Antibiotic-Induced Changes in Efflux Transporter Expression: A Key Factor in Pseudomonas aeruginosa Biofilm Resistance

Zara Imtiaz; Avinash Chatoo; Will Wang; Weiqi Li; Paramita Basu

doi:10.18006/2024.12(2).274.283

Authors

Zara Imtiaz Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027
Avinash Chatoo Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027
Will Wang Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027
Weiqi Li Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027
Paramita Basu Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027

DOI:

https://doi.org/10.18006/2024.12(2).274.283

Keywords:

Antimicrobial Resistance, Biofilm, Pseudomonas aeruginosa, Ceftazidime, Ofloxacin, Tobramycin, Antibiotic Sensitivity, Efflux transporters

Abstract

Listed by WHO as an antibiotic-resistant priority pathogen, Pseudomonas aeruginosa (P.A.) is a serious threat in nosocomial infections. Its high antibiotic resistance is attributed to major mechanisms that can be categorized into intrinsic, acquired, and adaptive resistance. This study tests the ability of three commonly used antibiotics to inhibit new biofilm formation and eradicate mature biofilm growth, as well as investigate changes in the expression levels of selected genes coding for multidrug efflux pumps in P.A. planktonic cells and biofilms before and after treatment with antibiotics to provide a conceptual estimate of the activity of the efflux transporters that work to extrude antibiotics leading to a reduction in their effectiveness. Antimicrobial susceptibility testing was conducted with Ofloxacin (OFLX), Tobramycin (TOB), and Ceftazidime (CAZ) to determine Mean Inhibitory Concentration (MIC) and Mean Bactericidal Concentration (MBC) using microtiter plate-based biofilm assay and spectrophotometric quantification. Extraction of total RNA was performed from planktonic cultures, inhibition phase, and eradication phase P.A. biofilms. Real-time quantitative reverse transcriptase PCR was utilized to analyze the changes in expression of the mexAB, mexXY, and oprM genes. Three (3) antibiotics that have proven to show less resistance are OFLX, TOB, and CAZ when tested against overnight cultures of P.A. strain PA01. Results showed that OFLX is best for bactericidal properties, which is also supported by the viability assay data obtained from Propidium Iodide staining. Our study showed that the PAO1 strain is susceptible to OFLX for both inhibition and eradication of mature biofilms. TOB was most effective at higher concentrations in the eradication phase.

Author Biography

Paramita Basu, Touro College of Pharmacy, 230 W 125 Street, New York, NY 10027

New York College of Podiatric Medicine, 53E 124 Street, New York, NY 10035

References

Alav, I., Sutton, J. M., & Rahman, K. M. (2018). Role of bacterial efflux pumps in biofilm formation. Journal of Antimicrobial Chemotherapy, 73(8), 2003-2020. doi:10.1093/jac/dky042 DOI: https://doi.org/10.1093/jac/dky042

Bhandari, S., Adhikari, S., Karki, D., Chand, A. B., Sapkota, S., et al. (2022). Antibiotic Resistance, Biofilm Formation and Detection of mexA/mexB Efflux-Pump Genes Among Clinical Isolates of Pseudomonas aeruginosa in a Tertiary Care Hospital, Nepal. Frontiers in Tropical Diseases, 17(2), 2021. https://doi.org/10.3389/fitd.2021.810863 DOI: https://doi.org/10.3389/fitd.2021.810863

Cavalieri, S. J. (2009). Manual of Antimicrobial Susceptibility Testing. United States: American Society for Microbiology.

Goli, H. R., Nahaei, M. R., Rezaee, M. A., Hasani, A., Kafil, H. S., et al. (2018). Role of MexAB-OprM and MexXY-OprM efflux pumps and class 1 integrons in resistance to antibiotics in burn and intensive care unit isolates of pseudomonas aeruginosa. Journal of Infection and Public Health, 11(3), 364-372. doi:10.1016/j.jiph.2017.09.016 DOI: https://doi.org/10.1016/j.jiph.2017.09.016

Hassuna, N. A., Darwish, M. K., Sayed, M., & Ibrahem, R. A. (2020). Molecular epidemiology and mechanisms of high-level resistance to meropenem and imipenem in Pseudomonas aeruginosa. Infection and Drug Resistance, 13, 285-293. doi:10.2147/IDR.S233808 DOI: https://doi.org/10.2147/IDR.S233808

Kishk, R.M., Abdalla, M.O., Hashish, A.A., Nemr, N.A., El Nahhas, N., et al. (2020). Efflux MexAB-Mediated Resistance in P. aeruginosa Isolated from Patients with Healthcare Associated Infections. Pathogens 9(6), 471. https://doi.org/10.3390/ pathogens9060471 DOI: https://doi.org/10.3390/pathogens9060471

Lorusso, A.B., Carrara, J.A., Barroso, C.D.N., Tuon, F.F., & Faoro, H. (2022). Role of Efflux Pumps on

Antimicrobial Resistance in Pseudomonas aeruginosa. International Journal of Molecular Sciences, 23, 15779. https://doi.org/10.3390/ijms232415779 DOI: https://doi.org/10.3390/ijms232415779

Lee, J., & Zhang, L. (2014). The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein & Cell, 6(1), 26-41. doi:10.1007/s13238-014-0100-x DOI: https://doi.org/10.1007/s13238-014-0100-x

Li, X., Plésiat, P., & Nikaido, H. (2015). The challenge of efflux-mediated antibiotic resistance in gram-negative bacteria. Clinical Microbiology Reviews, 28(2), 337-418. doi:10.1128/CMR.00117-14 DOI: https://doi.org/10.1128/CMR.00117-14

Lund-Palau, H., Turnbull, A. R., Bush, A., Bardin, E., Cameron, L., et al. (2016). Pseudomonas aeruginosa infection in cystic fibrosis: Pathophysiological mechanisms and therapeutic approaches Informa U.K. Limited. doi:10.1080/17476348.2016.1177460 DOI: https://doi.org/10.1080/17476348.2016.1177460

Mangiaterra, G., Cedraro, N., Vaiasicca, S., Citterio, B., Galeazzi, R., et al. (2020). Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model. Antibiotics, 9(7), 399. https://doi.org/10.3390/antibiotics9070399. DOI: https://doi.org/10.3390/antibiotics9070399

Marquez, B. (2005). Bacterial efflux systems and efflux pumps inhibitors. Biochimie, 87(12), 1137-1147. doi:10.1016/j.biochi.2005.04.012 DOI: https://doi.org/10.1016/j.biochi.2005.04.012

Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H., & Nishino, T. (2000). Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 44(9), 2242-2246. doi:10.1128/AAC.44.9.2242-2246.2000 DOI: https://doi.org/10.1128/AAC.44.9.2242-2246.2000

Morita, Y., Tomida, J., & Kawamura, Y. (2012). MexXY multidrug efflux system of Pseudomonas aeruginosa. Frontiers in Microbiology, 3, 408. doi:10.3389/fmicb.2012.00408 DOI: https://doi.org/10.3389/fmicb.2012.00408

O'Toole G. A. (2011). Microtiter dish biofilm formation assay. Journal of visualized experiments, 47, 2437. https://doi.org/10.3791/2437 DOI: https://doi.org/10.3791/2437-v

Patel, D., Sen, P., Hlaing, Y., Boadu, M., Saadeh, B., & Basu, P. (2021). Antimicrobial Resistance in Pseudomonas aeruginosa Biofilms. Journal of Pure and Applied Microbiology,15(4):2520-2528. DOI: https://doi.org/10.22207/JPAM.15.4.79

Poole, K. (2011). Pseudomonas aeruginosa: Resistance to the max. Frontiers in Microbiology, 2, 65. doi:10.3389/fmicb.2011.00065 DOI: https://doi.org/10.3389/fmicb.2011.00065

Pourakbari, B., Yaslianifard, S., Yaslianifard, S., Mahmoudi, S., Keshavarz-Valian, S., & Mamishi, S. (2016). Evaluation of efflux pumps gene expression in resistant Pseudomonas aeruginosa isolates in an Iranian referral hospital. Iranian journal of microbiology, 8(4), 249–256.

Qu, L., She, P., Wang, Y., Liu, F., Zhang, D., et al. (2016). Effects of norspermidine on pseudomonas aeruginosa biofilm formation and eradication. Microbiology Open (Weinheim), 5(3), 402-412. DOI: https://doi.org/10.1002/mbo3.338

Rutherford, S. T., & Bassler, B. L. (2012). Bacterial quorum sensing: Its role in virulence and possibilities for its control. Cold Spring Harbor Perspectives in Medicine, 2(11), a012427. DOI: https://doi.org/10.1101/cshperspect.a012427

Smith, R. S., & Iglewski, B. H. (2003). Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. The Journal of Clinical Investigation, 112(10), 1460-1465. doi:10.1172/JCI200320364 DOI: https://doi.org/10.1172/JCI200320364

Sousa, A. M., & Pereira, M. O. (2014). Pseudomonas aeruginosa diversification during infection development in cystic fibrosis Lungs—A review. Pathogens (Basel), 3(3), 680-703. DOI: https://doi.org/10.3390/pathogens3030680

Walker, J., & Moore, G. (2014). Pseudomonas aeruginosa in hospital water systems: Biofilms, guidelines, and practicalities. The Journal of Hospital Infection, 89(4), 324-327. doi:10.1016/j.jhin.2014.11.019 DOI: https://doi.org/10.1016/j.jhin.2014.11.019

World health organization. (2017). Retrieved from https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed

Zakhour, J., Sharara, S.L., Hindy, J.R., Haddad, S.F., & Kanj, S.S. (2022). Antimicrobial Treatment of Pseudomonas aeruginosa Severe Sepsis. Antibiotics, 11(10), 1432. DOI: https://doi.org/10.3390/antibiotics11101432