Michele Trucksis

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.

Molecular and Physiological Effects of Mycobacterial oxyR Inactivation

The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.

Goldfish as an animal model system for mycobacterial infection

Publisher Summary Humans are the primary natural host of M. tuberculosis infection; however, several animal models have been developed to study infection with this pathogen. The primary animal models used are the guinea pig, mouse, and rabbit models. Each of the commonly used mycobacterium models has its advantages and disadvantages. In addition to these small animal models, a primate model has been developed which most closely parallels human disease. It is uncommonly used because of its expense and the limited availability of primates. Tuberculosis is spread from an infected person to a new host through infected droplets made airborne by coughing and talking.In addition to a mycobactefial pathogenesis model, our animal model can be used as a vaccine challenge model. A challenge model is used to demonstrate whether a vaccine strain can elicit protective acquired immunity and therefore protect against a challenge with a fully virulent wild-type strain. This application was demonstrated in an experiment with two groups of fish inoculated intraperitoneally with M. marinurn ATCC 927 organisms at a dose of 10 3 or 105 cfu. However, animals immunized with 105 cfu of M. marinum were completely protected, with 100% of the fish surviving until the end of the experiment. This established that protective immunity develops following an M. marinum infection. This model can thus be used to evaluate the protective efficacy of candidate vaccine strains.