Expression of AcrAB Efflux Pump and Role of Mefloquine as Efflux Pump Inhibitor in MDR E.coli

Ghada F. Helaly¹, Sherine Shawky^1, , Rania Amer², OlaAbdel- kader¹, Gamal El-Sawaf¹ and Mohammed A. El Kholy³

¹Department of Microbiology, Medical Research Institute, Alexandria University, Alexandria, Egypt

²Department of Environmental Biotechnology, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Alexandria, Egypt

³Department of Microbiology and Immunology, Faculty of Pharmacy and Drug manufacturing, Pharos University, Alexandria, Egypt

Cite this paper:
Ghada F. Helaly, Sherine Shawky, Rania Amer, OlaAbdel- kader, Gamal El-Sawaf and Mohammed A. El Kholy. Expression of AcrAB Efflux Pump and Role of Mefloquine as Efflux Pump Inhibitor in MDR E.coli. American Journal of Infectious Diseases and Microbiology. 2016; 4(1):6-13. doi: 10.12691/ajidm-4-1-2

Abstract

Multi-drug resistant Escherichia coli (MDRE. coli), represents a major health problem. The AcrAB pump constitutes a major drug efflux system. Inhibition of efflux pumps is an approach to combat the drug resistance problem. Mefloquine is a bacterial RND efflux-pump inhibitor (EPI). This study aimed to investigate the expression levels of multidrug efflux genes acrAB, as well, to study the effect of mefloquine as an efflux pump inhibitor. acrA and acrB gene expression was measured using real-time PCR. Effect of mefloquine hydrochloride on minimum inhibitory concentration (MIC) of selected antibiotics against the tested isolates was determined using the broth microdilution technique. Overexpression of both genes was detected in all isolates. The differences between expression levels of both genes in MDR strains and an ATCC reference strain were statistically significant (p ˂ 0.001). There was a moderate correlation between acrA and acrB genes expression levels. (r = 0.593, p < 0.001). The isolates showed a decrease in MIC in presence of EPI. Susceptibility to levofloxacin was recovered in 95% of the tested isolates, 62.5% to ceftriaxone and in 5% each to ciprofloxacin and Gentamicin. In conclusion, Inhibition of acrAB efflux pump in MDR E. coli by mefloquine could be considered in the design of future antibiotics.

Keywords:
AcrAB Mefloquine EPI

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References:

[1]	Al-Assil, B., Mahfoud, M., Hamzeh, A.R., 2013. Resistance trends and risk factors of extended spectrum beta-lactamases in Escherichia coli infections in Aleppo, Syria. Am. J. Infect.Control.41, 597-600.
[2]	Aly, M.E.A., Essam, T.M., Amin, M.A., 2012. Antibiotic Resistance Profile of E. coli Strains Isolated from Clinical Specimens and Food Samples in Egypt. Int. J. Micobiol. Res. 3, 176-182.
[3]	Andersen JL, He GX, Kakarla P, K CR, Kumar S, Lakra WS, et al, 2015. Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens. Int J Environ Res Public Health. 12, 1487-547.
[4]	Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493-496.
[5]	Bhardwaj, A.K., Mohanty, P., 2012. Bacterial efflux pumps involved in multidrug resistance and their inhibitors: rejuvinating the antimicrobial chemotherapy. Recent. Pat. Anti. Infects. Drug. Discov. 7, 73-89.
[6]	Blair, J.M., Piddock, L.J., 2009. Structure, function and inhibition of RND efflux pumps in Gram-negative bacteria: an update. Curr. Opin. Microbiol. 12, 512-519.
[7]	Bolon, M.K., Wright, S.B., Gold, H.S., Carmeli, Y., 2004. The magnitude of the association between fluoroquinolone use and quinolone-resistant Escherichia coli and Klebsiella pneumoniae may be lower than previously reported. Antimicrob. Agents. Chemother. 48, 1934-1940.
[8]	Chen, S., Cui, S., McDermott, P.F., Zhao, S., White, D.G., Paulsen, I., Meng, J., 2007. Contribution of target gene mutations and efflux to decreased susceptibility of Salmonella enteric serovar typhimurium to fluoroquinolones and other antimicrobials. Antimicrob. Agents. Chemother. 51, 535-542.
[9]	Cornejo-Juarez P, Vilar-Compte D, Perez-Jimenez C, Namendys-Silva SA, Sandoval-Hernandez S, Volkow-Fernandez P., 2015. The impact of hospital-acquired infections with multidrug-resistant bacteria in an oncology intensive care unit. Int. J. Infect. Dis. 31, 31-4.
[10]	Drago, L., Nicola, L., Mattina, R., De Vecchi, E., 2010. In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations. BMC. Microbiol. 10, 119.
[11]	Eliopoulos, G.M., Mollering, R.C. Jr., 1996. Antimicrobial combinations.In: Lorain V (ed) Antibiotics in laboratory medicine, 4th edn. Williams & Wilkins, Baltimore, MD,pp330-396.
[12]	Elkins, C.A., Nikaido, H., 2002. Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops. J. Bacteriol. 184, 6490-6498.
[13]	Falagas, M.E., Karageorgopoulos, D.E., 2008. Pandrug resistance (PDR), extensive drug resistance (XDR), and multidrug resistance (MDR) among Gram-negative bacilli: need for international harmonization in terminology. Clin. Infect. Dis. 46, 1121-1122.
[14]	George, D.F., Gbedema, S.Y., Agyare, C., Adu, F., Boamah, V.E., Tawiah, A.A., Saana, S.B.B.M., 2012 Antibiotic Resistance Patterns of Escherichia coli Isolates from Hospitals in Kumasi, Ghana. ISRN. Microbiology. 2012, 5.
[15]	Hobbs, E.C., Yin, X., Paul, B.J., Astarita, J.L., Storz, G., 2012. Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance. Proc. Natl. Acad. Sci. U. S. A. 109, 16696-16701.
[16]	Hocquet, D., Patry, I., Dupont, P., Bize, M., Jeannot, K., Chavanet, P., Plesiat, P., 2005. Low-level resistance to fluoroquinolones conferred by efflux overproduction in Pseudomonas aeruginosa: clinical significance and routine detection. Pathol. Biol. (Paris). 53, 495-499.
[17]	Kumar, S.; Varela, M.F., 2013. Molecular mechanisms of bacterial resistance to antimicrobial agents. In Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education; Méndez-Vilas, A., Ed.; Formatex Research Center: Badajoz, Spain, 522-534.
[18]	Jakobsen, L., Kurbasic, A., Skjot-Rasmussen, L., Ejrnaes, K., Porsbo, L.J., Pedersen, K., Jensen, L.B., Emborg, H.D., Agerso, Y., Olsen, K.E., Aarestrup, F.M., Frimodt-Moller, N., Hammerum, A.M., 2010. Escherichia coli isolates from broiler chicken meat, broiler chickens, pork, and pigs share phylogroups and antimicrobial resistance with community-dwelling humans and patients with urinary tract infection. Foodborne. Pathog. Dis. 7, 537-547.
[19]	Jannes, G., De Vos, D., 2006. A review of current and future molecular diagnostic tests for use in the microbiology laboratory. Methods. Mol. Biol. 345, 1-21.
[20]	Jarvis, W.R., Martone, W.J., 1992. Predominant pathogens in hospital infections. J. Antimicrob .Chemother. Suppl A, 19-24.
[21]	Kaper, J.B., Nataro, J.P., Mobley, H.L., 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2, 123-140.
[22]	Keeney, D., Ruzin, A., McAleese, F., Murphy, E., Bradford, P.A., 2008. MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J. Antimicrob. Chemother. 61, 46-53.
[23]	Kunin, C.M., Ellis, W.Y., 2000. Antimicrobial activities of mefloquine and a series of related compounds. Antimicrob. Agents. Chemother. 44, 848-852.
[24]	Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods. 25, 402-408.
[25]	Lockhart, S.R., Abramson, M.A., Beekmann, S.E., Gallagher, G., Riedel, S., Diekema, D.J., Quinn, J.P., Doern, G.V., 2007. Antimicrobial resistance among Gram-negative bacilli causing infections in intensive care unit patients in the United States between 1993 and 2004. J. Clin. Microbiol. 45, 3352-3359.
[26]	Martins, M., McCusker, M.P., Viveiros, M., Couto, I., Fanning, S., Pages, J.M., Amaral, L., 2013. A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps. Open. Microbiol. J. 7, 72-82.
[27]	Mesaros, N., Glupczynski, Y., Avrain, L., Caceres, N.E., Tulkens, P.M., Van Bambeke, F., 2007. A combined phenotypic and genotypic method for the detection of Mex efflux pumps in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 59, 378-386.
[28]	Moreira, M.A., Rodrigues, P.P., Tomaz, R.S., de Moraes, C.A., 2009.Multidrug Efflux System in Escherichia coli and Enterobacter cloacae obtained from Wholesome Broiler Carcasses. Braz. J. Microbiol. 40, 241-247.
[29]	Namboodiri, S.S., Opintan, J.A., Lijek, R.S., Newman, M.J., Okeke, I.N., 2011. Quinolone resistance in Escherichia coli from Accra, Ghana. BMC. Microbiol. 11, 44.
[30]	Nguyen, R.N., Taylor, L.S., Tauschek, M., Robins-Browne, R.M., 2006. Atypical enteropathogenic Escherichia coli infection and prolonged diarrhea in children. Emerg. Infect. Dis. 12, 597-603.
[31]	Nikaido, H., 2011. Structure and mechanism of RND-type multidrug efflux pumps. Adv. Enzymol. Relat. Areas. Mol. Biol. 77, 1-60.
[32]	Nikaido, H., Pages, J.M., 2012. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS. Microbiol. Rev. 36, 340-363.
[33]	Oteo, J., Lazaro, E., de Abajo, F.J., Baquero, F., Campos, J., 2005. Antimicrobial-resistant invasive Escherichia coli, Spain. Emerg. Infect. Dis. 11, 546-553.
[34]	Pazhani, G.P., Chakraborty, S., Fujihara, K., Yamasaki, S., Ghosh, A., Nair, G.B., Ramamurthy, T., 2011. QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India. Indian. J. Med. Res. 134, 214-223.
[35]	Rahbar, M., Deldari, M., Hajia, M., 2006. Changing prevalence and antibiotic susceptibility patterns of different Shigella species in Tehran, Iran. Internet. J. Microbiol. 3
[36]	Rand, K.H., Houck, H.J., Brown, P., Bennett, D., 1993. Reproducibility of the microdilution checkerboard method for antibiotic synergy. Antimicrob. Agents. Chemother. 37, 613-615.
[37]	Saito, R., Sato, K., Kumita, W., Inami, N., Nishiyama, H., Okamura, N., Moriya, K., Koike, K., 2006. Role of type II topoisomerase mutations and AcrAB efflux pump in fluoroquinolone-resistant clinical isolates of Proteus mirabilis. J. Antimicrob. Chemother. 58, 673-677.
[38]	Schweizer, H.P., 2012. Understanding efflux in Gram-negative bacteria: opportunities for drug discovery. Expert. Opin. Drug. Discov. 7, 633-642.
[39]	Singh, R., Swick, M.C., Ledesma, K.R., Yang, Z., Hu, M., Zechiedrich, L., Tam,VH., 2012. Temporal interplay between efflux pumps and target mutations in development of antibiotic resistance in Escherichia coli. Antimicrob. Agents. Chemother. 56, 1680-1685.
[40]	Swick, M.C., Morgan-Linnell, S.K., Carlson, K.M., Zechiedrich, L., 2011. Expression of multidrug efflux pump genes acrAB-tolC, mdfA, and norEin Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrob. Agents. Chemother. 55, 921-924.
[41]	Tacconelli E, Cataldo MA, Dancer SJ, De Angelis G, Falcone M, Frank U, et al., 2014. ESCMID guidelines for the management of the infection control measures to reduce transmission of multidrug-resistant Gram-negative bacteria in hospitalized patients. Clin. Microbiol. Infect. 20, 1-55.
[42]	Tanwar J, Das S, Fatima Z, Hameed S., 2014. Multidrug resistance: an emerging crisis. Interdiscip Perspect. Infect. Dis., 2014:541340.
[43]	Tegos, G.P., Haynes, M., Strouse, J.J., Khan, M.M., Bologa, C.G., Oprea, T.I., Sklar, L.A., 2011. Microbial efflux pump inhibition: tactics and strategies. Curr. Pharm. Des. 17, 1291-1302.
[44]	Tikhonova, E.B., Zgurskaya, H.I., 2004. AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex. J. Biol. Chem. 279, 32116-32124.
[45]	Turnidge, J., 2004. Antibiotic use in animals--prejudices, perceptions and realities. J. Antimicrob. Chemother. 53, 26-27.
[46]	Vedel, G., 2005. Simple method to determine beta-lactam resistance phenotypes in Pseudomonas aeruginosa using the disc agar diffusion test. J. Antimicrob. Chemother. 56, 657-664.
[47]	Vidal-Aroca, F., Meng, A., Minz, T., Page, M.G.P., Dreier, J., 2009.Use of resazurin to detect mefloquine as an efflux-pump inhibitor in Pseudomonas aeruginosa and Escherichia coli. J. Microbiol. Methods. 79, 232-237.
[48]	Viveiros, M., Dupont, M., Rodrigues, L., Couto, I., Davin-Regli, A., Martins, M., Pages, J.M., Amaral, L., 2007. Antibiotic stress, genetic response and altered permeability of E. coli. PLoS. One. 2, e365.
[49]	Viveiros, M., Jesus, A., Brito, M., Leandro, C., Martins, M., Ordway, D., Molnar, A.M., Molnar, J., Amaral, L., 2005. Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes. Antimicrob. Agents. Chemother. 49, 3578-3582.
[50]	Yasufuku, T., Shigemura, K., Shirakawa, T., Matsumoto, M., Nakano, Y., Tanaka, K., Arakawa, S., Kinoshita, S., Kawabata, M., Fujisawa, M., 2011. Correlation of overexpression of efflux pump genes with antibiotic resistance in Escherichia coli Strains clinically isolated from urinary tract infection patients. J. Clin. Microbiol. 49, 189-194.
[51]	Zhang, R., Eggleston, K., Rotimi, V., Zeckhauser, R.J., 2006. Antibiotic resistance as a global threat: evidence from China, Kuwait and the United States. Global. Health. 2, 6.