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Background  The genus  Mycobacterium  ( M .) comprises highly pathogenic bacteria such as  M. tuberculosis  as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by  M. avium  subsp.  hominissuis  (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from  M. avium  have been defined among other things due to difficulties in generating  M. avium  mutants. More efforts in developing new methods for mutagenesis of  M. avium  and identification of virulence-associated genes are therefore needed.    Results  We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were  MAV_4334  (nitroreductase family protein),  MAV_5106  (phosphoenolpyruvate carboxykinase),  MAV_1778  (GTP-binding protein LepA) and  MAV_3128  (lysyl-tRNA synthetase LysS).    Conclusions  We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.

Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis