PCR-RFLP-Based Detection of Mutations in the Chromosomal Fluoroquinolone Targets gyrA and parC Genes of Acinetobacter baumanii Clinical Isolates from a Tertiary Hospital in Cairo, Egypt

Mahmoud M. Tawfick^{1, 2,} and Mervat I. El-Borhamy³

¹Microbiology and Immunology Department, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

²Microbiology and Immunology Department, Faculty of Pharmacy, MSA University, 6th October City, Giza, Egypt

³Clinical Pathology, International Medical Centre (IMC) and Misr International University, Cairo-Ismailia Desert Road, Cairo, Egypt

Cite this paper:
Mahmoud M. Tawfick and Mervat I. El-Borhamy. PCR-RFLP-Based Detection of Mutations in the Chromosomal Fluoroquinolone Targets gyrA and parC Genes of Acinetobacter baumanii Clinical Isolates from a Tertiary Hospital in Cairo, Egypt. American Journal of Microbiological Research. 2017; 5(2):37-43. doi: 10.12691/ajmr-5-2-3

Abstract

Background and Aim: Acinetobacter baumannii is one of the most antimicrobial resistant nosocomial pathogens encountered clinically worldwide. The emergence of fluoroquinolone resistance among A. baumannii isolates is currently of concern. This study aimed to investigate the antimicrobial susceptibility patterns among 49 clinical isolates of A baumannii collected from a tertiary care hospital in Egypt. These isolates were analysed for the mechanism of fluoroquinolone resistance based on the presence of mutations in the quinolone resistance-determining regions (QRDRs) of the chromosomal quinolone resistance determinants gyrA and parC genes. Methods: A. baumannii isolates were identified using conventional biochemical testing, VITEK 2 automated system and polymerase chain reaction (PCR) assay targeting the intrinsic bla_OXA-51-like gene of A. baumannii species. Antimicrobial susceptibility to different antimicrobial agents was tested using agar disk diffusion method and the minimum inhibitory concentration (MIC) of ciprofloxacin was determined using E-test (bioMérieux, France) on Mueller-Hinton agar medium following the Clinical and Laboratory Standards Institute guidelines. The QRDRs of the gyrA and parC genes in A. baumannii isolates were amplified by PCR using specific primers. Mutations in theses QRDRs were detected by HinfI restriction fragment length polymorphism (RFLP) of PCR products and sequencing. Results: The bla_OXA-51-like gene was detected in all isolates confirming identification as A. baumannii. Antimicrobial susceptibility study showed that all isolates (100 %) were MDR. They were 100 % resistant to the tested fluoroquinolones, ciprofloxacin and levofloxacin. The MIC of ciprofloxacin ranged from 4 to ≥ 32 μg/mL. All A. baumannii isolates (100 %) were found to harbour gyrA and parC genes. HinfI restriction analysis showed a detectable mutation in QRDRs at position Ser-83 of gyrA gene and Ser-80 of parC gene. There was single mutation in either gyrA or parC in 11 isolates showed ciprofloxacin MIC of < 32 μg/mL, while 38 isolates with MIC of ≥ 32 μg/mL had double mutations in QRDRs of both genes. Conclusions: Resistance of A. baumannii isolates to fluoroquinolones in Egypt is alarming as all MDR A. baumannii isolates in the current study were mostly highly resistant to ciprofloxacin with MIC ≥ 32 μg/mL, limiting the remaining therapeutic options and a public health policy on appropriate prescribing and thus the rational use of antimicrobial agents is required. Double mutation with substitutions at positions Ser-83 and Ser-80 of gyrA and parC genes, respectively, could lead to high‐level ciprofloxacin resistant phenotype than a single mutation in one of them. Further extensive studies including a larger number of isolates from different geographic areas in Egypt and investigating other fluoroquinolone resistance mechanisms are warranted.

Keywords:
PCR-RFLP A. baumannii gyrA parC fluoroquinolone

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Fournier, P.E., Richet, H. and Weinstein, R.A., “The epidemiology and control of Acinetobacter baumannii in health care facilities,” Clin Infect Dis, 42 (5). 692-699. Mar.2006.
[2]	Maragakis, L.L. and Perl, T.M., “Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options,” Clin Infect Dis, 46(8). 1254-1263. Apr.2008.
[3]	Doi, Y., Murray, G.L. and Peleg, A.Y., “Acinetobacter baumannii: Evolution of Antimicrobial Resistance-Treatment Options,” Semin Respir Crit Care Med, 36(1). 85-98. Feb.2015.
[4]	Callegan, M. C., Ramirez, R., Kane, S. T., Cochran, D. C. and Jensen, H. “Antibacterial activity of the fourth-generation fluoroquinolones gatifloxacin and moxifloxacin against ocular pathogens,” Advances in therapy, 20(5). 246-252. Sep.2003.
[5]	Ebbing, L., Metlay, J.P., Bilker, W.B., Edelstein, P.H. and Fishman, N.O., “Association between fluoroquinolone resistance and mortality in Escherichia coli and Klebsiella pneumoniae infections: the role of inadequate empirical antimicrobial therapy,” Clin Infect Dis, 41(7). 923-929. Oct.2005.
[6]	Drlica, K., Malik, M., Kerns, R.J. and Zhao, X., “Quinolone-mediated bacterial death,” Antimicrob Agents Chemother, 52(2). 385-392. Feb.2008.
[7]	Champoux, J.J., “DNA topoisomerases: structure, function, and mechanism,” Annu Rev Biochem, 70(1). 369-413. Jul.2001.
[8]	Naeem, A., Badshah, S.L., Muska, M., Ahmad, N. and Khan, K., “The current case of quinolones: synthetic approaches and antibacterial activity,” Molecules, 21(4). 268-287. Mar.2016.
[9]	Strahilevitz, J., Jacoby, G.A., Hooper, D.C. and Robicsek, A., “Plasmid-mediated quinolone resistance: a multifaceted threat,” Clin Microbiol Rev, 22(4). 664-689. Oct.2009.
[10]	Vila, J., Ruiz, J., Goni, P., Marcos, A. and de Anta, T.J., “Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii,” Antimicrob. Agents Chemother, 39(5). 1201-1203. May1995.
[11]	Vila, J., Ruiz, J., Goñi, P. and de Anta, T.J., “Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii,” J Antimicrob Chemother, 39(6). 757-762. Jun.1997.
[12]	Sáenz, Y., Zarazaga, M., Briñas, L., Ruiz-Larrea, F. and Torres, C., “Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans and animals,” J Antimicrob Chemother, 51(4). 1001-1005. Apr.2003.
[13]	Bauer, A., Kirby, W., Sherris, J. and Turck M., Bauer A., “Antibiotic susceptibility testing by a standardized single disk method,” Am J Clin Pathol, 45(4). 493-496. Apr.1966.
[14]	Clinical and Laboratory Standards Institute (CLSI) (2016): Performance standards for antimicrobial susceptibility testing; Twenty-sixth informational supplement. CLSI document M100-S26. Wayne, PA: Clinical and Laboratory Standards Institute.
[15]	Turton, J.F., Woodford, N., Glover, J., Yarde, S., Kaufmann, M.E. and Pitt, T.L., “Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species,” J Clin Microbiol, 44(8). 2974-2976. Aug.2006.
[16]	Lee, J.K., Lee, Y.S., Park, Y.K. and Kim, B.S., “Mutations in the gyrA and parC genes in ciprofloxacin‐resistant clinical isolates of Acinetobacter baumannii in Korea,” Microbiol Immunol, 49(7). 647-653. Jul.2005.
[17]	UK Standards for Microbiology Investigations, “Identification of Pseudomonas species and other Non-Glucose Fermenters,” Standards Unit, Microbiology Services, PHE, ID 17 (3). April 2015.
[18]	Lagamayo, E.N., “Antimicrobial resistance in major pathogens of hospital-acquired pneumonia in Asian countries,” Am J Infect Control, 36(4). 101-108. May 2008.
[19]	Huang, S.S., Lee, S.C., Lee, N., See, L.C., Tsai, M.H. and Shieh, W.B., “Comparison of in vitro activities of levofloxacin, ciprofloxacin, ceftazidime, cefepime, imipenem, and piperacillin-tazobactam against aerobic bacterial pathogens from patients with nosocomial infections,” J Microbiol Immunol Infect, 40(2). 134-140. Apr.2007.
[20]	Boroumand, M.A., Akhyani, H., Sheikhvatan, M., Yazdi, S.H., Saboorian, R., Hashemi, S.H. and Firouzkouhi, F., “Evaluation of antimicrobial resistance of Acinetobacter baumannii to imipenem, ciporofloxacin and ceftazidime using E Test,” Iranian J Publ Health, 38(2). 130-133. Apr.2009.
[21]	Joyanes, P., Conejo, M.D., Martı́nez-Martı́nez, L. and Perea, E.J., “Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting Gram-negative rods frequently isolated from clinical samples,” J Clin Microbiol, 39(9). 3247-3253. Sep.2001.
[22]	Mahdi, A., Mohammad, N., Morovat, T., Mhammad-Mahdi, F., Namam-Ali, A., Setareh, S., Mohammad, E., Amir, A., Abbas, M. and Ali, H., “Rapid identification of Iranian Acinetobacter baumannii strains by single PCR assay using BLA oxa-51-like carbapenemase and evaluation of the antimicrobial resistance profiles of the isolates” Acta microbiologica et immunologica Hungarica, 57(2). 87-94. Jun.2010.
[23]	Maleki, M.H., Jalilian, F.A., Khayat, H., Mohammadi, M., Pourahmad, F., Asadollahi, K., Pakzad, I., Sadeghifard, N., Soroush, S., Emaneini, M. and Taherikalani, M., “Detection of highly ciprofloxacin resistance Acinetobacter baumannii isolated from patients with burn wound infections in presence and absence of efflux pump inhibitor” Maedica (Buchar), 9(2). 162-167. Jun.2014.
[24]	Shakibaie, M.R., Adeli, S. and Salehi, M.H.,”Antibiotic resistance patterns and extended-spectrum β-lactamase production among Acinetobacter spp. isolated from an intensive care Unit of a hospital in Kerman, Iran,” Antimicrob Resist Infect Control, 1(1). Jan.2012.
[25]	Nowroozi, J., Sepahi, A.A., Kamarposhti, L.T., Razavipour, R. and Mazhar, F., “Evaluation of ciprofloxacin (gyrA, parC genes) and tetracycline (tetB gene) resistance in nosocomial Acinetobacter baumannii infections,” Jundishapur J Microbiol, 7(2). e8976. Feb.2014.
[26]	Prashanth, K. and Badrinath, S., “In vitro susceptibility pattern of Acinetobacter species to commonly used cephalosporins, quinolones, and aminoglycosides,” Indian J Med Microbiol, 22(2). 97-103. Apr-Jun.2004.
[27]	Redgrave, L.S., Sutton, S.B., Webber, M.A. and Piddock, L.J., “Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success. Trends in microbiology,” Trends in microbiology, 22(8). 438-445. Aug.2014.
[28]	Gomaa, F.M., Tawakol, W.M. and Abo El-Azm, F.I., “Phenotypic and genotypic detection of some antimicrobial resistance mechanisms among multidrug-resistant Acinetobacter baumannii isolated from immunocompromised patients in Egypt,” Egypt J Med Microbiol., 23. 99-111. Oct.2014.
[29]	Ardebili, A., Lari, A.R. and Talebi, M., “Correlation of ciprofloxacin resistance with the AdeABC efflux system in Acinetobacter baumannii clinical isolates,” Annals of laboratory medicine, 34(6). 433-8. Nov.2014.
[30]	Aly, M.M., Alsoud, N.A., Elrobh, M.S., Al Johani, S.M. and Balkhy, H.H. “High prevalence of the PER-1 gene among carbapenem-resistant Acinetobacter baumannii in Riyadh, Saudi Arabia,” European Journal of Clinical Microbiology & Infectious Diseases, 359110. 1759-66. Nov.2016.
[31]	Joshi, P.R., Acharya, M., Kakshapati, T., Leungtongkam, U., Thummeepak, R. and Sitthisak, S., “Co-existence of bla OXA-23 and bla NDM-1 genes of Acinetobacter baumannii isolated from Nepal: antimicrobial resistance and clinical significance,” Antimicrobial Resistance & Infection Control, 6(1). 21. Feb.2017.
[32]	Sohail, M., Rashid, A., Aslam, B., Waseem, M., Shahid, M., Akram, M., Khurshid, M. and Rasool, M.H., “Antimicrobial susceptibility of Acinetobacter clinical isolates and emerging antibiogram trends for nosocomial infection management,” Revista da Sociedade Brasileira de Medicina Tropical, 49(3). 300-4. Jun. 2016.
[33]	Chuang, Y.C., Sheng, W.H., Lauderdale, T.L., Li, S.Y., Wang, J.T., Chen, Y.C. and Chang, S.C., “Molecular epidemiology, antimicrobial susceptibility and carbapenemase resistance determinants among Acinetobacter baumannii clinical isolates in Taiwan,” Journal of Microbiology, Immunology and Infection, 47(4). 324-32. Aug. 2014.
[34]	Howard, W., Biedenbach, D.J. and Jones, R.N., “Comparative antimicrobial spectrum and activity of the desfluoroquinolone BMS284756 (T-3811) tested against non-fermentative Gram-negative bacilli,” Clin Microbiol Infect, 8(6). 340-344. Jun.2002.
[35]	Biedenbach, D.J., Croco, M.A., Barrett, T.J. and Jones, R.N., “Comparative in vitro activity of gatifloxacin against Stenotrophomonas maltophilia and Burkholderia species isolates including evaluation of disk diffusion and E test methods,” Eur J Clin Microbiol Infect Dis, 18(6). 428-431. Jun.1999.
[36]	Ruiz, J., “Mechanisms of resistance to quinolones: target alterations, decreased accumulation and DNA gyrase protection,” J Antimicrob Chemother, 51(5). 1109-1117. May2003.
[37]	Wisplinghoff, H., Decker, M., Haefs, C., Krut, O., Plum, G. and Seifert, H., “Mutations in gyrA and parC associated with resistance to fluoroquinolones in epidemiologically defined clinical strains of Acinetobacter baumannii,” J Antimicrob Chemother, 51(1). 177-180. Jan.2003.
[38]	Valentine, S.C., Contreras, D., Tan, S., Real, L.J., Chu, S. and Xu, H.H., “Phenotypic and molecular characterization of Acinetobacter baumannii clinical isolates from nosocomial outbreaks in Los Angeles County, California,” J Clin Microbiol, 46(8). 2499-2507. Aug.2008.
[39]	Barnard, F.M. and Maxwell, A., “Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser83and Asp87,” Antimicrob Agents Chemother, 45(7). 1994-2000. Jul.2001.
[40]	Park, S., Lee, K.M., Yoo, Y.S., Yoo, J.S., Yoo, J.I., Kim, H.S., Lee, Y.S. and Chung, G.T., “Alterations of gyrA, gyrB, and parC and Activity of Efflux Pump in Fluoroquinolone-resistant Acinetobacter baumannii,” Osong Public Health Res Perspect, 2(3). 164-170. Dec.2011.
[41]	Liu, Y.H., Kuo, S.C., Lee, Y.T., Chang, I.C., Yang, S.P., Chen, T.L. and Fung, C.P., “ Amino acid substitutions of quinolone resistance determining regions in GyrA and ParC associated with quinolone resistance in Acinetobacter baumannii and Acinetobacter genomic species 13TU,” J Microbiol Immunol Infect, 45(2). 108-112. Apr.2012.
[42]	Hamouda, A. and Amyes, S.G., “Novel gyrA and parC point mutations in two strains of Acinetobacter baumannii resistant to ciprofloxacin,” J Antimicrob Chemother, 54(3). 695-696. Sep. 2004.