Chapter 4 : Molecular Methods for Detection of Antibiotic Resistance

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

Molecular Methods for Detection of Antibiotic Resistance, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817534/9781555813062_Chap04-1.gif /docserver/preview/fulltext/10.1128/9781555817534/9781555813062_Chap04-2.gif


The combination of molecular characterization of resistant strains with precise identification of the antibiotic resistance gene(s) or mutation(s) and the genetic elements involved in the dissemination of these genes is an effective approach in the control of the spread of antibiotic resistance. Molecular methods also help to determine the location of the gene and to differentiate between horizontal gene transfer and clonal spread. This chapter describes polymerase chain reaction (PCR) and microarray analysis in detail, and specifies applications of these methods in the investigation of antibiotic-resistant strains. Numerous PCR assays for the detection of antibiotic resistance genes have been developed and the development of microarrays for the simultaneous detection of a large number of these genes and the genetic elements involved in their dissemination is in progress. The implementation of molecular methods in routine analysis can be achieved only when it is supported by the proper validation of the methods and the availability of the necessary controls, reference strains, and educated personnel. The molecular characterization of antibiotic-resistant strains can help to identify atypical resistant strains, describe new outbreak strains at an early stage, elucidate the epidemiology of resistant strains at a genotypic level, and explain the processes leading to the selection of resistant and virulent strains. In addition, molecular methods can allow proper risk assessment with respect to the use of antimicrobial substances. If the transcriptional and translational expression of antibiotic resistance genes can be better understood, molecular methods may replace phenotypic measurements.

Citation: Aarts H, Guerra B, Malorny B. 2006. Molecular Methods for Detection of Antibiotic Resistance, p 37-48. In Aarestrup F (ed), Antimicrobial Resistance in Bacteria of Animal Origin. ASM Press, Washington, DC. doi: 10.1128/9781555817534.ch4
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of Figure 1
Figure 1

Detection methods in real-time PCR. (A) SYBR Green emits a fluorescent signal when it binds to dsDNA. (B) Specific binding of a double-labeled hydrolysis, or TaqMan, probe. Q indicates the quencher dye and F the fluorescent (reporter) dye. (C) Specific binding of the hybridization FRET probes (acceptor and donor). The donor fluorescence (D) is transmitted to the acceptor fluorescence (AF) only when it is in close proximity, generating a fluorescence signal. See the text for further explanation.

Citation: Aarts H, Guerra B, Malorny B. 2006. Molecular Methods for Detection of Antibiotic Resistance, p 37-48. In Aarestrup F (ed), Antimicrobial Resistance in Bacteria of Animal Origin. ASM Press, Washington, DC. doi: 10.1128/9781555817534.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint


1.. Aarts, H. J. M.,, K. S. Boumedine,, X. Nesme,, and A. Cloeckaert. 2001. Molecular tools for the characterisation of antibiotic-resistant bacteria. Vet. Res. 32: 363 380.
2. Allignet, J.,, S. Aubert,, A. Morvan,, and N. El Solh. 1996. Distribution of genes encoding resistance to streptogramin A and related compounds among staphylococci resistant to these antibiotics. Antimicrob. Agents Chemother. 40: 2523 2528.
3. Altweg, M. 1995. General problems associated with diagnostic applications of amplification methods. J. Microbiol. Methods 23: 21 30.
4. Aminov, R. I.,, N. Garrigues-Jeanjean,, and R. I. Mackie. 2001. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins. Appl. Environ. Microbiol. 67: 22 32.
5.Anonymous. 2003. Microbiology of food and animal feeding stuffs—protocol for the validation of alternative methods (ISO 16140:2003). European Committee for Standardization, AFNOR, Paris, France.
6. Bergeron, M. G.,, and M. Ouellette. 1998. Preventing antibiotic resistance using rapid DNA-based diagnostic tests. Infect. Control Hosp. Epidemiol. 19: 560 564.
7. Boyd, D.,, A. Cloeckaert,, E. Chaslus-Dancla,, and M. R. Mulvey. 2002. Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob. Agents Chemother. 46: 1714 1722.
8. Bradford, P. A. 2001. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14: 933 951.
9. Butaye, P.,, A. Cloeckaert,, and S. Schwarz. 2003. Mobile genes coding for efflux-mediated antimicrobial resistance in Grampositive and Gram-negative bacteria. Int. J. Antimicrob Agents 22: 205 210.
10. Call, D. R.,, M. K. Bakko,, M. J. Krug,, and M. C. Roberts. 2003. Identifying antimicrobial resistance genes with DNA microarrays. Antimicrob. Agents Chemother. 47: 3290 3295.
11. Carattoli, A. 2001. Importance of integrons in the diffusion of resistance. Vet Res. 32: 243 259.
12. Carattoli, A.,, A. Dionisi,, and I. Luzzi. 2002. Use of a LightCycler gyrA mutation assay for identification of ciprofloxacinresistant Campylobacter coli. FEMS Microbiol. Lett. 214: 87 93.
13. Chu, C.,, C. H. Chiu,, W. Y. Wu,, C. H. Chu,, T. P. Liu,, and J. T. Ou. 2001. Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob. Agents Chemother. 45: 2299 2303.
14. Coenye, T.,, and P. Vandamme. 2004. Bacterial whole-genome sequences: minimal information and strain availability. Microbiology 150: 2017 2018. 15.
15. Doublet, B.,, R. Lailler,, D. Meunier,, A. Brisabois,, D. Boyd,, M. R. Mulvey,, E. Chaslus-Dancla,, and A. Cloeckaert. 2003. Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany. Emerg. Infect. Dis. 9: 585 591.
16. DuBois, D. B.,, and J. T. Brown,. 2004. Laboratory controls and standards, p. 697 703. In D. H. Persing,, F. C. Tenover,, J. Versalovic,, Y.-W. Tang,, E. R. Unger,, D. A. Relman,, and T. J. White (ed.), Molecular Microbiology: Diagnostic Principles and Practice. American Society for Microbiology, Washington, D.C.
17. Dutka-Malen, S.,, S. Evers,, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol. 33: 24 27.
18. Fang, H.,, and G. Hedin. 2003. Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay. J. Clin. Microbiol. 41: 2894 2899.
19. Fluit, A. C.,, M. R. Visser,, and F. J. Schmitz. 2001. Molecular detection of antimicrobial resistance. Clin. Microbiol. Rev. 14: 836 871.
20. Frech, G.,, C. Kehrenberg,, and S. Schwarz. 2003. Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources. J. Antimicrob. Chemother. 51: 180 182.
21. Frech, G.,, and S. Schwarz. 2000. Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes. J. Appl. Microbiol. 89: 633 641.
22. García de Viedma, D.,, M. D. S. Díaz Infantes,, F. Lasala,, F. Chaves,, L. Alcalá,, and E. Bouza. 2002. New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis. J. Clin. Microbiol. 40: 988 995.
23. Garcia de Viedma, D. G. 2002. Rapid detection of resistance in Mycobacterium tuberculosis: a review discussing molecular approaches. Clin. Microbiol. Infect. 9: 349 359.
24.Reference deleted.
25. Grimm, V.,, S. Ezaki,, M. Susa,, C. Knabbe,, R. D. Schmid,, and T. T. Bachmann. 2004. Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance. J. Clin. Microbiol. 42: 3766 3774.
26. Grisold, A. J.,, E. Leitner,, G. Muhlbauer,, E. Marth,, and H. H. Kessler. 2002. Detection of methicillin-resistant Staphylococcus aureus and simultaneous confirmation by automated nucleic acid extraction and real-time PCR. J. Clin. Microbiol. 40: 2392 2397.
27. Guerra, B.,, E. Junker,, A. Miko,, R. Helmuth,, and M. C. Mendoza. 2004. Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains. Microb. Drug Resist. 10: 83 91.
28. Guerra, B.,, S. Soto,, S. Cal,, and M. C. Mendoza. 2000. Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes. Antimicrob. Agents Chemother. 44: 2166 2169.
29. Guerra, B.,, S. M. Soto,, J. M. Arguelles,, and M. C. Mendoza. 2001. Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-]. Antimicrob. Agents Chemother. 45: 1305 1308.
30. Hoorfar, J.,, N. Cook,, B. Malorny,, M. Wagner,, D. De Medici,, A. Abdulmawjood,, and P. Fach. 2003. Making internal amplification control mandatory for diagnostic PCR. J. Clin. Microbiol. 41: 5835.
31. Hoorfar, J.,, B. Malorny,, A. Abdulmawjood,, N. Cook,, and P. Fach. 2003. Practical considerations when designing internal amplification control for diagnostic PCR assays. J. Clin. Microbiol. 42: 1863 1868.
32. Huletsky, A.,, R. Giroux,, V. Rossbach,, M. Gagnon,, M. Vaillancourt,, M. Bernier,, F. Gagnon,, K. Truchon,, M. Bastien,, F. J. Picard,, A. van Belkum,, M. Ouellette,, P. H. Roy,, and M. G. Bergeron. 2004. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J. Clin. Microbiol. 42: 1875 1884.
33. Jensen, L. B.,, A. M. Hammerum,, F. M. Aarestrup,, A. E. van den Bogaard,, and E. E. Stobberingh. 1998. Occurrence of satA and vgb genes in streptogramin-resistant Enterococcus faecium isolates of animal and human origins in The Netherlands. Antimicrob. Agents Chemother. 42: 3330 3331.
34. Kehrenberg, C.,, S. A. Salmon,, J. L. Watts,, and S. Schwarz. 2001. Tetracycline resistance genes in isolates of Pasteurella multocida, Mannheimia haemolytica, Mannheimia glucosida and Mannheimia varigena from bovine and swine respiratory disease: intergeneric spread of the tet(H) plasmid pMHT1. J. Antimicrob. Chemother. 48: 631 640.
35. Kim, S. H.,, A. B. Chang,, and M. H. Saier, Jr. 2004. Sequence similarity between multidrug resistance efflux pumps of the ABC and RND superfamilies. Microbiology 150: 2493 2495.
36. Lapierre, P.,, A. Huletsky,, V. Fortin,, F. J. Picard,, P. H. Roy,, M. Ouellette,, and M. G. Bergeron. 2003. Real-time PCR assay for detection of fluoroquinolone resistance associated with grlA mutations in Staphylococcus aureus. J. Clin. Microbiol. 41: 3246 3251.
37. Lee, Y.,, C. S. Lee,, Y. J. Kim,, S. Chun,, S. Park,, Y. S. Kim,, and B. D. Han. 2002. Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes. Mol. Cell 14: 192 197.
38. Levesque, C.,, L. Piche,, C. Larose,, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39: 185 191.
39. Livak, K. J.,, S. J. Flood,, J. Marmaro,, W. Giusti,, and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4: 357 362.
40. Marín, M.,, D. García de Viedma,, M. J. Ruiz-Serrano,, and E. Bouza. 2004. Rapid direct detection of multiple rifampin and isoniazid resistance mutations in Mycobacterium tuberculosis in respiratory samples by real-time PCR. Antimicrob. Agents Chemother. 48: 4293 4300.
41. Miko, A.,, K. Pries,, A. Schroeter,, and R. Helmuth. 2003. Multiple-drug resistance in d-tartrate-positive Salmonella enterica serovar Paratyphi B isolates from poultry is mediated by class 2 integrons inserted into the bacterial chromosome. Antimicrob. Agents Chemother. 47: 3640 3643.
42. Mohn, S. C.,, A. Ulvik,, R. Jureen,, R. J. Willems,, J. Top,, H. Leavis,, S. Harthug,, and N. Langeland. 2004. Duplex realtime PCR assay for rapid detection of ampicillin-resistant Enterococcus faecium. Antimicrob. Agents Chemother. 48: 556 560.
43. Morsczeck, C.,, D. Langendorfer,, and J. M. Schierholz. 2004. A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon. J. Biochem. Biophys. Methods 59: 217 227.
44.National Committee for Clinical Laboratory Standards. 1995. Molecular diagnostics methods for infectious diseases. Approved guideline MM3-A, vol. 15, no. 22. National Committee for Clinical Laboratory Standards, Wayne, Pa.
45. Ng, L. K.,, I. Martin,, M. Alfa,, and M. Mulvey. 2001. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell Probes 15: 209 215.
46. Noppe-Leclercq, I.,, F. Wallet,, S. Haentjens,, R. Courcol,, and M. Simonet. 1999. PCR detection of aminoglycoside resistance genes: a rapid molecular typing method for Acinetobacter baumannii. Res. Microbiol. 150: 317 322.
47. Nordmann, P. 1998. Trends in beta-lactam resistance among Enterobacteriaceae. Clin. Infect. Dis. 27(Suppl. 1): S100 S106.
48. Palladino, S.,, I. D. Kay,, A. M. Costa,, E. J. Lambert,, and J. P. Flexman. 2003. Real-time PCR for the rapid detection of v anA and vanB genes. Diagn. Microbiol. Infect. Dis. 45: 81 84.
49. Palladino, S.,, I. D. Kay,, J. P. Flexman,, I. Boehm,, A. M. Costa,, E. J. Lambert,, and K. J. Christiansen. 2003. Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay. J. Clin. Microbiol. 41: 2483 2486.
50. Perreten, V.,, and P. Boerlin. 2003. A new sulfonamide resistance gene ( sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob. Agents Chemother. 47: 1169 1172.
51. Poulsen, R. L.,, L. V. Pallesen,, N. Frimodt-Moller,, and F. Espersen. 1999. Detection of clinical vancomycin-resistant enterococci in Denmark by multiplex PCR and sandwich hybridization. APMIS 107: 404 412.
52. Reinert, R. R.,, C. Franken,, L. M. van der,, R. Lutticken,, M. Cil,, and A. Al Lahham. 2004. Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002-2003. Int. J. Antimicrob. Agents 24: 43 47.
53. Ruiz, M.,, M. J. Torres,, A. C. Llanos,, A. Arroyo,, J. C. Palomares,, and J. Aznar. 2004. Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auraminerhodamine- positive sputum specimens by real-time PCR. J. Clin. Microbiol. 42: 1585 1589.
54. Saier, M. H., Jr. 2000. Vectorial metabolism and the evolution of transport systems. J. Bacteriol. 182: 5029 5035.
55. Sandvang, D.,, and F. M. Aarestrup. 2000. Characterization of aminoglycoside resistance genes and class 1 integrons in porcine and bovine gentamicin-resistant Escherichia coli. Microb. Drug Resist. 6: 19 27.
56.. Schmitz, F. J.,, R. Sadurski,, A. Kray,, M. Boos,, R. Geisel,, K. Kohrer,, J. Verhoef,, and A. C. Fluit. 2000. Prevalence of macrolide-resistance genes in Staphylococcus aureus and Enterococcus faecium isolates from 24 European university hospitals. J. Antimicrob. Chemother. 45: 891 894.
57. Shortridge, V. D.,, R. K. Flamm,, N. Ramer,, J. Beyer,, and S. K. Tanaka. 1996. Novel mechanism of macrolide resistance in Streptococcus pneumoniae. Diagn. Microbiol. Infect. Dis. 26: 73 78.
58. Sutcliffe, J.,, T. Grebe,, A. Tait-Kamradt,, and L. Wondrack. 1996. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother. 40: 2562 2566.
59. Tan, T. Y.,, S. Corden,, R. Barnes,, and B. Cookson. 2001. Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR. J. Clin. Microbiol. 39: 4529 4531.
60. Tenover, F. C.,, and J. K. Rasheed,. 1999. Genetic methods for detecting antibacterial and antiviral resistance genes, p. 1578 1592. In P. R. Murray,, E. J. Baron,, M. A. Pfaller,, F. C. Tenover,, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
61. Tenover, F. C.,, and J. K. Rasheed,. 2004. Detection of antimicrobial resistance genes and mutations associated with antimicrobial resistance in microorganisms, p. 391 406. In D. H. Persing,, F. C. Tenover,, J. Versalovic,, Y.-W. Tang,, E. R. Unger,, D. A. Relman,, and T. J. White (ed.), Molecular Microbiology:Diagnostic Principles and Practice. American Society for Microbiology, Washington, D.C.
62. Thompson, J. D.,, T. J. Gibson,, F. Plewniak,, F. Jeanmougin,, and D. G. Higgins. 1997. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876 4882.
63. Torres, M. J.,, A. Criado,, M. Ruiz,, A. C. Llanos,, J. C. Palomares,, and J. Aznar. 2003. Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates. Diagn. Microbiol. Infect. Dis. 45: 207 212.
64. Trzcinski, K.,, B. S. Cooper,, W. Hryniewicz,, and C. G. Dowson. 2000. Expression of resistance to tetracyclines in strains of methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 45: 763 770.
64a.. van Hoek, A. H. A. M.,, I. M. J. Scholtens,, A. Cloeckaert,, and H. J. M. Aarts. 2005. Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. J. Microbiol. Methods 62: 13 23.
65. Vanhoof, R.,, C. Godard,, J. Content,, H. J. Nyssen,, and E. Hannecart-Pokorni. 1994. Detection by polymerase chain reaction of genes encoding aminoglycoside-modifying enzymes in methicillin-resistant Staphylococcus aureus isolates of epidemic phage types. J. Med. Microbiol. 41: 282 290.
66. Versalovic, J.,, and E. R. Unger,. 2004. External quality assessment and proficiency testing in diagnostic molecular microbiology, p. 691 696. In D. H. Persing,, F. C. Tenover,, J. Versalovic,, Y.-W. Tang,, E. R. Unger,, D. A. Relman,, and T. J. White (eds.), Molecular Microbiology: Diagnostic Principles and Practice. American Society for Microbiology, Washington, D.C.
67. Volkmann, H.,, T. Schwartz,, P. Bischoff,, S. Kirchen,, and U. Obst. 2004. Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan). J. Microbiol. Methods 56: 277 286.
68. Volokhov, D.,, V. Chizhikov,, K. Chumakov,, and A. Rasooly. 2003. Microarray analysis of erythromycin resistance determinants. J. Appl. Microbiol. 95: 787 798.
69. Wada, T.,, S. Maeda,, A. Tamaru,, S. Imai,, A. Hase,, and K. Kobayashi. 2004. Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR. J. Clin. Microbiol. 42: 5277 5285.
70. Walker, R. A.,, N. Saunders,, A. J. Lawson,, E. A. Lindsay,, M. Dassama,, L. R. Ward,, M. J. Woodward,, R. H. Davies,, E. Liebana,, and E. J. Threlfall. 2001. Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates. J. Clin. Microbiol. 39: 1443 1448.
71. Werner, G.,, and W. Witte. 1999. Characterization of a new enterococcal gene, satG, encoding a putative acetyltransferase conferring resistance to streptogramin A compounds. Antimicrob. Agents Chemother. 43: 1813 1814.
72. Westin, L.,, C. Miller,, D. Vollmer,, D. Canter,, R. Radtkey,, M. Nerenberg,, and J. P. O’Connell. 2001. Antimicrobial resistance and bacterial identification utilizing a microelectronic chip array. J. Clin. Microbiol. 39: 1097 1104.
73. Wittwer, C. T.,, M. G. Herrmann,, A. A. Moss,, and R. P. Rasmussen. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22: 130 138.
74. Yu, X.,, M. Susa,, C. Knabbe,, R. D. Schmid,, and T. T. Bachmann. 2004. Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates. J. Clin. Microbiol. 42: 4083 4091.
75. Yue, J.,, W. Shi,, J. Xie,, Y. Li,, E. Zeng,, L. Liang,, and H. Wang. 2004. Detection of rifampin-resistant Mycobacterium tuberculosis strains by using a specialized oligonucleotide microarray. Diagn. Microbiol. Infect. Dis. 48: 47 54.


Generic image for table
Table 1

Real-time PCR assays for the detection of antibiotic resistance genes

Citation: Aarts H, Guerra B, Malorny B. 2006. Molecular Methods for Detection of Antibiotic Resistance, p 37-48. In Aarestrup F (ed), Antimicrobial Resistance in Bacteria of Animal Origin. ASM Press, Washington, DC. doi: 10.1128/9781555817534.ch4

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error