Skip to main content
SpringerLink
Log in

Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Alguel Y, Lu D, Quade N, Sauter S, Zhang X (2010) Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa. J Struct Biol 172:305–310

    Article  PubMed  CAS  Google Scholar 

  • Ansari S, Dhital R, Shrestha S, Thapa S, Puri R, Chaudhary N, Khatiwada S, Gautam R (2016) Growing menace of antibacterial resistance in clinical isolates of Pseudomonas aeruginosa in Nepal: an insight of beta-lactamase production. Biomed Res Int 2016:8

    Article  CAS  Google Scholar 

  • Askoura M, Mottawea W, Abujamel T, Taher I (2011) Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa. Libyan J Med 6:5870. https://doi.org/10.3402/ljm.v6i0.5870

    Article  Google Scholar 

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1995) Short protocols in molecular biology, 3rd edn. Wiley, New York, pp 2.11–2.12

    Google Scholar 

  • Baron EJ, Peterson LR, Finegold SM (1994) Bailey and Scott’s diagnostic microbiology, 9th edn. The C V Mosby Company, St. Louis

    Google Scholar 

  • Black JA, Moland ES, Thomson KS (2005) AmpC disc test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 43:3110–3113

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Braz VS, Furlan JP, Fernandes AF, Stehling EG (2016) Mutations in NalC induce MexAB-OprM overexpression resulting in high level of Aztreonam resistance in environmental isolates of Pseudomonas aeruginosa. FEMS Microbiol Lett. https://doi.org/10.1093/femsle/fnw166.

    Article  PubMed  Google Scholar 

  • Cao L, Srikumar R, Poole K (2004) MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719. Mol Microbiol 53:1423–1436

    Article  PubMed  CAS  Google Scholar 

  • Chatterjee M, Anju C, Biswas L, Kumar VA, Mohan CG, Biswas R (2016) Antibiotic resistance in Pseudomonas aeruginosa and alternative therapeutic options. Int J Med Microbiol 306:48–58

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Hu J, Chen PR, Lan L, Li Z, Hicks LM, Dinner AR, He C (2008) The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism. Proc Natl Acad Sci USA 105:13586–13591

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen W, Wang D, Zhou W, Sang H, Liu X, Ge Z, Zhang J, Lan L, Yang C-G, Chen H (2016) Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa. Mol Microbiol 100:749–758

    Article  PubMed  CAS  Google Scholar 

  • Choudhury R, Panda S, Singh DV (2012) Emergence and dissemination of antibiotic resistance: a global problem. Indian J Med Microbiol 30:384–390

    Article  PubMed  CAS  Google Scholar 

  • Choudhury D, Ghose A, Chanda DD, Talukdar AD, Choudhury MD, Paul D et al (2016) Correction: Premature termination of MexR leads to overexpression of MexAB-OprM efflux pump in Pseudomonas aeruginosa in a tertiary referral hospital in India. PLoS ONE 11(3):e0151308

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Clinical and Laboratory Standards Institutes (CLSI) (2012) Performance standards for antimicrobial susceptibility testing: 22nd informational supplement, M100-S22. CLSI, Wayne

    Google Scholar 

  • Dumas JL, van Delden C, Perron K, Köhler T (2006) Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR. FEMS Microbiol Lett 254:217–225

    Article  PubMed  CAS  Google Scholar 

  • Easwaran S, Yerat RC, Ramaswamy R (2016) A study on detection of extended-spectrum beta-lactamases (ESBLs) and comparison of various phenotypic methods of AmpC detection in Pseudomonas aeruginosa from various clinical isolates in a tertiary care teaching hospital. Muller J Med Sci Res 7:35–39

    Article  Google Scholar 

  • Gadagkar SR, Rosenberg MS, Kumar S (2005) Inferring species phylogenies from multiple genes: concatenated sequence tree versus consensus gene tree. J Exp Zool B 304:64–74

    Article  CAS  Google Scholar 

  • Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755

    Article  PubMed  CAS  Google Scholar 

  • Islam S, Jalal S, Wretlind B (2004) Expression of the MexXY efflux pump in amikacin- resistant isolates of Pseudomonas aeruginosa. Clin Microbiol Infect 10:877–883

    Article  PubMed  CAS  Google Scholar 

  • Jahandideh S (2013) Diversity in structural consequences of MexZ mutations in Pseudomonas aeruginosa. Chem Biol Drug Des 81:600–606

    Article  PubMed  CAS  Google Scholar 

  • Jochumsen N, Marvig RL, Damkiær S, Jensen RL, Paulander W, Molin S, Jelsbak L, Folkesson A (2016) The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions. Nature Commun 7:13002

    Article  CAS  Google Scholar 

  • Khan NH, Ahsan M, Yoshizawa S, Hosoya S, Yokota A, Kogure K (2008) Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa isolates from the ocean. Appl Environ Microbiol 74:6194–6205

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Krumperman PH (1983) Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol 46:165–170

    PubMed  PubMed Central  CAS  Google Scholar 

  • Li X-Z, Plésiat P, Nikaido H (2015) The challenge of efflux-mediated antibiotic resistance in gram-negative bacteria. Clin Microbiol Rev 28:337–418

    Article  PubMed  PubMed Central  Google Scholar 

  • Lim D, Poole K, Strynadka NC (2002) Crystal structure of the MexR repressor of the mexRAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. J Biol Chem 277::29253–29259

    Article  CAS  Google Scholar 

  • Lister PD, Wolter DJ, Hanson ND (2009) Antibacterial- resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 22:582–610

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Llanes C, Hocquet D, Vogne C, Benali-Baitich D, Neuwirth C, Plésiat P (2004) Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob Agents Chemother 48:1797–1802

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Maseda H, Sawada I, Saito K, Uchiyama H, Nakae T, Nomura N (2004) Enhancement of the mexAB-oprM efflux pump expression by a quorum-sensing autoinducer and its cancellation by a regulator, MexT, of the mexEF-oprN efflux pump operon in Pseudomonas aeruginosa. Antimicrob Agents Chemother 48:1320–1328

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mataseje LF, Peirano G, Church DL, Conly J, Mulvey M, Pitout JD (2016) Colistin-nonsusceptible Pseudomonas aeruginosa sequence type 654 with bla NDM–1 arrives in North America. Antimicrob Agents Chemother 60:1794–1800

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Morita Y, Cao L, Gould VC, Avison MB, Poole K (2006) nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa. J Bacteriol 188:8649–8654

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Morita Y, Tomida J, Kawamura Y (2012) MexXY multidrug efflux system of Pseudomonas aeruginosa. Front Microbiol 3:408

    Article  PubMed  PubMed Central  Google Scholar 

  • Murugan N, Malathi J, Umashankar V, Madhavan HN (2016) Unraveling genomic and phenotypic nature of multidrug-resistant (MDR) Pseudomonas aeruginosa VRFPA04 isolated from keratitis patient. Microbiol Res 193:140–149

    Article  CAS  Google Scholar 

  • Nithya N, Remitha R, Jayasree PR, Faisal M, Manish Kumar PR (2017) Analysis of beta-lactamases, bla NDM–1 phylogeny & plasmid replicons in multidrug-resistant Klebsiella spp. from a tertiary care centre in south India. The Indian J Med Res 146(Suppl 1):S38–S45

    Google Scholar 

  • Pan YP, Xu YH, Wang ZX, Fang YP, Shen JL (2016) Overexpression of MexAB-OprM efflux pump in carbapenem resistant Pseudomonas aeruginosa. Arch Microbiol 198:565–571

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Piddock L (2006) Multidrug-resistance efflux pumps: not just for resistance. Nat Rev Microbiol 4:629–636

    Article  PubMed  CAS  Google Scholar 

  • Poole K (2001) Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. J Mol Microbiol Biotechnol 3:255–264

    PubMed  CAS  Google Scholar 

  • Poole K (2004) Efflux-mediated multiresistance in gram-negative bacteria. Clin Microbiol Infect 10:12–26

    Article  PubMed  CAS  Google Scholar 

  • Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2:65

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Poonsuk K, Tribuddharat C, Chuanchuen R (2014) Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates. Can J Microbiol 60:437–443

    Article  PubMed  CAS  Google Scholar 

  • Porras-Gómez M, Vega-Baudrit J, Núñez-Corrales S (2012) Overview of multidrug-resistant Pseudomonas aeruginosa and novel therapeutic approaches. J Biomater Nanobiotechnol 3:519–527

    Article  CAS  Google Scholar 

  • Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256

    Article  PubMed  CAS  Google Scholar 

  • Quale J, Bratu S, Gupta J, Landman D (2006) Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical Isolates. Antimicrob Agents Chemother 50:1633–1641

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sobel ML, McKay GA, Poole K (2003) Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical Isolates. Antimicrob Agents Chemother 47:3202–3207

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Sobel ML, Hocquet D, Cao L, Plesiat P, Poole K (2005) Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 49:1782–1786

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Strateva T, Yordanov D (2009) Pseudomonas aeruginosa–a phenomenon of bacterial resistance. J Med Microbiol 58:1133–1148

    Article  PubMed  CAS  Google Scholar 

  • Suman G, Khan M, Sabitha K, Jamil K (2006) Mutation in mexR-gene leading to drug resistance in corneal keratitis in human. Indian J Exp Biol 44:929–936

    PubMed  CAS  Google Scholar 

  • Tian Z-X, Yi X-X, Cho A, O’Gara F, Wang Y-P (2016) CpxR activates MexAB-OprM efflux pump expression and enhances antibiotic resistance in both laboratory and clinical nalB-type isolates of Pseudomonas aeruginosa. PLoS Pathog 12:e1005932

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • WHO (2017) http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics- needed/en/. Accessed 19 Sept 2017

  • Wilke MS, Heller M, Creagh AL, Haynes CA, McIntosh LP, Poole K, Strynadka NC (2008) The crystal structure of MexR from Pseudomonas aeruginosa in complex with its antirepressor ArmR. Proc Natl Acad Sci USA 105:14832–14837

    Article  PubMed  PubMed Central  Google Scholar 

  • Yoneda K, Chikumi H, Murata T, Gotoh N, Yamamoto H, Fujiwara H, Nishino T, Shimizu E (2005) Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction. FEMS Microbiol Lett 243:125–131

    Article  PubMed  CAS  Google Scholar 

  • Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y (2002) Imipenem-EDTA disc method for differentiation of metallo-beta-lactamase producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 40:3798–3801

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zavascki AP, Carvalhaes CG, Picão RC, Gales AC (2010) Multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: resistance mechanisms and implications for therapy. Expert Rev Anti Infect Ther 8:71–93

    Article  PubMed  CAS  Google Scholar 

  • Ziha-Zarifi I, Llanes C, Köhler T, Pechere J-C, Plesiat P (1999) In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM. Antimicrob Agents Chemother 43:287–291

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the Government of India, DST (Department of Science and Technology) - INSPIRE fellowship (Grant No. IF120394) to MS.

Author information

Authors and Affiliations

  1. Department of Biotechnology, University of Calicut, Malappuram, Kerala, 673635, India

    Manju Suresh, Nithya N. & Manish Kumar P. R.

  2. School of Health Sciences, University of Calicut, Malappuram, Kerala, 673635, India

    Jayasree P. R.

  3. Department of Botany, University of Calicut, Malappuram, Kerala, 673635, India

    Vimal K. P.

Authors
  1. Manju Suresh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nithya N.
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jayasree P. R.
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vimal K. P.
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manish Kumar P. R.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manish Kumar P. R..

Ethics declarations

Conflict of interest

All authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Suresh, M., Nithya, N., Jayasree, P.R. et al. Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa. World J Microbiol Biotechnol 34, 83 (2018). https://doi.org/10.1007/s11274-018-2465-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11274-018-2465-0

Keywords

Search

Navigation