Graphic representation of the experimental procedures of method 2.

<p>For method 2 the same non-selective starting cultures as for method 1 were used to inoculate one 10-ml culture for each strain used in this study. These cultures were incubated at 36°C in a shaking incubator until mid-logarithmic phase (ca. three weeks). Then four aliquots of 0.5 ml from each culture were plated on rifampicin-containing solid medium to select for resistant mutants. The plates were sealed and incubated at 36°C until sufficient bacterial growth of the mutants was visible (ca. four weeks) When mutant colonies were visible on the plates, DNA was extracted: one mutant colony corresponded to one DNA sample. Finally, DNA samples were screened by MLPA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029108#pone.0029108-Bergval2" target="_blank">[25]</a> or sequencing of the rifampicin resistance determining region in rpoB.