Identification of mycolic acid class ratios from Mycobacterium species using liquid chromatography-mass spectrometry

Alongside the recent technology advancements in liquid chromatography (LC) and mass spectrometry (MS) instrumentation there has also been steady progress made with respect to the systematic lipidomic studies of Mycobacterium tuberculosis (M. tb) where hyphenated LC and MS instrumentation were the main analytical technique employed. Although many reports have shown low limits of detection and high precision in analysing mycolic acids (MAs) and other M. tb lipids, few have reported efficient LC selectivity, even when employing high resolution mass spectrometry (HRMS) to determine the accurate mass of a lipid molecule for identification purposes. MAs are one of the most widely reported lipid classes that are found in the cell wall of M. tb and consist of a diversity of high mass branched chain of fatty acids that could potentially be used as diagnostic biomarkers for determining active M. tb infections. The chemical diversity of MAs can be used for taxonomic identification of mycobacterial species, but their analysis is complicated due to their extreme hydrophobic properties and homologous chemistry. Three dominant subclasses of MA molecules are found in mycobacteria: alpha-(α-), ketoand methoxy-MAs. When analysing MAs using MS, the product ions obtained from the infusion of a purified mixture of MAs, extracted from M. tb H37Rv strain, on a tandem mass spectrometer (MS/MS) are m/z 367.3 and 395.4, which correlate with published data but are not unique to single MA classes and therefore cannot be used for the identification of a specific MA subclass molecule. The ratios of the diverse MA class precursor ions can however be used to predict the identity of M. tb strains. Further genetic assays are however required to confirm the taxonomic identification. In this study, self-extracted MAs (M. tb H37Rv strain) were compared to that of commercially available MAs (M. bovis strain) and by integrating the chromatographic peak area of each dominant precursor ion within a MA class, a peak area ratio between the α-, keto- and methoxy- MAs were determined. The resulting percentage ratios between α-, keto- and methoxy- MA classes were found to be 53:8:38 for M. tb H37Rv and 53:15:32 for M. bovis respectively. The LC data dependent acquisition (DDA) precursor ion method, developed here specifically for the MA ratio comparison, has also shown the ability to resolve isomers within a MA class that has not previously been reported. Two other classes of mycobacterial lipids, phthiocerol dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. dimycocerosates (DIMa) and phthiodiolone dimycocerosates (DIMb), collectively called PDIMs, have recently emerged as lipids which play a significant role in the virulence of drug resistant M. tb. Hence, a robust LC-HRMS method was developed for the analysis of extractable non-polar lipids extracted from three different drug resistant phenotypes of M. tb from clinical isolates and compared to a drug susceptible M. tb H37Rv clinical isolate. In future, the feasibility of using LC-HRMS as a routine technique to phenotype M. tb drug resistant strains may require a mass analyser with a mass resolution in excess of 100 000, as well as chromatographic technologies that are capable of resolving non-derivatized complex mixtures of large (C60-C100) nonpolar and/or polar lipid molecules.Dissertation (MSc)--University of Pretoria, 2019.PharmacologyMScUnrestricte

A follow-up cross-sectional study of environmental lead exposure in early childhood in urban South Africa

Background. Lead exposure has significant detrimental effects on the health and wellbeing of children. In resource-poor countries, information on the extent of lead exposure is often inadequate owing to the lack of surveillance and screening programmes.Objective. To determine the degree of lead exposure in children residing in South African urban areas.Methods. A cross-sectional survey was conducted in schools in Johannesburg, Cape Town and Kimberley in 2007 - 2008. Blood lead levels were assessed in a total of 1 349 grade 1 children using the LeadCare Analyser system. Parents completed a structured questionnaire on sociodemographic profiles and risk factors to provide information about socioeconomic status and other risk factors for lead exposure. Results. Blood lead levels ranged from 0.8 - 32.3 μg/dl. The mean blood lead level in the total sample was 7.97 μg/dl; 74% had blood lead levels ≥5 μg/dl. The highest proportion (84%) of children with blood lead levels ≥5 μg/dl was in Johannesburg. In the multivariate analysis, socioeconomic status was significantly associated with blood lead levels ≥5 μg/dl. Conclusion. Lead exposure in South African urban areas remains widespread. The risk of lead poisoning in some areas and certain groups of children may be increasing despite the phasing out of lead-containing petrol. Children living in poverty continue to be the most vulnerable.

Mycobacterium malmesburyense sp. nov : a novel non-tuberculous Mycobacterium species revealed by multiple gene sequence characterization

Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and an increasing number of NTM species have been isolated and characterized from both humans and animals, highlighting the zoonotic potential of these bacteria. Host exposure to NTM may impact on cross-reactive immune responsiveness which may affect diagnosis of bovine tuberculosis and may also play a role in the variability of the efficacy of Mycobacterium bovis BCG vaccination against tuberculosis. In this study we characterized 10 NTM isolates originating from water, soil, nasal swabs of cattle and African buffalo as well as bovine tissue samples. These isolates were previously identified during an NTM survey and were all found, using 16S rDNA sequence analysis to be closely-related to Mycobacterium moriokaense. A polyphasic approach that includes phenotypic characterization, antibiotic susceptibility profiling, mycolic acid profiling and phylogenetic analysis of four gene loci, viz 16S rDNA, hsp65, sodA, and rpoB was employed to characterize these isolates. Sequence data analysis of the four gene loci revealed that these isolates belong to a unique Mycobacterium species. This evidence was further supported by several differences in phenotypic characteristics between the isolates and the closely related species. We proposed the name, Mycobacterium malmesburyense sp. nov. for this new species. The type strain is WCM 7299T (ATCC® BAA- 2759TM =CIP 110822T). The Genbank accession numbers for the partial gene sequences [16S rDNA , hsp65, rpoB and sodA] for the type strain are as follows: 16S rRNA= KJ 873241; hsp65=KJ 873243; rpoB= KJ 873245; sodA= KJ 873247.WOTRO Science for global development; grant number W01.65.321.00.http://ijs.sgmjournals.orghb2017Veterinary Tropical Disease

Functionalization of PLGA nanoparticles with 1,3-β-glucan enhances the intracellular pharmacokinetics of rifampicin in macrophages

Purpose Mycobacterium tuberculosis which causes tuberculosis, is primarily resident within macrophages. 1,3-β-glucan has been proposed as a ligand to target drug loaded nanoparticles (NPs) to macrophages. In this study we characterized the intracellular pharmacokinetics of the anti-tubercular drug rifampicin delivered by 1,3-β-glucan functionalized PLGA NPs (Glu-PLGA). We hypothesized that Glu-PLGA NPs would be taken up at a faster rate than PLGA NPs, and consequently deliver higher amounts of rifampicin into the macrophages. Methods Carbodiimide chemistry was employed to conjugate 1,3-β-glucan and rhodamine to PLGA. Rifampicin loaded PLGA and Glu-PLGA NPs as well as rhodamine functionalized PLGA and Glu-PLGA NPs were synthesized using an emulsion solvent evaporation technique. Intracellular pharmacokinetics of rifampicin and NPs were evaluated in THP-1 derived macrophages. A pharmacokinetic model was developed to describe uptake, and modelling was performed using ADAPT 5 software. Results The NPs increased the rate of uptake of rifampicin by a factor of 17 and 62 in case of PLGA and Glu-PLGA, respectively. Expulsion of NPs from the macrophages was also observed, which was 3 fold greater for Glu-PLGA NPs than for PLGA NPs. However, the ratio of uptake to expulsion was similar for both NPs. After 24 h, the amount of rifampicin delivered by the PLGA and Glu-PLGA NPs was similar. The NPs resulted in at least a 10-fold increase in the uptake of rifampicin. Conclusions Functionalization of PLGA NPs with 1,3-β-glucan resulted in faster uptake of rifampicin into macrophages. These NPs may be useful to achieve rapid intracellular eradication of Mycobacterium tuberculosi

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020T (=CIP 110823T=ATCC BAA-2758).WOTRO Science for global development; grant number W01.65.321.00.http://ijs.sgmjournals.org2019-05-01hj2018Veterinary Tropical Disease

Curdlan-Conjugated PLGA Nanoparticles Possess Macrophage Stimulant Activity and Drug Delivery Capabilities

Research Article published by SpringerPurpose There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells.We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system. Methods Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using 1H and 13C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTTassay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug. Results Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca2+ dependent uptake of nanoparticles by the macrophages. Conclusions PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy

A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics