Mycobacteria: Bugs and bugbears (two steps forward and one step back)

The use of molecular techniques to study the mycobacteria has advanced greatly since the first genomic libraries of Mycobacterium tuberculosis and M. leprae were constructed in 1985. However, there are still pitfalls for the unwary. Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems: slow growth rate causing problems with contamination; the formation of macroscopic clumps when grown in culture; resistance to standard chemical lysis procedures; the requirement for containment facilities for pathogenic species; the lack of suitable genetic vectors; and the problems of spontaneous antibiotic resistance. Despite these problems, considerable progress has been made and standard techniques have been developed for the preparation of protein, nucleic acids (DNA and RNA) and cell wall components, chemical and transposon mutagenesis and gene replacement methods, the use of reporter genes and expression vectors, and improved detection and drug sensitivity testing.