Mycobacterium kansasii in a rhesus monkey

The incidence of pulmonary disease caused by "atypical" mycobacteria has been increasing gradually in the human population since the 1950s. Mycobacterium kansasii and Mycobacterium intracellulare are the two organisms most responsible for this trend. A rhesus monkey was euthanatized and necropsied after reacting positive to mammalian Old Tuberculin on semi-annual testing. Histopathology demonstrated the presence of small numbers of acid fast organisms in pulmonary lesions. Further microbiological testing identified the causative organism as M. kansasii.     

Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis

Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen.     

Nutritional Characteristics of the Atypical Mycobacteria

The ability of Mycobacterium kansasii and groups II and III of the atypical mycobacteria to utilize oleic acid, as well as selected carbohydrates and other compounds, as sources of carbon for growth was compared with that of the H37Rv and H37Ra strains of M. tuberculosis. The highest rate of growth of all of the mycobacteria examined occurred in the medium containing oleic acid as the carbon source when single substrates were tested. The H37Ra strain of M. tuberculosis and all of the atypical mycobacteria examined, except the P-8 strain of M. kansasii, displayed a deficiency in ability to utilize glucose for growth. The deficiency was manifested as delayed appearance of growth, suboptimal growth, or complete inability to utilize the sugar. Variant substrains of organisms that possessed an enhanced ability to utilize glucose for growth were isolated from representative strains of M. kansasii and groups II and III atypical mycobacteria inoculated on modified Kirchner glucose-agar and incubated for an extended period of time.     

Activation of an NLRP3 inflammasome restricts Mycobacterium kansasii infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Trehalose-containing lipooligosaccharides from mycobacteria: structures of the oligosaccharide segments and recognition of a unique N-acylkanosamine-containing epitope

The structures of the oligosaccharide segments of nine trehalose-containing lipooligosaccharides (LOS) of Mycobacterium kansasii have been established by positive and negative fast-atom bombardment mass spectrometry, acetolysis, partial acid hydrolysis, methylation analyses, and nuclear magnetic resonance. Upon acetolysis, all produce the alpha,alpha-trehalose-containing tetraglucose (Glc4) "core" -beta-D-Glcp(1----3)-beta-D-Glcp(1----4)-alpha-D-Glcp(1----1)-alpha-D-Gl cp. The simplest (LOS I') contains an additional alpha 1----3-linked 3-O-methyl-L-rhamnopyranose (3-O-Me-L-Rhap) unit; those of intermediate complexity (LOS I-III) contain an additional D-xylopyranose (D-Xylp) residue or xylobiose in beta 1----4 linkage; and those of ultimate complexity (LOS IV-VIII) contain further D-Xylp residues and the distal N-acylkanosamine- (KanNAcyl) and fucopyranosyl- (Fucp) containing disaccharide KanNAcyl(1----3)Fucp. Thus, the structure of the oligosaccharide from LOS VII is KanNAcyl(1----3)Fucp(1----4) [-beta-D-Xylp(1----4)]6-alpha-L-3-O-Me-Rhap(1----3)Glc4++ +. Polyclonal rabbit and murine monoclonal antibodies react only with the more complex KanNAcyl-Fucp-containing lipooligosaccharides, indicating that the KanNAcyl distal end, not the trehalose end, contains the antibody binding site unique to M. kansasii and is responsible for the serological distinctiveness of M. kansasii among mycobacterial species.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Molecular characterization of Mycobacterium kansasii isolates in the State of São Paulo between 1995-1998

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of the gene enconding for the 65 kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of Sao Paulo was almost exclusively subtype I regardless of HIV status.     

Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis. Relation to trehalose-based oligosaccharides attached to lipid.

A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.     

Fatty acid composition and mycolic acid pattern of some chromogenic mycobacteria

Twenty-nine strains of chromogenic mycobacteria belonging to the species Mycobacterium aurum (5 strains), M. duvalii (2), M. flavescens (1), M. gordonae (6), M. kansasii (3), M. obuense (1), M. parafortuitum (3), M. phlei (2), M. rhodesiae (1), M. vaccae (2) and Mycobacterium spp. (3) were studied for fatty acid composition and mycolic acid patterns by gas-liquid chromatography and thin-layer chromatography respectively. Fatty acids found ranged from those with 12-24 carbon atoms and were saturated and monounsaturated straight chain fatty acids, along with 10-methyl branched of 16, 17 and 18 (tuberculostearic acid) carbon atoms. Moreover, 2-methyl tetradecanoic acid was found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains), and 2,4-dimethyl tetradecanoic acid in M. kansasii and Mycobacterium spp. (2 strains). Nonadecenoic acid was found only in M. flavescens and tuberculostearic acid was not detected in M. gordonae. Three patterns of mycolic acids were obtained: the first, found in M. aurum, M. flavescens, M. phlei, M. rhodesiae and Mycobacterium spp. (1 strain), was characterized by the presence of several spots assigned to alpha-mycolates, keto-mycolates and wax-ester mycolates (omega-carboxy-mycolates and 2-eicosanol and related alcohols); the second, found in M. duvalii, M. obuense, M. parafortuitum and M. vaccae was similar to the first, but it contained an additional spot of alpha'-mycolates; the third pattern, found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains) contained three spots considered to be alpha-mycolates, methoxy-mycolates and keto-mycolates. The results obtained confirm previously reported data on the fatty and mycolic acid composition of the species studied.     

Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls

The long-posed question of the nature of the link between the mycolylarabinogalactan and the underlying peptidoglycan of the cell walls of Mycobacterium sp. has been addressed. The insoluble cell wall matrix of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis was partially hydrolyzed with acid either before or after per-O-methylation and the resulting oligosaccharides further derivatized and analyzed by gas chromatography/mass spectrometry. The structures of fragments arising from the reducing end of arabinogalactan demonstrated the existence of the terminal sequence----5)-D-Galf-(1----4)-L-Rhap-(1---3)-D-GlcNAc. Other analyses confirmed the presence of muramyl-6-P within the peptidoglycan of these mycobacteria. Based on the acid lability of the 3-linked GlcNAc unit, the presence of about equimolar amounts of Rhap-(1----3)-D-GlcNAc and muramyl-6-P in an isolated cell wall fragment, and 31P NMR analysis, it was concluded that the GlcNAc residue of the terminal triglycosyl unit of arabinogalactan is joined by 1-O-phosphoryl linkage to the 6-position of some muramyl residues within the peptidoglycan. Thus, it is reasoned that the massive mycolylarabinogalactan of mycobacteria, responsible for aspects of disease pathogenesis and much of the antibody response in infections, is attached to the peptidoglycan framework by the actinomycete-specific diglycosylphosphoryl bridge, L-Rhap-(1----3)-D-GlcNAc-(1----P, perhaps thereby providing a unique target for site-directed chemotherapy of mycobacterial infections.     

Structural and immunological properties of the phenolic glycolipids from Mycobacterium gastri and Mycobacterium kansasii.

Mycobacterial species-specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose-containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali-labile epitopes were found to characterize the lipooligosaccharide antigen type. In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K-I was delineated as the distal monoacetylated disaccharidic residue: 2,6-dideoxy-4-O-methyl-alpha-D-arabino-hexopyranosyl-(1----3)-2-O-methyl -4-O- acetyl-alpha-L-fucopyranose. This acetoxy group is required for K-I epitope recognition demonstrating that alkali-labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K-I epitope was localized around the electron-transparent layer on the M. kansasii cell-wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K-III and Phe Gl K-IV were discovered in minute amounts. They were purified and characterized by their retention time in direct-phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K-I. The complete family of phenolic glycolipids Phe Gl K-I, K-II, K-III and K-IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.     

Trehalose-containing lipooligosaccharide antigens of Mycobacterium sp.: presence of a mono-O-methyltri-O-acyltrehalose "core"

We have described the surface antigens of Mycobacterium kansasii as trehalose-containing lipooligosaccharides (LOS) which at the nonreducing "epitope" end bear a unique amino sugar containing diglycosyl unit, whereas the putative reducing end consists of an acylated alpha, alpha-trehalose-containing tetraglucosyl "core" [Hunter, S. W., Jardine, I., Yanagihara, D. L., & Brennan, P. J. (1985) Biochemistry 24, 2798-2805]. The presence of a new variation on this core, in Mycobacterium szulgai, is now reported, ----3)beta-D-Glcp-(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O- Me-alpha-D-Glcp, representing the first example of an O-methyltrehalose unit in nature. The simplest of the LOS class of glycolipids in M. szulgai was defined as alpha-L-2-O-Me-Fucp(1----3)alpha-L-Rhap(1----3)alpha-L-Rh ap(1----3) beta-D-Glcp(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O-Me-alpha-D-G lcp. Further glycosylation of this nonantigen, by an incompletely defined 6-deoxyhexosyl residue, confers specific antigenicity on the organism. Thus, these extraordinary structures, in a manner analogous to the better known lipopolysaccharides from rough variants of Enterobactericiae, are highly amphipathic and display variability not only in the immunogenic, distal region but also in the "invariant" lipophilic core. The contribution of these glycolipids to the hydrophobic barrier, the pseudo outer membrane of mycobacteria, is discussed.     

Fatty acid composition and mycolic acid pattern of some chromogenic mycobacteria

Twenty-nine strains of chromogenic mycobacteria belonging to the species Myco-bacterium aurum (5 strains), M. duvalii (2), M. flavescens (1), M. gordonae (6), M. kansasii (3), M. obuense (1), M. parafortuitum (3), M. phlei (2), M. rhodesiae (1), M. vaccae (2) and Mycobacterium spp. (3) were studied for fatty acid composition and mycolic acid patterns by gas-liquid chromatography and thin-layer chromatog-raphy respectively. Fatty acids found ranged from those with 12–24 carbon atoms and were saturated and monounsaturated straight chain fatty acids, along with 10-methyl branched of 16, 17 and 18 (tuberculostearic acid) carbon atoms. Moreover, 2-methyl tetradecanoic acid was found in M. gordonae, M. kansasii and Mycobacterium spp. (2 strains), and 2,4-dimethyl tetradecanoic acid in M. kansasii and Mycobacterium spp. (2 strains). Nonadecenoic acid was found only in M. flavescens and tuberculostearic acid was not detected in M. gordonae . Three patterns of mycolic acids were obtained: the first, found in M. aurum, M. flavescens, M. phlei, M. rhodesiae and Mycobacterium spp. (1 strain), was characterized by the presence of several spots assigned to α-mycolates, keto-mycolates and wax-ester mycolates (ω-carboxy-rnycolates and 2-eicosanol and related alcohols); the second, found in M. duvalii, M. obuense, M. parafortuitum and M. vaccae was similar to the first, but it contained an additional spot of α-mycolates; the third pattern, found in M. gordonae, M. kansasii and Mycohacterium spp. (2 strains) contained three spots considered to be α-mycoiates, methoxy-mycolates and keto-mycolates. The results obtained confirm previously reported data on the fatty and mycolic acid composition of the species studied.     

Biochemical characteristics and fatty acid compositions of some armadillo-derived mycobacteria and their relation to Mycobacterium gordonae

The long-chain components of 75 strains of mycobacteria, cultivated from Mycobacterium leprae-infected or non-infected armadillos, and of eight clinical and 15 environmental isolates of M. gordonae, were compared. Four major groups could be distinguished based on the presence of 10-methyloctadecanoic (tuberculostearic) and 2-methyl 3-hydroxyeicosanoic acids and secondary alcohols (2-octadecanol and 2-eicosanol). Some heterogeneity was found in strains assigned to M. gordonae: the characteristic absence of tuberculostearic acid and secondary alcohols and the presence of the branched C14 and the hydroxylated C20 acids were seen in only 34 of the 49 strains studied. Three strains were identified as M. malmoense, one as M. kansasii, ten as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex and eight as belonging to new groups of armadillo-derived mycobacteria (ADM 1, ADM 2 and ADM 3) by conventional bacteriological tests and fatty acid compositions, though M. malmoense was heterogeneous in its fatty acids composition. Four strains, identified as M. avium by conventional tests, differed from this species by their fatty acid compositions. Thirteen strains showed some similarity to M. simiae and ten strains differed from all other known mycobacteria.     

Carbohydrate epitope structural elucidation by 1H-NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen.

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K-IV from Mycobacterium kansasii was successfully established from only one- and two-dimensional 1H-NMR data. Among the scalar two-dimensional techniques, correlated spectroscopy with a 45 degree mixing pulse and phase-sensitive double-quantum-filtered correlated spectroscopy were selected, combined with two-dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K-IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part: 4-O-Me-alpha-Manp-(1----3)-4-O-Ac-2-O-Me-alpha-Fucp-(1----3) -2-O-Me-alpha-Rhap- (1----3)-2,4-di-O-Me-alpha-Rhap. The PheGl K-IV shares, with the other phenolic glycolipids isolated from M. kansasii (K-I, K-II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.     

Structural elucidation of the major phenolic glycolipid from Mycobacterium kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety

Data from the literature indicate the presence, in the Mycobacterium kansasii wall, of a phenolic glycolipid called mycoside A. A tentative trisaccharide structure was proposed for the oligosaccharide part, whereas the aglycone was found to correspond to a phenol phthiocerol residue esterified by two mycocerosic acids. In the present work, structural information mainly arising from fast atom bombardment-mass spectrometry, tandem mass spectrometry, and 1H NMR of native and chemically degraded phenolic glycolipid indicates a tetrasaccharide structure for the inherent oligosaccharide. This structure is now determined as: 2,6-dideoxy-4-O-methyl-arabino-hexopyranosyl(1 alpha----3)2-O-methyl-4-O- acetylfucopyranosyl(1 alpha----3)2-O-methyl-rhamnopyranosyl(1 alpha----3)2-4-di- O-methylrhamnopyranosyl-1 alpha----phenolglycol. Evidence for the structure of the dideoxyhexose, unique in mycobacteria, is presented in the following paper (Fournié, J.-J., Rivière, M., Papa, F., and Puzo, G. (1987) J. Biol. Chem. 262, 3180-3184).     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

A glycosyltransferase involved in biosynthesis of triglycosylated glycopeptidolipids in Mycobacterium smegmatis: impact on surface properties

The cell envelope of mycobacteria is a complex structure that plays an important role in the interactions of the cell with its environment and in the protection against the antimicrobial activity of the immune system. Glycopeptidolipids (GPLs) are species- or type species-specific glycolipids that are present at the surface of a number of mycobacteria and that are characterized by a high variability in glycosylation patterns. These GPLs possess various biological activities that depend mostly on the sugars capping the core molecule. In Mycobacterium smegmatis, the GPL core can be substituted by either two or three deoxyhexoses. In this study, we show that Gtf3 is a glycosyltransferase responsible for the synthesis of the triglycosylated GPLs. Biochemical analysis of these molecules, with a combination of mass spectrometry and chemical degradation methods, has shown that they contain three deoxyhexose moieties. The presence of the triglycosylated GPLs is associated with cell surface modifications that lead to a decrease in sliding motility as well as a modification in cellular aggregation and colony appearance on Congo red. Phylogenetic analysis indicated that Gtf3 is a member of a yet-uncharacterized glycosyltransferase family conserved among the mycobacteria.