Detection of Novel gyrB Mutation in Fluoroquinolone-Resistant Salmonella and Escherichia coli using PCR-RFLP

Abstract

Background Emergence of fluoroquinolone resistance in gut pathogens is a cause of concern. Resistant to quinolone is mainly due to the point mutations at the quinolone-resistance determining regions (QRDR). The aim of the study was to develop polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) to detect QRDR mutations in gyrA and gyrB regions in enteric pathogens.

Methodology PCR-RFLP was done for gyrA 83 region using HinfI and for gyrB 447 using AcuI for fluoroquinolone resistant and susceptible gut pathogens. The products were also sequenced to confirm the presence of restriction sites.

Results In this study, a PCR-RFLP technique was developed to detect gyrA 83 mutations in Salmonella typhi and Escherichia coli. A first of its kind PCR-RFLP was also developed to detect gyrB 447 mutation using a restriction enzyme AcuI. Restriction digestion of gyrA using HinfI resulted in three bands for resistant S. typhi isolates due to the presence of mutation at gyrA 83 and four bands were seen for sensitive S. typhi isolates, while two bands for resistant and three bands were seen in sensitive E. coli isolates. Similarly, restriction digestion of gyrB using AcuI resulted in no digestion for resistant S. typhi isolates and two bands for resistant E. coli isolates. This suggest that there is mutation at gyrB 447 region ofE. coli, while no mutation was found in S. typhi isolates.

Conclusion The PCR-RFLP developed in the present study could successfully detect gyrA 83 and gyrB 447 mutations in fluoroquinolone-resistant S. typhi and E. coli. The technique can be efficiently used in epidemiological studies instead of a cost-intensive sequencing method to detect the status of multiple point mutations in gut pathogens.

Keywords

Escherichia coli - fluoroquinolone resistance - QRDR mutations - Salmonella typhi

Publikationsverlauf

Artikel online veröffentlicht:
10. Oktober 2022

• References

• 1 D'Aoust JY, Maurer J. Salmonella species. In Food Microbiology: Fundamentals and Frontiers. 3rd edition. United States:. American Society of Microbiology; 2007:187–236
• 2 Gordon MA. Salmonella infections in immunocompromised adults. J Infect 2008; 56 (06) 413-422
• 3 Shrestha KL, Pant ND, Bhandari R, Khatri S, Shrestha B, Lekhak B. Re-emergence of the susceptibility of the Salmonella spp. isolated from blood samples to conventional first line antibiotics. Antimicrob Resist Infect Control 2016; 5: 22
• 4 Goettsch W, van Pelt W, Nagelkerke N. et al. Increasing resistance to fluoroquinolones in Escherichia coli from urinary tract infections in the Netherlands. J Antimicrob Chemother 2000; 46 (02) 223-228
• 5 Nakaya H, Yasuhara A, Yoshimura K, Oshihoi Y, Izumiya H, Watanabe H. Life-threatening infantile diarrhea from fluoroquinolone-resistant Salmonella enterica typhimurium with mutations in both gyrA and parC. Emerg Infect Dis 2003; 9 (02) 255-257
• 6 Stapleton AE, Wagenlehner FME, Mulgirigama A, Twynholm M. Escherichia coli resistance to fluoroquinolones in community-acquired uncomplicated urinary tract infection in women: a systematic review. Antimicrob Agents Chemother 2020; 64 (10) e00862-e20
• 7 Hooper DC. Emerging mechanisms of fluoroquinolone resistance. Emerg Infect Dis 2001; 7 (02) 337-341
• 8 Redgrave LS, Sutton SB, Webber MA, Piddock LJ. Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success. Trends Microbiol 2014; 22 (08) 438-445
• 9 Martínez-Martínez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998; 351 (9105): 797-799
• 10 Hooper DC. Mechanisms of action and resistance of older and newer fluoroquinolones. Clin Infect Dis 2000; 31 (Suppl. 02) S24-S28
• 11 Jazeela K, Chakraborty G, Shetty SS, Rohit A, Karunasagar I, Vijaya Kumar D. Comparison of mismatch amplification mutation assay PCR and PCR-restriction fragment length polymorphism for detection of major mutations in gyrA and parC of Escherichia coli associated with fluoroquinolone resistance. Microb Drug Resist 2019; 25 (01) 23-31
• 12 Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing; Twenty ninth Informational Supplement M100-S29, Clinical and Labo ratory Standards Institute, CLSI, Wayne, Pa, USA
• 13 Bej AK, DiCesare JL, Haff L, Atlas RM. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl Environ Microbiol 1991; 57 (04) 1013-1017
• 14 Rahn K, De Grandis SA, Clarke RC. et al. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella. Mol Cell Probes 1992; 6 (04) 271-279
• 15 Britto CD, Wong VK, Dougan G, Pollard AJ. A systematic review of antimicrobial resistance in Salmonella enterica serovar Typhi, the etiological agent of typhoid. PLoS Negl Trop Dis 2018; 12 (10) e0006779
• 16 Deekshit VK, Kumar BK, Rai P, Karunasagar I, Karunasagar I. Differential expression of virulence genes and role of gyrA mutations in quinolone resistant and susceptible strains of Salmonella Weltevreden and Newport isolated from seafood. J Appl Microbiol 2015; 119 (04) 970-980
• 17 Shetty SS, Deekshit VK, Jazeela K. et al. Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples. Indian J Med Res 2019; 149 (02) 192-198
• 18 Yoshida H, Bogaki M, Nakamura M, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob Agents Chemother 1990; 34 (06) 1271-1272
• 19 Alonso R, Galimand M, Courvalin P. An extended PCR-RFLP assay for detection of parC, parE and gyrA mutations in fluoroquinolone-resistant Streptococcus pneumoniae. J Antimicrob Chemother 2004; 53 (04) 682-683
• 20 Gopal M, Elumalai S, Arumugam S. et al. GyrA ser83 and ParC trp106 mutations in Salmonella enterica serovar Typhi isolated from typhoid fever patients in tertiary care hospital. J Clin Diagn Res 2016; 10 (07) DC14-DC18
• 21 Al-Emran HM, Heisig A, Dekker D. et al. Detection of a novel gyrB mutation associated with fluoroquinolone-nonsusceptible Salmonella enterica serovar Typhimurium isolated from a bloodstream infection in Ghana. Clin Infect Dis 2016; 62 (Suppl. 01) S47-S49