotics (Basel) 2021; 11: 30.
38. Lamut A, Peterlin Mašič L, Kikelj D, Tomašič T. Efflux pump inhibitors of clinically relevant multidrug resistant bacteria. Med Res Rev 2019; 39: 2460-2504.
39. Mech P (2024). Modulation of antibacterial resistance with special reference to inhibition of efflux pump in Staphylococcus aureus: Indian Veterinary Research Institute.
40. Srichaiyapol O, Thammawithan S, Siritongsuk P, Nasompag S, Daduang S, Klaynongsruang S, et al. Tannic acid-stabilized silver nanoparticles used in biomedical application as an effective antimelioidosis and prolonged efflux pump inhibitor against melioidosis causative pathogen. Molecules 2021; 26: 1004.
41. Khameneh B, Iranshahy M, Vahdati-Mashhadian N, Sahebkar A, Fazly Bazzaz BS. Non-antibiotic adjunctive therapy: a promising approach to fight tuberculosis. Pharmacol Res 2019; 146: 104289.
42. Talebi-Taher M, Gholami A, Rasouli-Kouhi S, Adabi M. Role of efflux pump inhibitor in decreasing antibiotic cross-resistance of Pseudomonas aeruginosa in a burn hospital in Iran. J Infect Dev Ctries 2016; 10: 600-604.
43. Kuok C-F, Hoi S-O, Hoi C-F, Chan C-H, Fong I-H, Ngok C-K, et al. Synergistic antibacterial effects of herbal extracts and antibiotics on methicillin-resistant Staphylococcus aureus: A computational and experimental study. Exp Biol Med (Maywood) 2017; 242: 731-743.
44. Kalishwaralal K, BarathManiKanth S, Pandian SRK, Deepak V, Gurunathan S. Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis. Colloids Surf B Biointerfaces 2010; 79: 340-344.
45. Ilyas M, Niaz F, Ishaq R, Khanum A. Detection of KatG Mutation in MDR Mycobacterium tuberculosis isolates by PCR-RFLP and DNA Sequencing. Bangladesh J Med Sci 2023; 22: 804-808.
| Files | ||
| Issue | Vol 17 No 4 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i4.19271 | |
| Keywords | ||
| Anti-bacterial agents Checkerboard assay Drug resistance Efflux pump Pseudomonas aeruginosa | ||
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How to Cite
1.
Ullah F, Ali M, Niaz F, Ali Khan I, Wali Khan S, Khan M, Ishaq R, Manan A, Yu Y, Ilyas M. Exploring novel amides as efflux pump inhibitors for overcoming antibiotic resistance in multidrug-resistant Pseudomonas aeruginosa. Iran J Microbiol. 2025;17(4):569-576.
