Mechanisms of Quinolone Resistance in Vibrio parahemolyticus strains Isolated from Imported Shrimp

Mohamed Nawaz, Kidon Sung, Kuppan Gokulan, Sangeeta Khare, Saeed Khan, Safia Nawaz, Ashraf Khan, Roger Steele


Sixty-one quinolone-resistant strains of Vibrio parahaemolyticus were isolated from 247 shrimp samples. These isolates had MICs of 256 and 4-8 µg/mL for nalidixic acid and ciprofloxacin respectively. Template DNA of the 61 isolates were screened by PCR for the quinolone resistance genes. Purified PCR amplicons of gyrA, gyrB and parC from these isolates were sequenced and analyzed for point mutations that confer resistance to these antibiotics. Point mutations in the quinolone resistance determining region (QRDR) of GyrA at positions 68, 83, 85 and 89 and in ParC at positions 85 as well as in the non QRDR of GyrA at positions 48 along with 4 different point mutations in GyrB at positions 311, 354, 360 and 374 conferred resistances to these antibiotics.  Structural analysis were undertaken to determine the role of these four novel point mutations in V. parahaemolyticus at codons 48, 68, 83 and 89 on GyrA in enhanced MICs to quinolone antibiotics. Homology analysis indicated that all four mutations are localized in the vicinity of quinolone binding and DNA binding major grooves. These four mutations are known to play pivotal roles in readjusting the position of α-helices, stabilization of GyrA, loss of electrical charges or the formation of A2 dimers resulting in enhanced MICs to the quinolone antibiotics. Ethidium bromide uptake experiments indicated higher efflux pump activities in drug resistant V. parahaemolyticus than their sensitive counterparts. Our results indicate that imported shrimp is a reservoir of quinolone-resistant V. parahaemolyticus.


Quinolone resistance Vibrio parahaemolyticus Imported shrimp

Full Text:


Arnold K, Bordoli L, Kopp J, and Schwede, T. 2006. The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22:195-201.

Baranwal S, Dey K, Ramamurthy T, Nair GB, Kundu M. 2002. Role of active efflux in association with target gene mutations in fluoroquinolone resistance in clinical isolates of Vibrio cholerae. Antimicrob. Agent Chemother. 46: 2676-2678.

Bauer AW, Kirby WM, Sherris JC, Turck, M. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45: 493-496.

Bax BD, Chan PF, Eggleston D.S, Fosberry A, Gentry DR, Gorrec F, Giordano I, Hann MM, Hennessy A, Hibbs M, Huang J, Jones E, Jones J, Brown KK, Lewis CJ, May EW, Saunders MR, Singh O, Spitzfaden CE, Shen C, Shillings A, Theobald AJ, Wohlkonig A, Pearson ND, Gwynn, MN. 2010. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 19, 466:935-40.

Biao X, Kaijin Y. 2007. Shrimp farming in China: operating characteristics, environmental impact and perspectives. Ocean Coastal Management 50: 538-550.

Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, Tan Z, Shariff M. 2005. Disease and health management in Asian aquaculture. Vet. Parasitology. 132: 249-272.

Emsley P, Lohkamp B, Scott WG, Cowtan, K. 2009. Features and development of Coot. Acta Cryst. 66: 486-501.

Ghosh AS, Ahamed J, Chauhan KK, Kundu M. 1998. Involvement of an efflux system in high-level fluoroquinolone resistance of Shigella dysenteriae. Biochem. Biophys. Res. Commun. 242: 54-56.

Hopkins KL, Davies RH, Threlfall EJ. 2005. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int. J. Antimicrob. Agents. 25: 358-373.

Karczmarczyk M, Martins M, Quinn T, Leonard N, Fanning S. 2011. Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food producing animals. Appl. Environ. Microbiol.77:7113-20.

Kitiyodom S, Khemtong S, Wongtavatchai J, Chuanchuen R. 2010. Characterization of antibiotic resistance in Vibrio spp. isolated from farmed marine shrimps (Penaeus monodon). FEMS Microbiol. Ecol. 72: 219-227.

Laponogov I, Sohi MK, Veselkov DA, Pan XS, Sawhney R, Thompson AW, McAuley KE, Fisher LM, Sanderson MR. 2009. Structural insight into the quinolone-DNA cleavage complex of type IIA topoisomerases. Nature Struct Mol Biol.16: 667-9.

Lawrence AK, Sternberg MEJ. 2009. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols..4: 363–371.

Lee CY, Chen MF, Yu MS, Pan MJ. 2002. Purification and characterization of putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol. Lett. 209, 31-37.

Liu M, Wong MHY, Chen S. 2013. Mechanisms of fluoroquinolone resistance in Vibrio parahaemolyticus. Int. J. Antimicrob. Agents. 42: 187-193.

National Committee for Clinical Standards. 2002. Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. Approved guidance M37-A2. National Committee for Clinical Standards, Wayne, PA.

Nawaz MS, Khan SA, Tran Q, Sung K, Khan AA, Steele RS. 2012. Isolation and characterization of multidrug-resistant Klebsiella spp. isolated from shrimp imported from Thailand. Int. J. Food Microbiol. 155: 179-184.

Okuda J, Hayakawa E, Nishibuchi M, Nishino T. 1999. Sequence analysis of the gyrA and parC homologues of a wild type strain of Vibrio parahaemolyticus and its fluoroquinolone-resistant mutants. Antimicrob.Agents. Chemother. 43: 1156-1162.

Ouabdesselam S, Hooper DC, Tankovic J, Soussy CJ. 1995. Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations. Antimicrob. Agents Chemother. 39:1667-70.

Poole K. 2000. Efflux-mediated resistance to fluoroquinolones in Gram-negative bacteria. Antimicrob. Agent Chemother. 44: 2233-2241.

Ragunath P, Karunasagar I, Karunasagar I. 2009. Improved isolation and detection of pathogenic Vibrio parahaemolyticus from seafood using a new enrichment broth. Int. J. Food Microbiol. 129: 200-203.

Ruiz J. 2003. Mechanism of resistance to quinolones: target alteration, decreased accumulation and DNA gyrase protection. J. Antimicrob. Chemother. 51: 1109-1117.

Shakir Z, Khan S, Sung K, Khare S, Khan A, Steele R and Nawaz, M. 2012. Molecular characterization of fluoroquinolone-resistant Aeromonas spp. isolated from imported shrimp. Appl. Environ. Microbiol. 78: 8137-8141.

US Food and Drug Administration. 1997. Fluoroquinolones and glycopeptides-

order of prohibition. Fed. Regist. 62: 27944-27947.

Webber M.A, Piddock, L.J.V. 2003. The importance of efflux pumps in bacterial antibiotic resistance. J. Antimicrob. Chemother. 51: 9-11.

Yoshida H, Bogaki M, Nakamura M, Yamanaka LM, Nakamura S. 1991. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli. Antimicrob.Agents Chemother. 35:1647-50.

DOI: http://dx.doi.org/10.18103/mra.v0i3.118


  • There are currently no refbacks.