Molecular techniques offer the possibility for more timely antibiotic resistance profiles of both slow-growing microorganisms and microorganisms that are difficult to culture. For severe infections, molecular techniques may provide more rapid determination of resistance profiles. This becomes more important with increasing antibiotic resistance, which compromises adequate options for empiric therapy. Molecular techniques have been described for the detection of antibiotic resistance for a large number of resistance determinants and a wide range of bacterial species. The development of new molecular techniques always led to quick adaptation for the detection of antibiotic resistance. Despite the possibilities offered by molecular techniques, their use is frequently limited to a research setting and implementation in routine diagnostics can be problematic. There are a number of reasons for this:
The drawbacks mentioned often limit the application of molecular techniques to epidemiological studies for one or a few genes. These are usually detected by a conventional PCR. Conventional PCR has come within the grasp of more and more laboratories. The technique is straightforward and simple, especially with the development of software programs that help design appropriate primers for any gene for which a sequence is known. This simplicity also represents a danger since the PCR protocol itself may lead to unexpected amplification products and contamination by other samples or previous amplification products. This requires rigorous laboratory procedures and quality control. Unfortunately, the required expertise is not present everywhere and even when present the molecular techniques may provide unexpected challenges. This is underscored by a study by
Noordhoek et al. , who demonstrated that a considerable number of laboratories had difficulty in correctly identifying samples containing M. tuberculosis. More complicated techniques increase the risk for false-negative and false-positive results. However, new techniques may help reduce the risks by improved concepts and instrumentation.
New techniques also offer possibilities for more effectively coping with large numbers of resistance determinants or point mutations. Advances in two areas— sequencing and microarrays—are potentially important in this respect. Pyro-sequencing is likely to become an important tool for the detection of point mutations. Its ability to inexpensively sequence small (40-50 bp) stretches of DNA make it ideal to detect point mutations or single nucleotide polymorphisms (SNPs). Its impact on routine diagnostics is difficult to predict, but the technique has significant potential. Pyrosequencing will certainly become an important tool for research, not only for the detection of SNPs, but in conjunction with the new technology introduced by 454 Life Sciences (U.S.A.) for sequencing of large DNA fragments, such as resistance plasmids. Briefly, DNA is fragmented to random pieces of appropriate size and ligated with primers for amplification and sequencing. The fragments are made single-stranded and bound to microbeads in such a way that only one fragment per bead is obtained. The beads are emulsified in buffer oil. Each bead is encapsulated in its own buffer capsule. The buffer contains all ingredients for amplification, the products of which become bound to the beads. The beads are then prepared for pyrosequencing, which is performed in a massively parallel fashion. Up to 200,000 sequences are generated. Special computer software determines the final sequence based on overlapping fragments. The other technique that has the potential to become more prominent is microarray technology. Microarrays offer the ability to interrogate thousands of genes simultaneously, but relatively large numbers of bacteria are required to obtain sufficient amounts of labeled DNA. Another issue is quality control, which becomes more complicated with increasing numbers of genes.
Currently, rtPCR, reverse line blot, and heteroduplex analyses are the most important techniques for the detection of antibiotic resistance in routine settings. Most studies presented here to detect antibiotic resistance among multidrug-resistant isolates do not cover all possible mechanisms. When epidemiological studies show that certain mechanisms are either absent or have a very low prevalence, it may be a cost-effective approach to ignore rare mechanisms, although the risk exists that some of these mechanisms may become more important. The consequences of non-detection of resistant isolates are not certain, but it may be expected that these will replace other strains and become responsible for new outbreaks of (multi)resistant strains. It will therefore be necessary to design surveillance studies to capture resistance mechanisms not included on a routine basis.
Molecular assays have a place in routine diagnosis of antibiotic resistance. In the future, with the development of new technologies and improvement of current technologies, the importance of molecular assays for routine detection of antibiotic resistance will only increase, although the implementation of these techniques may be slower than desirable.
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