Various Species Of Mycobacteria Research Articles

Identification of a Potential Antimycobacterial Drug Sensitizer Targeting a Flavin-Independent Methylenetetrahydrofolate Reductase.

The increasing antibiotic resistance of Mycobacterium tuberculosis and pathogenic nontuberculosis mycobacteria highlights the urgent need for new prevention and treatment strategies. Recently, the cocrystal structure of a Mycobacterium smegmatis flavin-independent 5,10-methylenetetrahydrofolate reductase (MsmMTHFR) that binds with a reduced nicotinamide adenine dinucleotide (NADH) has been well-determined, providing a structural basis for the screening of antimycobacterial leads targeting MsmMTHFR, a new enzyme involved in tetrahydrofolic acid (THF) biosynthesis. In this study, we identified compound AB131 as a promising candidate that fits well into the NADH binding pocket of MsmMTHFR through virtual screening. We discovered that AB131 and its derivatives (13 and 14) can sensitize the antimycobacterial activity of the antitubercular drug para-aminosalicyclic acid (PAS) by 2-5-fold against various species of mycobacteria. Although the compounds themselves do not exhibit any antimycobacterial activity, the high binding affinity of AB131 with MsmMTHFR or Rv2172c was evaluated by microscale thermophoresis analysis. Additionally, we predicted and validated the key residues (V115, V117, P118, and R163) of MsmMTHFR that are involved in the interaction with AB131 by using molecular docking and mutagenesis analysis. These findings offer a potential exploitable target for developing potent and specific antimycobacterial drug sensitizers.

Synthesis of trehalose glycolipids.

Trehalose containing glycolipids have been isolated from various species of mycobacteria, fungi and worms. Owing to their versatile biological roles, and heterogenicity of the natural isolates, there is great interest in the synthesis of trehalose glycolipids. In this review we discuss recent developments in the total synthesis of biologically important and structurally complex trehalose glycoconjugates and oligosaccharides.

Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions

is a cutaneous mycobacterial disease that occurs in tropical countries in sub-Saharan Africa, South-East Asia, Australia and America. The responsible pathogen is Mycobacterium ulcerans. Cote d'Ivoire is the most affected country, with more than 30 endemic health districts reporting a large number of chronic skin lesions. The clinical forms and the severity of ulcers vary from one patient to another. Samples from suspected patients were analyzed by PCR at Pasteur Institute of Cote d'Ivoire, as recommended by WHO. IS2404 sequence was detected in 61% of cases, incriminating M. ulcerans in chronic cutaneous lesions. For the other cases the etiology was not identified, thus raising several questions. Are all reported Buruli ulcer cases really caused by M. ulcerans? Would other mycobacteria be involved in the occurrence of chronic skin lesions considered as Buruli ulcer? BU suspected patients were enrolled in endemic areas of Cote d’Ivoire. Samples were collected from cutaneous lesions and transported to the lab at +4°C in 2 ml of Middlebrook 7H9 medium supplemented by Cetylpiridium chloride. The centrifugation pellet was taken with saline buffer to perform microscopic examination and mycobacteria isolation on Lowenstein-Jensen medium. Biochemical characteristics were described by the nicotinic acid detection according to Konno protocol, by the Nitrates reduction test, by the catalase activity detection at 22° and 68°C and the Wayne's Tween 80 hydrolysis test. Genotypic characteristics were determined by PCR with 1 ml of bacterial suspension targeting the insertion sequences (IS6110, IS2404, and IS2606), the plasmid virulence genes and Miru-VNTR loci (Miru-1, VNTR 6, VNTR 19, ST-1). A total of 47 mycobacterial strains were isolated with 3 different types of colonies whose microscopic examination showed Acid-Alcohol-Resistant Bacilli. 65.9% of isolates expressed biochemical characters in favor of M. ulcerans strains and 6.4% in favor of M. marinum strains. For 29.8% of isolates, the characteristics were related to atypical mycobacterial species. The genotyping targeting the IS6110, IS2606 and IS2404 insertion sequences allowed simultaneous amplification in 53.2% of isolates. IS2404 was amplified in 93.6% of isolates; IS6110 was amplified in 74.5% of isolates and IS2606 was amplified in 70.2% of isolates. Five genotypes were identified corresponding to various species of mycobacteria: genotypes 1 and 2 accounted for 63.8% with all the 3 insertion sequences and biochemical characteristics in favor of M. ulcerans strains; genotype 4 accounted for 6.4% of isolates with insertion sequences and biochemical characteristics in favor of M. marinum strains; The strains of genotypes 3 and 5 expressed molecular and biochemical characters relating to various non-M. ulcerans mycobacteria. Virulence genes were found in 72.3% of isolates corresponding to 90% of M. ulcerans strains and 60.7% of non-M. ulcerans mycobacteria. This study confirmed the involvement of several genotypes of M. ulcerans and other mycobacteria in chronic cutaneous lesions suspected as cases of in Cote d’Ivoire.

Antibacterial effect of "Ecocide C" disinfectant against mycobacteria

We presented the results of experimental studies on the bactericidal properties of the disinfectant "Ecocide С" (produced by "KRKA", Slovenia) with respect to various types of mycobacteria. The objects of research were 15 cultures of various species of mycobacteria from the collection of pathogens of infectious diseases of animals of the National Scientific Center "Institute of Experimental and Clinical Veterinary Medicine" (Kharkov). The disinfectant "Ecocide C" had bactericidal properties with respect to various species of mycobacteria: M. avium, M. bovis, M. kansasii, M. gordonae, M. scrofulaceum, M. intracellulare, M. terrae, M. triviale, M. xenopi, M. diernhoferi, M. flavescens, M. fortuitum, M. phlei, M. smegmatis, M. thamnopheos. When exposed to bactericidal concentrations, the disinfectant "Ecocide C" caused destruction of the cell wall and cytoplasmic membrane of mycobacteria, destruction of the granular component of the cytoplasm with the formation of osmiophilic fine granular inclusions, and electron-transparent vacuoles. When we applied the disinfectant "Ecocide C" on mycobacterial cells it caused the irreversible changes in the structural elements of mycobacteria and finally the death of microorganisms. Disinfectant "Ecocide C" shown bactericidal properties against atypical mycobacteria and pathogens of tuberculosis of farm animals and poultry at a concentration of 5.0 % with exposure to the action of 24 hours and can be applied in a common complex of antituberculous activities.

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Multicenter Cross-Sectional Study of Nontuberculous Mycobacterial Infections among Cystic Fibrosis Patients, Israel

This 2-year cross-sectional evaluation of nontuberculous mycobacterial (NTM) infections involved all Israeli medical centers that treat cystic fibrosis patients. The study comprised 186 patients whose sputum was analyzed for NTM. The prevalence of NTM isolates was 22.6%, and 6.5% and 10.8% of the patients fulfilled the 1997 and 2007 American Thoracic Society criteria for NTM lung disease, respectively. Mycobacterium simiae (40.5%), M. abscessus (31.0%), and M. avium complex (14.3%) were the most prevalent. Presence of Aspergillus spp. in sputum and the number of sputum specimens processed for mycobacteria were the most significant predictors for isolation of NTM (odds ratio [OR] = 5.14, 95% confidence interval [CI] 1.87-14.11 and OR = 1.47, 95% CI 1.17-1.85, respectively). The incidence of NTM pulmonary infections is increasing among cystic fibrosis patients, reflecting the increase in longevity of such patients as well as environmental exposure to various species of mycobacteria.

Fatty Acid Analysis Tool (FAAT): An FT-ICR MS Lipid Analysis Algorithm

Electrospray ionization mass spectrometry is becoming an established tool for the investigation of lipids. As the methods for lipid analysis become more mature and their throughput increases, computer algorithms for the interpretation of such data will become a necessity. Toward this end, an algorithm dedicated to the analysis of Fourier transform mass spectral data from lipid extracts has been developed. The algorithm, Fatty Acid Analysis Tool, termed FAAT, has been successfully used to investigate complex lipid extracts containing thousands of components, from various species of mycobacteria including M. tuberculosis and M. abscessus. FAAT is rapid, generally taking tens of seconds to interpret multiple spectra, and accessible to most users as it is implemented in Microsoft Excel Visual Basic Software. In the reduction of data, FAAT begins by scaling spectra (i.e., to account for dilution factors), identifying monoisotopic ions, and assigning isotope packets. Unique features of FAAT include the following: (1) overlapping saturated and unsaturated lipid species can be distinguished, (2) known ions are assigned from a user-defined library including species that possess methylene heterogeneity, (3) and isotopic shifts from stable isotope labeling experiments are identified and assigned (up to a user-defined maximum). In addition, abundance differences between samples grown under normal and stressed conditions can be determined. In the analysis of mycobacterial lipid extracts, FAAT has successfully identified isotopic shifts from incorporation of 15N in M. abscessus. Additionally, FAAT has been used to successfully determine differences in lipid abundances between M. tuberculosis wild-type and mutant strains.

Identification of two Mycobacterium avium subspecies paratuberculosis gene products differentially recognised by sera from rabbits immunised with live mycobacteria but not heat-killed mycobacteria.

The investigation of environmentally regulated proteins has led to a better understanding of host-pathogen interactions and identified novel vaccine candidate antigens for several bacterial pathogens. In an effort to identify such proteins in Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), a genomic expression library was differentially screened with sera from rabbits that had been immunised with live M. paratuberculosis (alpha-live) as well as sera from rabbits immunised with heat-killed M. paratuberculosis (alpha-killed). These experiments identified seven recombinant plaques that were uniquely recognised by the alpha-live sera. Sequence data showed that five of these clones overlapped with each other and contained a common open-reading frame encoding a 25-kDa protein, termed Csp1. The 25-kDa antigen shows weak similarity to a secreted Corynebacterium glutamicum protein. The remaining two clones overlapped with each other and contained two partial open-reading frames, both encoding proteins with strong homology to polyketide synthase from various species of mycobacteria. Antisera were produced against a peptide of the polyketide synthase gene product designated Pks7. Csp1-specific antibodies were affinity purified from the alpha-live sera. These purified antibodies demonstrated that Csp1 was present within infected macrophages. Collectively, these data identify novel M. paratuberculosis antigens that may be important in pathogenesis.

The mycobacteria: an introduction to nomenclature and pathogenesis.

Tuberculosis, caused by Mycobacterium tuberculosis, and leprosy, caused by M. leprae, are diseases known since antiquity. In developing countries, tuberculosis is still the leading cause of mortality due to an infectious disease. Taxonomically, mycobacteria belong to the genus Mycobacterium, which is the single genus within the family of Mycobacteriaceae, in the order Actinomycetales. Actinomycetales include diverse micro-organisms, but mycobacteria and allied taxa are easily distinguished on the basis of the ability to synthesise mycolic acids. Mycobacterial species are traditionally differentiated on the basis of phenotypic characteristics, and the authors provide an updated list of the biochemical tests currently employed and the culture properties that help to discriminate among various species of mycobacteria. However, as the phenotypic characteristics do not allow precise identification of all species, recent molecular taxonomical approaches for mycobacterial classification and phylogeny are also described. Mycobacteria are also a leading cause of infection in various domesticated animals and wildlife. The authors briefly describe the mycobacteria involved in animal infections, the wildlife reservoirs and strategies to control bovine tuberculosis, and the use of molecular tools for diagnostics and epidemiology of mycobacterial infections in animals. The characteristic of intracellular parasitism is discussed, in addition to the fate of pathogenic mycobacteria that have the ability to grow inside phagosomes and phagolysosomes of infected host macrophages. The mycobacterial cell envelope, which is a complex tripartite structure containing a high proportion of lipids (approximately 30% to 40% of the total weight) could play a crucial role in the adaptation of mycobacteria to intracellular growth and survival, immune modulation and drug resistance.

THERAPEUTIC HYPERBARIC OXYGENATION AS AN ADJUNCT TO MULTIDRUG THERAPY IN HANSEN'S DISEASE

Hansen's disease is a chronic infection caused by Mycobacterium leprae. Current therapy for this disease is with the WHO recommended Multi Drug Therapy (MDT) with DDS, rifampicin and clofazimine. Hyper Baric Oxygen Therapy (HBOT) has been used to treat many medical conditions including infections with a great deal of success. It's efficacy on various species of mycobacteria and other bacteria have been studied in vitro and in vivo and it is found to be an effective antimicrobial agent under specified conditions. HBOT has been used to treat Hansen's disease, including drug resistant lepromatous leprosy cases with good results. However there is a paucity of literature on this form of therapy. We have treated twenty cases of Hansen's disease with HBOT prior to exhibiting MDT as per the WHO schedule. There has been a marked regression in skin signs especially decrease in erythema and flattening of raised lesions. This correlated well with the histopathological picture which showed a reduction in the inflammatory exudate.

THE ORIGIN AND ERRATIC GLOBAL SPREAD OF TUBERCULOSIS: How the Past Explains the Present and Is the Key to the Future

Although tuberculosis is a disease known in antiquity, it was not distributed equally or simultaneously throughout the world. Recent genetic studies of the various species of mycobacteria give strong evidence of evolution of M. tuberculosis from saprophytic soil bacteria to M. bovis, which attacks a wide spectrum of lower animals, and then to M. tuberculosis, with the pathogenicity largely limited to humans. The great discrepancies in the time of arrival of this organism to diverse parts of the world, and in its ability to kill the young, account for significant differences in the emergence of innate resistance to tuberculosis in various populations. Innate resistance to particular infections are highly specific, and are derived from whatever scourge one's ancestors had to survive.

The Bcg Gene Story

The genetic influences on the course of mycobacterial infections during epidemics and in endemic areas have always been suspected, but the precise nature of such genetic control and of the inherited mechanisms of susceptibility have been unknown. We have used methods of population genetics in the mouse to discover a single dominant autosomal gene ( Bcg), which controls the susceptibility to various species of mycobacteria as well as to other intracellular parasites. The phenotypic expression of the Bcg gene has been defined as nonspecific Inacrophage activation for bactericidal function, resulting in the destruction of ingested intracellular parasites early following infection. Using recombinant inbred strains of mice, we have mapped this gene to the centromeric part of chromosome 1 and we have created a high resolution linkage map and, subsequently, a physical map in the close vicinity of this locus. A 400 kb bacteriophage and coslnid contig assembled within the genomic interval overlapping Bcg contained six novel transcription units. RNA expression studies showed that one of these genes (designated Nramp for «natural resistance associated Inacrophage protein»), was expressed exclusively in macrophages. Nramp encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems, suggesting a macrophagespecific membrane transport function. Susceptibility to infection ( Bcg s ) in 27 Bcg s and Bcg r strains tested is associated with a Gly-10S to Asp-lOS substitution within predicted transmembrane domain 2 of Nramp, making this gene a strong candidate for Bcg. The chromosomal segment in the vicinity of the Bcg gene has been conserved in the human genome (chromosome 2q). Linkage analysis between the phenotype of disease during a tuberculosis outbreak in an extended multisib Canadian Indian family and allelic variants of chromosome 2 has revealed a significant LOD score. This finding, together with the emerging information on almost total sequence homology between the murine and human Nramp genes suggests that this gene may be responsible for the phenotype of resistance or susceptibility to tuberculosis.

Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Differences in uptake of mycobacteria by human monocytes: a role for complement.

We investigated the influence of serum factors on the uptake of various species of mycobacteria by human peripheral blood monocytes (PBM). On the basis of the percentage of PBM involved during in vitro uptake, the mycobacteria were of two distinct groups. The mycobacteria of one group, which consisted of Mycobacterium avium complex and M. chelonae, were taken up by many PBM; the other group, consisting of M. tuberculosis, M. kansasii, M. fortuitum, and M. gordonae, were taken up by fewer PBM. M. scrofulaceum was intermediate to these two groups on the basis of its uptake by PBM. Serum depleted of complement by heating or treatment with cobra venom factor significantly reduced the extent of PBM involvement with M. avium complex, indicating that complement is an important serum component mediating the uptake of M. avium complex organisms. Preincubation of mycobacteria with serum containing 10 mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and 10 mM MgCl2 resulted in uptake by a high percentage of PBM, while preincubation in heated serum or serum containing 10 mM EDTA resulted in a significantly reduced percentage of PBM involved in uptake of M. avium complex organisms, indicating that these organisms are activators of the alternative pathway of complement. Incubation of M. avium complex organisms in human serum consumed 51% of the hemolytic complement activity. Parallel experiments indicated that serum had a lesser effect on the uptake of M. tuberculosis. Thus, serum is important in in vitro M. avium complex uptake by PBM; complement has a major role in the effect of serum, but this role is less important with M. tuberculosis.

Skin test responsiveness to four new tuberculins in patients with chronic obstructive airways disease receiving short term high doses of, or long term maintenance treatment with, prednisolone: clinical appearances and histometric studies.

The response to skin testing with tuberculins extracted from various species of mycobacteria was studied in 49 patients from Dundee with chronic obstructive airways disease. Seventeen had never been treated with steroids (group 1), 17 were receiving short term high doses of prednisolone (group 2) and did not have impaired Synacthen tests; 15 were receiving long term maintenance treatment and did have impaired Synacthen tests (group 3). Erythematous and indurated reactions were seen in a few patients, more commonly to antigens from Mycobacterium tuberculosis than to the other species: neither of the latter treatment groups showed appreciable reduction in reactivity compared with that of the group 1 patients. The number and microanatomical distribution of the T4 and T8 lymphocytes and the M3 bearing monocytes and macrophages was studied immunocytochemically in cryostat sections of biopsy specimens from the antigen injection sites. The density of these cells was significantly less in clinically negative reactions than in those with erythema or induration, but was unrelated to the presence or absence of a history of treatment with prednisolone. The T4:T8 ratio in the section as a whole was similar to that of the peripheral blood, but T8 cells were relatively more common in the perivascular and periappendicular foci, and T4 lymphocytes were predominant in the diffuse component of the infiltrate. I12 receptor bearing lymphocytes were uncommon: such cells were least common in the clinically negative reactions, but the number and distribution were apparently unrelated to the presence or absence of prednisolone treatment. It was concluded that currently accepted regimens of treatment with prednisolone did not reduce the effector arm of type IV (delayed type hypersensitivity) responses and so are unlikely to compromise this aspect of protective immunity.

Metabolism of mycobacteria

The metabolism of mycobacteria have been studied with reference to carbohydrate, lipids, nitrogen metabolism and oxidative phosphorylation. Some of the enzymes of glycolytic pathway, tricarboxylic acid cycle and lypogenic enzymes were purified, characterized and their kinetic properties investigated. The effect of age of the culture and environmental factors on different aspects of metabolism of mycobacteria were also studied. A comparison of lipid profile in various species of mycobacteria grown in different culture conditions were made. The metabolism of spheroplasts isolated from mycobacteria has been established with respect to their energy charge and to synthesize peptidoglycan using D-alanine as the precursor

Antigenic relationships among mycobacterial species studied by modified-rocket and crossed immunoelectrophoresis.

The complexity of antigens from various species of mycobacteria was explored with use of variations in the techniques of fused-rocket immunoelectrophoresis (IEP) and two-dimensional IEP. Resolution and formation of lines of identity depended on shape and arrangement of antigen wells, concentration of reagents, and procedure for electrophoresis. Rocket IEP of a sonicate into heterologous antiserum in one dimension followed by IEP into homologous antiserum in a second dimension permitted ready identification of heterologous reactions, with direct identity to homologous precipitin bands. Variations in procedures provided versatility that permitted comparison of antigens of unknown or different species, expression of relatedness, comparison of different antisera, and progress of purification or fractionation.

Specific skin-reactive protein from culture filtrate of Mycobacterium bovis BCG.

A highly purified protein, named MPB70, was isolated from the culture filtrate of Mycobacterium bovis BCG. This protein accounted for more than 10% of the proteins secreted into the culture medium. MPB70 was purified by precipitation with ammonium sulfate, followed by treatment with diethylaminoethyl ion exchanger, with or without 3 M urea, and by gel filtration. The final MPB70 preparation was homogenous as judged by several analyses. The molecular weight was estimated to be 18,000 by electrophoresis or molecular sieve and 15,100 by sedimentation equilibrium. The preparation did not contain sugars. The amino acid composition did not include cysteine or tryptophan. In skin reaction, MPB70 was a strictly BCG-specific antigen and, among the guinea pigs sensitized with the heat-killed cells of the various species of mycobacteria--Mycobacterium tuberculosis strains H37Rv and Aoyama B, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium phlei, and BCG, it elicited a delayed cutaneous reaction only in the guinea pigs sensitized with BCG. The potency of MPB70 in the skin reaction was about one-twentieth of the standard purified protein derivative.

Deoxyribonucleic acid hybridization between different species of mycobacteria.

The homology percentages between DNA from M. smegmatis ATCC 607 and DNA from nine various species of mycobacteria have been determined. DNA-DNA hybridization was measured in a spectrophotometer. The technique, calculation of results and the uncertainty of the method are described.

The effect of synthetic and naturally occurring trehalose fatty acid esters in tumor regression

Dimycolates of trehalose, purified cord factor (P3), or monomycolates of trehalose (P4) isolated from various species of mycobacteria, when emulsified with oil droplets and endotoxins from 0 antigen-deficient Re mutants of Salmonella typhimurium or S. minnesota, were found to be effective in regressing transplantable line-10 tumors in strain-2 guinea pigs. Each of these naturally occurring mono- and diesters contains mixtures of several different mycolic acids. To help determine the structural requirements for tumor regression, 6-monoesters and 6,6′-diesters of α,α-D-trehalose were prepared synthetically and tested. Esters formed with single types of mycolic acids isolated from Mycobacterium tuberculosis strain Brevannes, when combined with endotoxin and oil droplets, had tumor regressive potency equal to that of the more complex natural products. The synthetic 6,6′ trehalose diester of 2-eicosyl-3-hydroxy-tetracosanoic (behenylbehenic) acid, whose structure resembles that of mycolic acid but is of lower molecular weight, provided a cure rate of 60%. However, a similar ester prepared with straight chain docosanoic (behenic) acid was essentially inactive.