Plasposon mutagenesis in Pseudomonas aeruginosa isolates illustrates the role of ABC transporter in intrinsic resistance to antibiotics
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Abstract
Pseudomonas aeruginosa is an opportunist pathogen most commonly related to nosocomial infections. P. aeruginosa infection therapy poses a significant challenge due to its ability to resist various antibiotics currently available. As a result, excessive use of antibiotics during therapy expedites the development of multidrug resistant P. aeruginosa. Hence, this study aimed to identify novel genes involved in multiple antibiotic resistance using plasposon mutagenesis technique. One hundred and ten P. aeruginosa isolates were collected from various clinical sources involving urine, burns and wound’s pus. An antimicrobial susceptibility test was performed to detect their resistance to 18 antibiotics. Results showed that all isolates were resistant to ampicillin and tetracycline, and the highest resistance ras were detected for nitrofurantoin and sulfamethoxazole (99%), followed by amoxiclav, cefotaxime, cefoxitin, ceftriaxone, and kanamycin (98%). While the lowest resistance rate was towards imipenem (49%). Plasposon mutagenesis was used to detect the genes involved in multi-antibiotic resistance. The pTnMod-Gm was introduced to the recipient P. aeruginosa PA4 isolate via triparental mating using E. coli HB101/ pRK2013 as a helper strain. Mutants were screened for resistance defects by plating them on nutrient agar supplemented with different antibiotics. Two mutants were identified; one (M1) exhibited susceptibility to tetracycline, cefotaxime, and ceftazidime, and the other (M9) to ceftazidime and ceftriaxone. The analysis of these mutants revealed the insertion of the plasposon into an open reading frame for the ABC transporter in P. aeruginosa, which plays a distinctive role in extruding antibiotics out of cells.
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Keywords
Antibiotic resistance, cloning, P. aeruginosa, plasposon mutagenesis
References
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Akhtar, A. A. & Turner, D. P. J. (2022). The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microbial Pathogenesis, 171, 105734. Doi: 10.1016/j.micpath.2022.105734
Blanco, P., Hernando-Amado, S., Reales-Calderon, J. A., Corona, F., Lira, F., Alcalde-Rico, M. & Martinez, J. L. (2016). Bacterial multidrug efflux pumps: much more than antibiotic resistance determinants. Microorganisms, 4 (14). Doi: 10.3390/microorganisms4010014
Chen, L., Yang, L., Zhao, X., Shen, L. & Duan, K. (2010). Identification of Pseudomonas aeruginosa genes associated with antibiotic susceptibility. Science China Life Sciences, 53,1247–1251. Doi.org/10.1007/s11427-010-4071-8
Chen, L. & Duan, K. (2016). A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 60 (5), 3016-3024. Doi: 10.1128/AAC.02986-15
Choi, C. C. & Ford, R.C. (2021). ATP binding cassette importers in eukaryotic organisms. Biological Reviews, 96, 1318–1330. Doi.org/10.1111/brv.12702
Davidson, A. L., Dassa, E., Orelle, C. & Chen, J. (2008). Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems. Microbiology and Molecular Biology Reviews, 72(2), 317–364. Doi: 10.1128/MMBR.00031-07.
Dawson, R. J. P., Hollenstein, K. & Locher, K. P. (2007). Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism. Molecular Microbiology, 65(2), 250–257. Doi.org/10.1111/j.1365-2958.2007.05792.x
de Sousa, T., Silva, C., Alves, O., Costa, E., Igrejas, G., Poeta, P. & Hébraud, M. (2023). Determination of Antimicrobial Resistance and the Impact of Imipenem + Cilastatin Synergy with Tetracycline in Pseudomonas aeruginosa Isolates from Sepsis. Microorganisms, 11, 2687. Doi.org/10.3390/microorganisms11112687
Delmar, J.A., Su, C.C. & Yu, E.W. (2014). 'Bacterial multidrug efflux transporters'. Annual review of biophysics, 43, 93-117. Doi.org/10.1146/annurev-biophys-051013-0228 55
Dennis, J. J. & Zylstra, G. J. (1998). Plasposons: modular self-cloning mini transposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Applied and Environmental Microbiology, 64(7), 2710-2715. Doi: 10.1128/aem.64.7.2710-2715.1998
Du, D., van Veen, H. W., Murakami, S., Pos, K. M. & Luisi, B. F. (2015). Structure, mechanism and cooperation of bacterial multidrug transporters. Current Opinion in Structural Biology, 33, 76–91. Doi: 10.1016/j.sbi.2015.07.015
Faisal, R. M. (2019). Understanding the role of dibenzofuran 4, 4a dioxygenase reveals a silent pathway for biphenyl degradation in Sphingomonas wittichii RW1. Furthermore, it helps to engineer dioxin-degrading strains (Doctoral dissertation, Rutgers University-School of Graduate Studies).
Faisal, R. M. & Rasol, A. H. (2022). Physiological role of isocitrate lyase in dibenzo-p-dioxin and dibenzofuran metabolism by Sphingomonas wittichii RW1. Journal of Genetic Engineering and Biotechnology, 20(1), 52. Doi.org/10.1186/s43141-022-00334-3
Grossman, T. H. (2016). Tetracycline Antibiotics and Resistance. Cold Spring Harbor Perspective Medicine, 6, a025387. Doi: 10.1101/cshperspect.a025387
Hernando-Amado, A. & Martínez, J. L. (2023). Special Issue: “Antimicrobial Resistance in Pseudomonas aeruginosa”. Microorganisms, 11, 744. Doi: 10.3390/microorganisms11030744
Hocquet, D., Muller, A. & Bertrand, X. (2016). What happens in hospitals does not stay in hospitals: antibiotic-resistant bacteria in hospital wastewater systems. Journal of Hospital Infection, 93 (4), 395–402. Doi: 10.1016/j.jhin.2016.01.010.
Hulen, C., Racine, P., Chevalier, S., Feuilloley, M. & Lomer, N. (2020). Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in Pseudomonas aeruginosa PAO1. Antibiotics, 9, 596. Doi: 10.3390/antibiotics9090596.
Hulen, C., Racine, P., Feuilloley, M., Elomri, A. & Lomr, N. (2022). Effects of Verapamil and Two Bisbenzylisoquinolines,Curine and Guattegaumerine Extracted from Isolona hexaloba, on the Inhibition of ABC Transporters from Pseudomonas aeruginosa. Antibiotics, 11(5), 700. Doi: 10.3390/antibiotics11050700
Kanagaratnam, R., Sheikh, R., Alharbi, F. & Kwon, D. H. (2017). An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG). Phytomedicine, 36, 194–200. Doi: 10.1016/j.phymed.2017.10.010.
Khan, Z. A., Siddiqui, M. F. & Park, S. (2019). Current and emerging methods of antibiotic susceptibility testing. Diagnostics, 9 (2), 49. Doi: 10.3390/diagnostics9020049.
Locher, K. (2016). Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nature Structural and Molecular Biology, 23(6), 487–493. Doi.org/10.1038/nsmb.3216
Nasser, M., Palwe, S., Bhargava, R. N., Feuilloley, M. G. J. & Kharat, A. S. (2020). Retrospective Analysis on Antimicrobial Resistance Trends and Prevalence of β-lactamases in Escherichia coli and ESKAPE Pathogens Isolated from Arabian Patients during 2000–2020. Microorganisms, 8(10),1626. Doi: 10.3390/microorganis ms8101626
Orelle, C., Mathieu, K. & Jault, J. (2019). Multidrug ABC transporters in bacteria. Research in Microbiology, 170(8), 381-391. Doi: 10.1016/j.resmic.2019.06.001
Pachori, P., Gothalwal, R. & Gandhi, P. (2019). Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes & Diseases, 6(2), 109-119. Doi: 10.1016/j.gendis.2019.04.001
Park, A. J., Surette, M. D. & Khursigara, C. M. (2014). Antimicrobial targets localize to the extracellular vesicle-associated proteome of Pseudomonas aeruginosa grown in a biofilm. Frontiers in Microbiology, 5, 464. Doi.org/10.3389/fmicb.2014.00464
Pletzer, D., Braun, Y., Dubiley, S., Lafon, C., Kohler, T., Page, M.G., Mourez, M., Severinov, K. & Weingart, H. (2015). The Pseudomonas aeruginosa PA14 ABC transporter NppA1A2BCD is required for uptake of peptidyl nucleoside antibiotics. Journal of Bacteriology, 197(13), 2217–2228. Doi: 10.1128/JB.00234-15.
Rasheed, M. N., Latteef, N. S., Jassim, A. B. & Nader, M. I. (2016). Rapid Identification of Pseudomonas aeruginosa by Using Real Time PCR. Kufa Journal for Veterinary Medical Sciences, 7 (1B), 80-86. Doi.org/10.36326/kjvs/2016/v7i1B4275
Roulová, N., Mot’ková, P., Brožková, I. & Pejchalová. M. (2022). Antibiotic resistance of Pseudomonas aeruginosa isolated from hospital wastewater in the Czech Republic. Journal of Water and Health, 20 (4), 693. Doi: 10.2166/wh.2022.101
Saleh, B. H., Al-Jumaily, E. F. & Shalal M. Hussain, S. M. (2012). Production and purification of exotoxin A extracted from social strain of Pesudomonas aeruginosa in Iraq. Trends in Biology Research, 1 (1), 15-23.
Sambrook, J. (1989). Molecular cloning: a laboratory manual. Synthetic oligonucleotides.
Shaikh, S., Fatima, J., Shakil, S., Rizvi, S. M. D. & Kamal, M. A. (2015). Prevalence of multidrug resistant and extended spectrum beta lactamase producing Pseudomonas aeruginosa in a tertiary care hospital. Saudi Journal of Biological Sciences, 22 (1), 62-4. Doi: 10.1016/j.sjbs.2014.06.001.
Shbaita, S., Abatli, S., Sweileh, M. W., Aiesh, B. M., Sabateen, A., Salameh, H. T., AbuTaha, A. & Zyoud, S. H. (2023). Antibiotic resistance profiles and associated factors of Pseudomonas Infections among patients admitted to large tertiary care hospital from a developing country. Antimicrobial Resistance & Infection Control, 12,149. Doi.org/10.1186/s13756-023-01355-4
Streeter, K. & Katouli, M. (2016). Pseudomonas aeruginosa: A review of their Pathogenesis and Prevalence in Clinical Settings and the Environment. Infection Epidemiology and Microbiology, 2 (1), 25- 32. Doi: 10.18869/modares.iem.2.1.25
Tacconelli, E., Magrini, N., Kahlmeter, G. & Singh, N. (2017). Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. World Health Organization, 27, 318–327.
Tanakaa, K. J., Songa, S., Masonb, K. & Pinkett, H. W. (2018). Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis. BBA- Biomembranes, 1860(4), 868- 877. Doi: 10.1016/j.bbamem.2017.08.011.
Winn, W., Allen, S., & Janda, W. (2006). Koneman's color atlas and textbook of diagnostic microbiology. 6th ed. Lippincott Williams and Wilkins, Philadelphia.
Winsor, G.L., Lam, D.K., Fleming, L., Lo, R., Whiteside, M.D., Yu, N.Y., Hancock, R.E. & Brinkman, F.S. (2011). Pseudomonas genome database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Research, 39, D596 –D600.Doi: 10.1093/nar/gkq869
Zhou, J., Hao, D., Wang, X., Liu,T., He, C., Xie, F., Sun, Y. & Zhang, J. (2006). An important role of a “probable ATP-binding component of ABC transporter” during the process of Pseudomonas aeruginos resistance to fluoroquinolone. Proteomics, 6, 2495–2503.
Akhtar, A. A. & Turner, D. P. J. (2022). The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microbial Pathogenesis, 171, 105734. Doi: 10.1016/j.micpath.2022.105734
Blanco, P., Hernando-Amado, S., Reales-Calderon, J. A., Corona, F., Lira, F., Alcalde-Rico, M. & Martinez, J. L. (2016). Bacterial multidrug efflux pumps: much more than antibiotic resistance determinants. Microorganisms, 4 (14). Doi: 10.3390/microorganisms4010014
Chen, L., Yang, L., Zhao, X., Shen, L. & Duan, K. (2010). Identification of Pseudomonas aeruginosa genes associated with antibiotic susceptibility. Science China Life Sciences, 53,1247–1251. Doi.org/10.1007/s11427-010-4071-8
Chen, L. & Duan, K. (2016). A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 60 (5), 3016-3024. Doi: 10.1128/AAC.02986-15
Choi, C. C. & Ford, R.C. (2021). ATP binding cassette importers in eukaryotic organisms. Biological Reviews, 96, 1318–1330. Doi.org/10.1111/brv.12702
Davidson, A. L., Dassa, E., Orelle, C. & Chen, J. (2008). Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems. Microbiology and Molecular Biology Reviews, 72(2), 317–364. Doi: 10.1128/MMBR.00031-07.
Dawson, R. J. P., Hollenstein, K. & Locher, K. P. (2007). Uptake or extrusion: crystal structures of full ABC transporters suggest a common mechanism. Molecular Microbiology, 65(2), 250–257. Doi.org/10.1111/j.1365-2958.2007.05792.x
de Sousa, T., Silva, C., Alves, O., Costa, E., Igrejas, G., Poeta, P. & Hébraud, M. (2023). Determination of Antimicrobial Resistance and the Impact of Imipenem + Cilastatin Synergy with Tetracycline in Pseudomonas aeruginosa Isolates from Sepsis. Microorganisms, 11, 2687. Doi.org/10.3390/microorganisms11112687
Delmar, J.A., Su, C.C. & Yu, E.W. (2014). 'Bacterial multidrug efflux transporters'. Annual review of biophysics, 43, 93-117. Doi.org/10.1146/annurev-biophys-051013-0228 55
Dennis, J. J. & Zylstra, G. J. (1998). Plasposons: modular self-cloning mini transposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Applied and Environmental Microbiology, 64(7), 2710-2715. Doi: 10.1128/aem.64.7.2710-2715.1998
Du, D., van Veen, H. W., Murakami, S., Pos, K. M. & Luisi, B. F. (2015). Structure, mechanism and cooperation of bacterial multidrug transporters. Current Opinion in Structural Biology, 33, 76–91. Doi: 10.1016/j.sbi.2015.07.015
Faisal, R. M. (2019). Understanding the role of dibenzofuran 4, 4a dioxygenase reveals a silent pathway for biphenyl degradation in Sphingomonas wittichii RW1. Furthermore, it helps to engineer dioxin-degrading strains (Doctoral dissertation, Rutgers University-School of Graduate Studies).
Faisal, R. M. & Rasol, A. H. (2022). Physiological role of isocitrate lyase in dibenzo-p-dioxin and dibenzofuran metabolism by Sphingomonas wittichii RW1. Journal of Genetic Engineering and Biotechnology, 20(1), 52. Doi.org/10.1186/s43141-022-00334-3
Grossman, T. H. (2016). Tetracycline Antibiotics and Resistance. Cold Spring Harbor Perspective Medicine, 6, a025387. Doi: 10.1101/cshperspect.a025387
Hernando-Amado, A. & Martínez, J. L. (2023). Special Issue: “Antimicrobial Resistance in Pseudomonas aeruginosa”. Microorganisms, 11, 744. Doi: 10.3390/microorganisms11030744
Hocquet, D., Muller, A. & Bertrand, X. (2016). What happens in hospitals does not stay in hospitals: antibiotic-resistant bacteria in hospital wastewater systems. Journal of Hospital Infection, 93 (4), 395–402. Doi: 10.1016/j.jhin.2016.01.010.
Hulen, C., Racine, P., Chevalier, S., Feuilloley, M. & Lomer, N. (2020). Identification of the PA1113 Gene Product as an ABC Transporter Involved in the Uptake of Carbenicillin in Pseudomonas aeruginosa PAO1. Antibiotics, 9, 596. Doi: 10.3390/antibiotics9090596.
Hulen, C., Racine, P., Feuilloley, M., Elomri, A. & Lomr, N. (2022). Effects of Verapamil and Two Bisbenzylisoquinolines,Curine and Guattegaumerine Extracted from Isolona hexaloba, on the Inhibition of ABC Transporters from Pseudomonas aeruginosa. Antibiotics, 11(5), 700. Doi: 10.3390/antibiotics11050700
Kanagaratnam, R., Sheikh, R., Alharbi, F. & Kwon, D. H. (2017). An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG). Phytomedicine, 36, 194–200. Doi: 10.1016/j.phymed.2017.10.010.
Khan, Z. A., Siddiqui, M. F. & Park, S. (2019). Current and emerging methods of antibiotic susceptibility testing. Diagnostics, 9 (2), 49. Doi: 10.3390/diagnostics9020049.
Locher, K. (2016). Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nature Structural and Molecular Biology, 23(6), 487–493. Doi.org/10.1038/nsmb.3216
Nasser, M., Palwe, S., Bhargava, R. N., Feuilloley, M. G. J. & Kharat, A. S. (2020). Retrospective Analysis on Antimicrobial Resistance Trends and Prevalence of β-lactamases in Escherichia coli and ESKAPE Pathogens Isolated from Arabian Patients during 2000–2020. Microorganisms, 8(10),1626. Doi: 10.3390/microorganis ms8101626
Orelle, C., Mathieu, K. & Jault, J. (2019). Multidrug ABC transporters in bacteria. Research in Microbiology, 170(8), 381-391. Doi: 10.1016/j.resmic.2019.06.001
Pachori, P., Gothalwal, R. & Gandhi, P. (2019). Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review. Genes & Diseases, 6(2), 109-119. Doi: 10.1016/j.gendis.2019.04.001
Park, A. J., Surette, M. D. & Khursigara, C. M. (2014). Antimicrobial targets localize to the extracellular vesicle-associated proteome of Pseudomonas aeruginosa grown in a biofilm. Frontiers in Microbiology, 5, 464. Doi.org/10.3389/fmicb.2014.00464
Pletzer, D., Braun, Y., Dubiley, S., Lafon, C., Kohler, T., Page, M.G., Mourez, M., Severinov, K. & Weingart, H. (2015). The Pseudomonas aeruginosa PA14 ABC transporter NppA1A2BCD is required for uptake of peptidyl nucleoside antibiotics. Journal of Bacteriology, 197(13), 2217–2228. Doi: 10.1128/JB.00234-15.
Rasheed, M. N., Latteef, N. S., Jassim, A. B. & Nader, M. I. (2016). Rapid Identification of Pseudomonas aeruginosa by Using Real Time PCR. Kufa Journal for Veterinary Medical Sciences, 7 (1B), 80-86. Doi.org/10.36326/kjvs/2016/v7i1B4275
Roulová, N., Mot’ková, P., Brožková, I. & Pejchalová. M. (2022). Antibiotic resistance of Pseudomonas aeruginosa isolated from hospital wastewater in the Czech Republic. Journal of Water and Health, 20 (4), 693. Doi: 10.2166/wh.2022.101
Saleh, B. H., Al-Jumaily, E. F. & Shalal M. Hussain, S. M. (2012). Production and purification of exotoxin A extracted from social strain of Pesudomonas aeruginosa in Iraq. Trends in Biology Research, 1 (1), 15-23.
Sambrook, J. (1989). Molecular cloning: a laboratory manual. Synthetic oligonucleotides.
Shaikh, S., Fatima, J., Shakil, S., Rizvi, S. M. D. & Kamal, M. A. (2015). Prevalence of multidrug resistant and extended spectrum beta lactamase producing Pseudomonas aeruginosa in a tertiary care hospital. Saudi Journal of Biological Sciences, 22 (1), 62-4. Doi: 10.1016/j.sjbs.2014.06.001.
Shbaita, S., Abatli, S., Sweileh, M. W., Aiesh, B. M., Sabateen, A., Salameh, H. T., AbuTaha, A. & Zyoud, S. H. (2023). Antibiotic resistance profiles and associated factors of Pseudomonas Infections among patients admitted to large tertiary care hospital from a developing country. Antimicrobial Resistance & Infection Control, 12,149. Doi.org/10.1186/s13756-023-01355-4
Streeter, K. & Katouli, M. (2016). Pseudomonas aeruginosa: A review of their Pathogenesis and Prevalence in Clinical Settings and the Environment. Infection Epidemiology and Microbiology, 2 (1), 25- 32. Doi: 10.18869/modares.iem.2.1.25
Tacconelli, E., Magrini, N., Kahlmeter, G. & Singh, N. (2017). Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. World Health Organization, 27, 318–327.
Tanakaa, K. J., Songa, S., Masonb, K. & Pinkett, H. W. (2018). Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis. BBA- Biomembranes, 1860(4), 868- 877. Doi: 10.1016/j.bbamem.2017.08.011.
Winn, W., Allen, S., & Janda, W. (2006). Koneman's color atlas and textbook of diagnostic microbiology. 6th ed. Lippincott Williams and Wilkins, Philadelphia.
Winsor, G.L., Lam, D.K., Fleming, L., Lo, R., Whiteside, M.D., Yu, N.Y., Hancock, R.E. & Brinkman, F.S. (2011). Pseudomonas genome database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Research, 39, D596 –D600.Doi: 10.1093/nar/gkq869
Zhou, J., Hao, D., Wang, X., Liu,T., He, C., Xie, F., Sun, Y. & Zhang, J. (2006). An important role of a “probable ATP-binding component of ABC transporter” during the process of Pseudomonas aeruginos resistance to fluoroquinolone. Proteomics, 6, 2495–2503.
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Plasposon mutagenesis in Pseudomonas aeruginosa isolates illustrates the role of ABC transporter in intrinsic resistance to antibiotics. (2024). Journal of Applied and Natural Science, 16(3), 1256-1264. https://doi.org/10.31018/jans.v16i3.5856
