Abstract
The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla OXA-2, strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Andersson DI, Levin BR (1999) The biological cost of antibiotic resistance. Curr Opin Microbiol 2:489–493
Bjorkman J, Andersson DI (2000) The cost of antibiotic resistance from a bacterial perspective. Drug Resist Updat 3:237–245
Lenski RE, Simpson RC, Nguyen TT (1994) Genetic analysis of a plasmid-encoded, host genotype-specific enhancement of bacterial fitness. J Bacteriol 176:3140–3147
Schrag SJ, Perrot V, Levin BR (1997) Adaptation to the fitness cost of antibiotic resistance in Escherichia coli. Proc R Soc Lond B Biol Sci 264:1287–1291
Wu SW, de Lancastre H, Tomasz A (2001) Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus. J Bacteriol 183:2417–2424
Podglajen I, Breuil J, Collatz E (1994) Insertion of a novel DNA sequence IS1186, upstream of the silent carbapenemase gene, cfiA, promotes expression of carbapenem resistance in clinical isolates of Bacteroides fragilis. Mol Microbiol 12:105–114
Enne VI, Delsol AA, Roe JM, Bennett PM (2006) Evidence of antibiotic resistance gene silencing in Escherichia coli. Antimicrob Agents Chemother 50:3003–3010
Enne VI, Cassar C, Sprigings K, Woodward MJ, Bennett PM (2008) A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter. FEMS Microbiol Lett 278:193–199
Steward CD, Rasheed JK, Hubert SK, Biddle JW, Raney PM, Anderson GJ, Williams PP, Brittain KI, Oliver A, McGowan JE, Tenover FC (2001) Characterisation of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the national committee for clinical laboratory standards extended-spectrum β-lactamase detection methods. J Clin Microbiol 39:2864–2872
Livermore DM, Warner M, Hall LMC, Enne VI, Projan SJ, Dunman PM, Wooster SL, Harrison G (2001) Antibiotic resistance in bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) from west Wales. Environ Microbiol 3:658–661
Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL (2001) Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67:5675–5682
CLSI (2006) Performance standards for antimicrobial susceptibility testing; 16th informational supplement, vol 26. Clinical and Laboratory Standards Institute, Wayne, PA, pp M100–S16
Andrews JM (2005) BSAC standardized disc susceptibility testing method. J Antimicrob Chemother 56:60–76
Acknowledgments
The work described here was funded by the Department of Environment, Food and Rural Affairs under project code OD2007.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Enne, V.I., Bennett, P.M. (2010). Methods to Determine Antibiotic Resistance Gene Silencing. In: Gillespie, S., McHugh, T. (eds) Antibiotic Resistance Protocols. Methods in Molecular Biology, vol 642. Humana Press. https://doi.org/10.1007/978-1-60327-279-7_3
Download citation
DOI: https://doi.org/10.1007/978-1-60327-279-7_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-278-0
Online ISBN: 978-1-60327-279-7
eBook Packages: Springer Protocols