Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly→Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants

Molecular Detection of Antimicrobial Resistance

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