Genomic Degeneration and Reduction in the Fish Pathogen Mycobacterium shottsii.

Mycobacterium shottsii is a dysgonic, nonpigmented mycobacterium originally isolated from diseased striped bass (Morone saxatilis) in the Chesapeake Bay, USA. Genomic analysis reveals that M. shottsii is a Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) member, but unlike the superficially similar M. pseudoshottsii, also isolated from striped bass, it is not an M. ulcerans ecovar, instead belonging to a transitional group of strains basal to proposed "Aronson" and "M" lineages. Although phylogenetically distinct from the human pathogen M. ulcerans, the M. shottsii genome shows parallel but nonhomologous genomic degeneration, including massive accumulation of pseudogenes accompanied by proliferation of unique insertion sequences (ISMysh01, ISMysh03), large-scale deletions, and genomic reorganization relative to typical M. marinum strains. Coupled with its observed ecological characteristics and loss of chromogenicity, the genomic structure of M. shottsii is suggestive of evolution toward a state of obligate pathogenicity, as observed for other Mycobacterium spp., including M. ulcerans, M. tuberculosis, and M. leprae. IMPORTANCE Morone saxatilis (striped bass) is an ecologically and economically important finfish species on the United States east coast. Mycobacterium shottsii and Mycobacterium pseudoshottsii were originally described in the early 2000s as novel species from outbreaks of visceral and dermal mycobacteriosis in this species. Biochemical and genetic characterization place these species within the Mycobacterium ulcerans/M. marinum clade (MuMC), and M. pseudoshottsii has been proposed as an ecovar of M. ulcerans. Here, we describe the complete genome of M. shottsii, demonstrating that it is clearly not an M. ulcerans ecovar; however, it has undergone parallel genomic modification suggestive of a transition to obligate pathogenicity. As in M. ulcerans, the M. shottsii genome demonstrates widespread pseudogene formation driven by proliferation of insertion sequences, as well as genomic reorganization. This work clarifies the phylogenetic position of M. shottsii relative to other MuMC members and provides insight into processes shaping its genomic structure.

In search of virulence factors of human bacterial disease.

Traditional genetic techniques and a variety of animal and tissue-culture model systems have sustained the study of bacterial virulence mechanisms for several decades. However, the recent application of newly developed molecular and cellular techniques has brought our understanding of bacterial pathogenesis to new heights by permitting the identification and analysis of previously unknown constitutively and differentially expressed virulence-associated factors.

Rapid identification of Legionella pneumophila zinc metalloprotease using chromogenic detection.

Strains of Legionella spp. produce extracellular proteases than can be detected using synthetic chromogenic peptides. Chromogenic tri- and tetrapeptides show a high degree of sensitivity, specificity and reagent stability when linked to para-nitroaniline (pNA). For example, SucOMe-Arg-Pro-Tyr.pNa (S-2586) is specifically hydrolysed by proteases of Legionella pneumophila and some other Legionella species. A paper disc method to sample protease directly from agar plates has been used to evaluate chromogenic peptides as reagents for diagnostic purposes. Strains of Legionella spp., Pseudomonas spp. and Enterobacteriaceae were examined, together with a recombinant Escherichia coli strain containing the cloned 38 kDa zinc metalloprotease from L. pneumophila, S-2586 was hydrolysed by 282 out of 283 L. pneumophila strains, and by the recombinant E. coli. Two of the six strains representing other Legionella species, and 22 of the 50 strains from the Pseudomonas group were also positive. No reaction was seen with any of the Enterobacteriaceae strains. Although there was functional homology between proteases from several bacterial groups, the high prevalence of S 2586-hydrolysing proteases within L. pneumophila indicates a potential usefulness for phenotypic identification.

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs.

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Molecular biology, virulence, and pathogenicity of mycobacteria.

The diseases resulting from infections with Mycobacterium species are important sources of morbidity and mortality throughout the world today, with particularly devastating effects in tropical and developing countries. Almost 2 billion people have been infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, and approximately 3 million people die each year from this disease. Tuberculosis also has re-emerged as an important public health problem in the United States, and this resurgence has been accompanied by an increased incidence of tuberculosis resistant to the standardly used anti-tuberculosis drugs. Researchers' ability to investigate the molecular basis of the pathogenicity and drug resistance of the mycobacteria has been hampered by a lack of appropriate experimental tools. However, during the past 5 years, tremendous progress has been made in the development of the molecular biology of mycobacteria, and molecular tools are now available for detailed analysis of their genetics and for elucidation of the molecular mechanisms of their pathogenicity. The development of these tools is briefly reviewed, and the uses of the tools to investigate drug resistance in Mycobacterium tuberculosis, to identify mycobacterial virulence factors, and to explore intracellular survival strategies are described.

Mycobacterium xenopi multiplies within human macrophages and enhances HIV replication in vitro.

Mycobacterium xenopi can cause opportunistic infections, particularly in persons infected with human immunodeficiency virus type 1 (HIV-1). The primary focus of this effort was to determine if M. xenopi isolates could survive and grow in human peripheral blood macrophage (MPhi), and if these isolates could promote the replication of HIV-1 in vitro. M. xenopi bacilli survived and replicated 10-fold within 48 h in human MPhi while avirulent Mycobacterium smegmatis, did not grow within the MPhi. M. xenopi bacilli when cultured with peripheral blood mononuclear cells enhanced HIV-1 replication 30- and 50-fold with the macrophage-tropic HIV-1(Ba-L) and 50- and 75-fold with T-cell-tropic strain HIV-1(LAI) by 6 days post-infection when compared to M. smegmatis. The enhanced HIV replication was associated with increased production of TNF-alpha. Partial inhibition of HIV-1 induction was observed using a neutralizing anti-TNF-alpha monoclonal antibody, pentoxifylline, and matrix metalloproteinase (MMP) inhibitor I. Similar mechanisms of pathogenesis among mycobacterial species may help elucidate better treatment approaches in HIV co-infected persons.