Isolates of the Mycobacterium avium-intracellulare complex (MAC) have long been known to segregate into transparent, opaque and rough colony morphotypes that differ from each other in clinically important parameters including drug susceptibility and virulence. Here the authors report additional morphotypic variation that occurs on two levels: interspecific (between M. avium and M. intracellulare) and intraspecific (within individual M. avium isolates). Clinical isolates of M. avium grown on Congo red (CR) plates formed red, pink or mixed (red and white) opaque colonies, while M. intracellulare isolates formed purely white opaque colonies. A quantitative CR binding assay showed that this interspecific differential applies to transparent as well as opaque colony variants; however, it was less pronounced among laboratory reference strains than among recent clinical isolates. Opaque colonies of M. avium isolates with 'mixed' phenotypes segregated into stable opaque red and white variants with shared IS1245 banding patterns (intraspecific segregation). White segregants of M. avium were more flocculent and significantly more resistant to ciprofloxacin and rifamycin drugs than were red segregants. Thus, cultivation on CR agar revealed a previously unknown multidrug resistant colony morphotype of M. avium.
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.
Mycobacterium avium undergoes reversible morphotypic switching between the virulent transparent colony type and the less virulent opaque colony type. A new morphotypic switch in M. avium, termed red-white, that becomes visible when opaque colonies of clinical isolates are grown on agar media containing Congo red, was recently described. White opaque (WO) variants were found to be more resistant to multiple antibiotics than were red opaque (RO) variants. The present paper reports that transparent derivatives of RO and WO clones retain the differential Congo red binding properties of their opaque parents, indicating that the opaque-transparent switch operates independently of the red-white switch. White transparent variants were more resistant to clarithromycin and rifampin in vitro, and better able to survive within human macrophages, than their red transparent counterparts. Neither red nor white variants were markedly favoured during growth in vitro ; however, red variants were better able to spread on soft agar (sliding motility), a potential selective advantage under some environmental circumstances. White-to-red switching was frequently observed in vitro and was accompanied by decreased antibiotic resistance and increased motility. Red-to-white switching has yet to be observed in vitro, indicating that the red morphotype is very stable. Significantly, some widely studied laboratory reference strains of M. avium, including strain 2151 and the genome sequence strain 104, are stable red clones. These strains are intrinsically antibiotic resistant and virulent in animal models, but they may not express genes encoding the elevated levels of antibiotic resistance and intracellular survival observed in white variants.
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