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Chapter 12.5 : Molecular Methods for Antimicrobial Agent Resistance Determination

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Abstract:

Antimicrobial agent resistance is an increasing problem worldwide, particularly among critically ill hospitalized patients. For this reason, there is a renewed interest in monitoring the development and spread of antimicrobial agent resistance and a recognition of the need for effective interventions to limit the spread of resistance to prolong the therapeutic life of the available antimicrobial agents. Unfortunately, conventional methods to perform antimicrobial agent susceptibility testing may be too slow and insensitive in detecting antimicrobial agent resistance to be of much use clinically. The techniques of molecular biology have been used to characterize resistance at the DNA level and may provide rapid, sensitive, and specific information to the clinician for use in therapeutic decision making ( ). Genetic material that confers antimicrobial agent resistance may be carried on the bacterial chromosome or on transposons or plasmids and has been detected by probe hybridization or by DNA amplification with PCR (Table 12.1-6). Molecular detection of resistance has potential value for decisions directly related to patient care and is useful for calibration of conventional susceptibility tests and for precise definition of the mechanisms of resistance to selected antimicrobial agents. Molecular techniques have been used to detect genes encoding several different mechanisms of resistance against antimicrobial agents, e.g., β-lactam agents, aminoglycosides, macrolides, and fluoroquinolones, after isolation of a clinical isolate (Table 12.1-6).

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
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Figures

Image of Figure 12.5.2-1
Figure 12.5.2-1

Agarose gel electrophoresis pattern of PCR products. Lanes: A, size marker ϕX174 phage DNA III digest; B, product (783 bp); C, product (297 bp); D, product (822 bp); E, product (439 bp). PCR products for and resulted from the reaction described above; and products were obtained with single primer sets per reaction mixture. Figure from Free and Sahm ( ).

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
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References

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3. Persing, D. H.,, D. A. Relman,, and F. C. Ten-over,. 1996. Genotypic detection of antimicrobial resistance, p. 3357. In D. H. Persing (ed.), PCR Protocols for Emerging Infectious Diseases. ASM Press, Washington, DC.
4. Rasheed, J. K.,, F. Cockerill,, and F. C. Ten-over,. 2007. Detection and characterization of antimicrobial resistance genes in pathogenic bacteria, p. 12481251. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. Pfaller (ed.), Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
1.CLSI. 2004. Quality Assurance for Commercially Prepared Microbiological Culture Media, 3rd ed. Approved standard M22-A3. CLSI, Wayne, PA.
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1.. CLSI. 2004. Quality Assurance for Commercially Prepared Microbiological Culture Media, 3rd ed. Approved standard M22-A3. CLSI, Wayne, PA.
2.. Murakami, K.,, and W. Minamide,. 1993. PCR identification of methicillin-resistant Staphylococcus aureus, p. 539542. In D. H. Persing,, T. F. Smith,, F. C. Tenover,, and T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. American Society for Microbiology, Washington, DC.
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11. Marshall, S. A.,, W. W. Wilke,, M. A. Pfaller,, and R. N. Jones. 1998. Staphylococcus aureus and coagulase-negative staphylococci from blood stream infections: frequency of occurrence, antimicrobial susceptibility, and molecular (mecA) characterization of oxacillin resistance in the SCOPE program. Diagn. Microbiol. Infect. Dis. 30:205214.
12. Ramotar, K.,, M. Bobrowska,, P. Jessamine,, and B. Toye. 1998. Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods. Diagn. Microbiol. Infect. Dis. 30:267273.
13. Anderson, K. F.,, D. R. Lonsway,, J. K. Rasheed,, J. Biddle,, B. Jensen,, L. K. McDougal,, R. B. Carey,, A. Thompson,, S. Stocker,, B. Limbago,, and J. B. Patel. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45:27232725.
14. Conrad, S.,, M. Oethinger,, K. Kaifel,, G. Klotz,, R. Marre,, and W. V. Kern. 1996. gyrA mutations in high-level fluoroquinolone-resistant clinical isolates of Escherichiacoli. J. Antimicrob. Chemother. 38:443455.
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17. Naas, T.,, G. Cuzon,, M. Villegas,, M. Lartigue,, J. P. Quinn,, and P. Nordmann. 2008. Genetic structures at the origin of acquisition of the β-lactamase blaKPC gene. Antimicrob. Agents Chemother. 52:12571263.
18. Queenan, A. M.,, and K. Bush. 2007. Carbapenemases: the versatile β-lactamases. Clin. Microbiol. Rev. 20:440458.
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Tables

Generic image for table
Table 12.5.2-1

PCR primers for detection of , and in enterococci

GenBank accession numbers: , X56895; , U00456; , M75132; , L29638

Primer sequences for VanC1-1, VanC1-2, VanC2-1, and VanC2-2 from Dutka-Malen et al. ( ).

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
Generic image for table
Table 12.5.3-1

PCR primers for detection of in staphylococci

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
Generic image for table
Table 12.5.4-1

PCR primers and probe for the detection of KPC

*Store at 2 to 8°C after initial thaw for up to 2 months.

R = A or G.

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
Generic image for table

Ct, cycle threshold.

Undetected, no Ct detected within 40 cycles.

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
Generic image for table

Ct, cycle threshold.

Undetected, no Ct detected within 40 cycles.

If the 16S rRNA gene is not detected, the sample result is not valid. Review results to determine the possible problem(s). Bad primers, probes, or reagents or even overinoculation may be the cause. Prepare new lysates or try different reagents, as recommended by the supervisor.

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
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Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5
Generic image for table
Untitled

Citation: Garcia L. 2010. Molecular Methods for Antimicrobial Agent Resistance Determination, p 410-427. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.5

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