Tuberculostearic acid, a potential parameter for scoring system construction for Tuberculous meningitis diagnosis

This study aimed to validate the value of tuberculostearic acid (TBSA) whether it could implicate the existence of M. tuberculosis and assist for clinical diagnosis of Tuberculous Meningitis (TBM). Gas Chromatography/mass spectrometry was used to detect TBSA in the chemically pretreated cerebrospinal fluid of suspected TBM patients. In total, 140 patients were admitted for our study included 27 confirm TBM patients and 50 TBSA positive patients. Sensitivity of 0.7407 (CI 95%: 0.5372-0.8889) and specificity of 0.7345 (CI 95%: 0.6432-0.8132) were calculated. The Lancet consensus scoring system was also applied to evaluate the possibility of TBM in suspected patients, finding that TBSA positive patients showed a similar distributive grouping as the definite TBM patients. Our study implicates that the prospective use of TBSA is worth combining into a scoring system for characterizing the features of Mtb, showing a great potential of TBM diagnosis by TBSA in the future.

Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determine Mycobacterium tuberculosis bacterial burden

It is estimated that approximately one-fourth of the world's population is infected with strains of the Mycobacterium tuberculosis complex (MTBC), the causative agents of tuberculosis (TB). In this study, we present rationally developed molecular markers for bacterial burden, which are derived from mycobacterial phospholipids. Using lipidomic approaches, we show that tuberculostearic acid (TSA)-containing phosphatidylinositols (PI) are present in all clinically relevant MTBC lineages investigated. For the major abundant lipid PI 16:0_19:0 (TSA), a detection limit equivalent to 102 colony forming units (CFU) was determined for bacterial cultures and approximately 103 for cell culture systems. We further developed a mass spectrometry based targeted lipid assay, which – in contrast to bacterial quantification on solid medium – can be performed within several hours including sample preparation. Translation of this indirect and culture-free detection approach allowed the determination of pathogen loads in infected murine macrophages, human neutrophils and murine lung tissue. We show that marker lipids inferred from the mycobacterial PIs are increased in peripheral blood mononuclear cells (PBMCs) of TB patients beyond the lipid metabolic background in comparison to healthy controls. In a small cohort of drug-susceptible TB patients elevated levels of these marker molecules were detected at therapy start and declined following successful anti-tuberculosis treatment. The concentration of TSA-containing PIs can be used as correlate for reliable and rapid quantification of Mycobacterium tuberculosis (Mtb) burden in experimental in vitro model systems and may also provide a clinically relevant tool for monitoring TB therapy.One Sentence SummaryTuberculostearic acid containing phosphatidylinositols represent a novel, fast to measure, reliable correlate of Mycobacterium tuberculosis bacterial burden in experimental model systems, which makes a future clinical application conceivable.

Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

Our study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

Description of the bacterial RNA polymerase inhibitor GE23077-producer Actinomadura sp. NRRL B-65521T as Actinomadura lepetitiana sp. nov.

The filamentous actinomycete that produces the antibiotic GE23077 was isolated by the Lepetit Research Group from a soil sample collected in Thailand, and it was classified as a member of the genus Actinomadura on the basis of its morphology and cell-wall composition. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this strain formed a distinct monophyletic line within the genus Actinomadura, and it was most closely related to Actinomadura bangladeshensis DSM 45347T (99.31 % similarity) and Actinomadura mexicana DSM 44485T (98.94 %). The GE23077-producing strain formed an extensively branched, non-fragmented vegetative mycelium; no pseudosporangia were formed and the arthrospores were organized in slightly twisted chains. The cell wall contained meso-2,6-diaminopimelic acid and the diagnostic sugar was madurose. The predominant menaquinone was MK-9(H6), with minor amounts of MK-9(H8) and MK-9(H4). The diagnostic phospholipids were phosphatidylinositol and diphosphatidylglycerol. The major cellular fatty acids were C16 : 0 and tuberculostearic acid (10-methyloctadecanoic acid), followed by minor amounts of C18:1ω9c, C16:1ω7c and 10-methylheptadecanoic acid. The genomic DNA G+C content was 71.77 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, and the low DNA–DNA relatedness between the GE23077-producing strain and closely related type strains clearly demonstrate that it represents a novel species of the genus Actinomadura , for which the name Actinomadura lepetitiana sp. nov. is proposed. The type strain is NRRL B-65521T(=LMG 31258T=DSM 109019T).

Total synthesis and mass spectrometric analysis of a Mycobacterium tuberculosis phosphatidylglycerol featuring a two-step synthesis of (R)-tuberculostearic acid

A two-step synthesis of (R)-tuberculostearic acid enables the total synthesis of a Mycobacterium tuberculosis phosphatidylglycerol. Mass spectrometric fragmentation of synthetic PG regioisomers of acylation patterns.

Biochemical characterization of an S-adenosyl-l-methionine-dependent methyltransferase (Rv0469) of Mycobacterium tuberculosis

Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of mycobacterial membrane phospholipids, and its biosynthesis involves the direct methylation of oleic acid esterified as a component of phospholipids. The methyltransferases of mycobacteria were long proposed to be involved in the synthesis of methyl-branched short-chain fatty acids, but direct experimental evidence is still lacking. In this study, we identified the methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase capable of catalyzing the conversion of olefinic double bond of phospholipid-linked oleic acid to biologically essential TSA. Therefore, UmaA, catalyzing such modifications, offer a viable target for chemotherapeutic intervention.

Motilibacter peucedani gen. nov., sp. nov., isolated from rhizosphere soil

A novel actinomycete strain, designated RP-AC37T, was isolated from rhizosphere soil collected on Mara Island of Jeju, Republic of Korea. Cells were aerobic, Gram-positive, oxidase-negative, catalase-positive, non-mycelium-forming and motile rods (0.6–0.7×1.9–2.4 µm). Phylogenetic analysis based on 16S rRNA gene sequences showed that the organism formed a distinct clade within the radiation occupied by the suborder Frankineae. 16S rRNA gene similarity values were less than 93.2 % to members of the suborder Frankineae and related taxa. The diamino acid isomer in the cell-wall peptidoglycan was ll-diaminopimelic acid. The major whole-cell sugars were glucose, galactose and xylose. The major menaquinone was MK-9(H4). The polar lipids were diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. The cellular fatty acids were straight-chain, unsaturated and saturated, with a significant amount of tuberculostearic acid (10-methyl-C18 : 0). The DNA G+C content was 73.2 mol%. The combination of morphological, chemotaxonomic and phylogenetic data clearly separate the isolate from members of known genera of the suborder Frankineae and related taxa, suggesting that the isolate represents a novel species in a new genus in this suborder, for which the name Motilibacter peucedani gen. nov., sp. nov. is proposed; the type strain is RP-AC37T ( = KCTC 19630T = DSM 45328T).