Molecular detection of antimicrobial resistance

Abstract

The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art.

FIG. 1

Schematic representation of 5′→3′ exonuclease cleavage of a 5′-labeled (small black star) probe with a 3′-phosphate (grey circle) extension blocker. After cleavage of the label from the probe by DNA polymerase (large black star), the small labeled fragments generated can be separated from the larger probe.

FIG. 2

Principle of molecular beacons. The stem of the hairpin is less stable than the hybridization of the specific probe (loop region) with its target (top). Hybridization leads to denaturation of the stem and the physical separation of the fluorophore (white star) and its quencher (black star), allowing fluorecence to occur (bottom).

FIG. 3

The Scorpions primer is an extension of molecular beacons. To a molecular beacon, a blocker (grey circle) and PCR primer are added (top). The blocker prevents the copying of the molecular beacon part of the molecule. After one round of amplification (middle), the molecular beacon extension of the primer is able to hybridize with the newly synthesized DNA strand, allowing fluorescence (bottom).

FIG. 4

Schematic presentation of bDNA amplification. For details, see the text. Reprinted from M. N. Widjojoatmodjo, Diagnosis of infections based on DNA amplification: obstacles and solutions, Academic thesis, University of Utrecht, Utrecht, The Netherlands, 1995, with permission of the author.

FIG. 5

Principle of oligonucleotide array sequencing. Alignment of the overlapping probes reconstructs the complement of the original target (see the text for details).