Genotyping Techniques for Determining the Diversity of Microorganisms

Typing of microbial pathogens, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. The part that molecular methods have to play in elucidating bacterial diversity is increasingly important. The shortcomings of phenotypically based typing methods (generally these methods are viewed as being too time consuming and lacking in sufficient resolution amongst related strains) have led to the development of many DNA – based techniques. A suitable typing method must have high discrimination power combined with good to moderate interand intra-laboratory reproducibility. In addition, it should be easy to set up, to use and to interpret, and inexpensive (Olive & Bean, 1999). In this chapter, we review the current bacterial genotyping methods and classify them into six main categories: (1 and 2) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated respectively by enzymatic digestion of genomic/plasmid DNA, and PCR amplification, (3) DNA hybridization–based methods using nucleotidic probes, (4) DNA sequencing-based methods, which study the polymorphism of DNA sequences, (5) differentiation of isolates on the basis of presence or absence of particular genes and (6) high resolution melting analysis–real–time monitoring of melting process of PCR amplified polymorphic DNA fragment. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing and described the current trends of genotyping methods.