Genes required for intrinsic multidrug resistance by Mycobacterium avium were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations. Two genes, pks12 and Maa2520, were disrupted in multiple drug-susceptible mutants. The pks12 gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes. Its ortholog in Mycobacterium tuberculosis is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid. Mutants of M. avium with insertions into pks12 exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells. A pks12 mutant of M. tuberculosis was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered. M. avium complex (MAC) and M. tuberculosis appear to have different genetic mechanisms for resisting the effects of these antibiotics, with pks12 playing a relatively more significant role in MAC. The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in M. tuberculosis and Mycobacterium leprae. It is immediately upstream of Maa2521, which may code for an exported protein. Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells. Like pks12 mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications. Maa2520 and pks12 are the first genes to be linked by mutation to intrinsic drug resistance in MAC.

The cell wall of the environmental pathogen Mycobacterium avium is important to its virulence and intrinsic antimicrobial resistance. To identify genes involved in cell wall biosynthesis, "transposome" insertion libraries were screened for mutants with altered colony morphology on medium containing the lipoprotein stain Congo red. Nineteen such mutants were isolated and mapped, including 10 with insertions in a functional island of cell wall biosynthetic genes that spans approximately 40 kb of the M. avium genome.The Mycobacterium avium complex is an environmental pathogen that causes serious disease in susceptible individuals (10,14,15,21). The most well-characterized member of the group is M. avium subsp. avium. The virulence and intrinsic multidrug resistance of this pathogen have been attributed in part to its unique cell wall (6,13,15,16,19,20). The M. avium subsp. avium cell wall is a complex array of hydrocarbon chains containing the arabinogalactan-peptidoglycan-mycolic acid core found in all mycobacteria, surrounded by a second electron-dense layer made up, in part, of serovar-specific glycopeptidolipids (ssGPL) found only in M. avium complex (2-4, 15, 22). ssGPL consist of core nonspecific GPL (nsGPL) common to many environmental mycobacteria, modified by the addition of serovar-specific oligosaccharide side chains.Most clinical isolates of M. avium subsp. avium form multiple colony-type variants (morphotypes). Analysis of the irreversible switch that results in the rough colony type has yielded good information about the genetics of cell wall biosynthesis (2-4, 8). Rough mutants fall into two categories: those that lack all traces of GPL and those that produce a lipopeptide core of GPL that is not glycosylated. Both categories result from spontaneous deletions within a cluster of genes involved in ssGPL biosynthesis. The ssGPL gene cluster, named ser2, is 17 to 27 kb long, depending upon polymorphisms related to insertion elements (2,3,8).Additional morphotypic switches in M. avium subsp. avium are less well understood. Smooth transparent variants are more virulent and more drug resistant than their smooth opaque counterparts (15). A separate switch, termed redwhite, becomes visible when clinical isolates are grown on agar media containing the lipoprotein stain Congo red (CR) (6,7,17). Compared to red variants, white variants are more resistant to multiple antibiotics in vitro, more common in patient samples, and more virulent in disease models (6,17).We have taken a mutational approach to identifying genes involved in cell wall biosynthesis and colony morphotype. Genetic analysis of M. avium subsp. avium is challenging due to the organism's low growth rate, genetic instability, and intrinsic multidrug resistance. Transposon mutagenesis, a powerful tool for analyzing gene function, has not previously been applied to M. avium subsp. avium (a system was reported for M. avium subspecies paratuberculosis, a more stable subspecies of M. avium) (11). Therefore, we developed and applied a protocol for rando...

The N and W-Beijing families of Mycobacterium tuberculosis are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS6110 restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages

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