An improved extraction and nonradioactive thin-layer chromatography detection method of mycolic acid / 生物工程学报

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, 14C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of 14C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids

Non-bilayer phospholipid arrangements are three-dimensional structures that form when anionic phospholipids with an intermediate structure of the tubular hexagonal phase II are present in a bilayer of lipids. Antibodies that recognise these arrangements have been described in patients with antiphospholipid syndrome and/or systemic lupus erythematosus and in those with preeclampsia; these antibodies have also been documented in an experimental murine model of lupus, in which they are associated with immunopathology. Here, we demonstrate the presence of antibodies against non-bilayer phospholipid arrangements containing mycolic acids in the sera of lepromatous leprosy (LL) patients, but not those of healthy volunteers. The presence of antibodies that recognise these non-bilayer lipid arrangements may contribute to the hypergammaglobulinaemia observed in LL patients. We also found IgM and IgG anti-cardiolipin antibodies in 77% of the patients. This positive correlation between the anti-mycolic-non-bilayer arrangements and anti-cardiolipin antibodies suggests that both types of antibodies are produced by a common mechanism, as was demonstrated in the experimental murine model of lupus, in which there was a correlation between the anti-non-bilayer phospholipid arrangements and anti-cardiolipin antibodies. Antibodies to non-bilayer lipid arrangements may represent a previously unrecognised pathogenic mechanism in LL and the detection of these antibodies may be a tool for the early diagnosis of LL patients.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

INTRODUCTION: Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE: This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS: The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS: Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (Rf) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959 bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS: The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

Evaluation of mycolic acid analysis in identification of Mycobacterium species through SMIS / 中华检验医学杂志

Objective To evaluate the usefulness of mycolic acid for identification of Mycobacterium species using SMIS. Methods One hundred and eighteen clinical Mycobacterium isolates collected from Beijing Tuberculosis and Thoracic Tumor Research Institute through whole year of 2007 were analyzed. The 118 isolates contain 25 isolates of Mycobacterium tuberculosis and 93 non tuberculosis Mycobacterium identified by PNB method. Mycolic acid analysis using SMIS is evaluated for identification of a broad range of Mycobacteria in comparison with 16S rDNA , 16-23S rDNA ITS sequencing to measure the concordance rate and agreement, and verify the concordance rate and agreement among results of mycolic acid, sequencing and PNB in identifying Mycobacterium tuberculosis and non tuberculosis Mycobacterium. Results The concordance rate between mycolic acid method analysis and DNA sequencing is 92% ( 108/118), of which concordance rate in Mycobacterium tuberculosis complex and non tuberculosis Mycobacterium are 95% (35/37) and 90% (73/81) respectively, agreement of both is great( agreement Kappa value is 0. 96). Through retrospective analysis, the concordance of results between SMIS and PNB method analysis is 90% (106/118)and agreement is well( agreement Kappa value is 0. 73 ), the concordance of results between sequencing and PNB method analysis is also 90% ( 106/118 ) and agreement is well (agreement Kappa value is 0. 74 ),despite the identification results of 11 isolates by PNB method are discordant. Conclusion Mycolic acid analysis by SMIS enables rapid identification of a broad range of clinical Mycobacterium species, which could play an important role in polyphasic identification of Mycobacterium species.

Abordagem racional no planejamento de novos tuberculostáticos: inibidores da InhA, enoil-ACP redutase do M. tuberculosis: [revisão] / Rational approach in the new antituberculosis agent design: inhibitors of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis: [review]

Associada à disseminação da infecção causada pelo HIV, a tuberculose (TB) é considerada, atualmente, problema mundial de saúde pública devido às proporções que vem assumindo. A resistência micobacteriana aos fármacos utilizados na terapêutica é a principal causa da reincidência da TB. Diante deste quadro alarmante, o desenvolvimento de novos e seletivos fármacos anti-TB se faz urgente e necessário. A biossíntese de ácidos graxos é um processo bioquímico realizado por procariotos e eucariotos, o qual fornece precursores essenciais à montagem de componentes celulares importantes, tais como fosfolipídeos, lipoproteínas, lipopolissacarídeos, ácidos micólicos e envelope celular. As diferenças bioquímicas e funcionais entre o mecanismo biossintético de ácidos graxos em bactérias e mamíferos tornam-no alvo relevante ao planejamento de novos antibacterianos, mais seletivos e menos tóxicos. As enoil-ACP redutases são enzimas cruciais à etapa de alongamento de ácidos graxos, considerados produtos intermediários na biossíntese de ácidos micólicos - os principais componentes da parede celular micobacteriana. Portanto, tais enzimas são tidas como alvos moleculares no planejamento racional de novos tuberculostáticos. Avanços recentes no processo de descoberta de novos agentes anti-TB, particularmente os inibidores da enoil-ACP redutase, serão discutidos nesta revisão.

In conjunction with the spread of HIV infection, tuberculosis (TB) has been among the worldwide health threats. Mycobacteria resistance to the drugs currently used in the therapeutics is the main cause of TB resurgence. In view of this severe situation, the new and selective anti-TB design is of utmost importance. Fatty acid biosynthesis is a prokariontes and eucariontes biochemical process that supplies essential precursors for the assembly of important cellular components, such as phospholipids, lipoproteins, lipopolysaccharides, mycolic acids and cellular envelope. However, the biochemical and functional differences between the bacterial and mammals' fatty acid synthetic pathway have endowed the mycobacterial enzymes with distinct properties. These provide valuable opportunities for structure- or catalytic mechanism-based design of selective inhibitors as novel anti-TB drugs with improved properties. The enoyl-reductases are essential enzymes in the fatty acids elongation pathway towards the mycolic acids, the main mycobacteria cell wall constituents, biosynthesis and so they are potential targets to the rational new antimycobacteria drug design. This paper highlights recent approaches regarding the design of new anti-TB agents, particularly, the enoyl-ACP reductase inhibitors.

Identificación de micobacterias no tuberculosas en pacientes VIH/SIDA por métodos convencionales y de fracciones de ácidos micólicos / Identification of non tuberculosis mycobacteria from HIV/AIDS patients by conventional methods and by study of mycolic acid fractions

Las micobacterias no tuberculosas son patógenos oportunistas capaces de producir infecciones pulmonares y extrapulmonares. El aumento de su incidencia se ha acelerado después de la aparición del Síndrome de Inmunodeficiencia Adquirida (SIDA). En este trabajo se estudiaron 40 cepas aisladas de pacientes infectados por el Virus de Inmunodeficiencia Humana, A los aislamientos con significación patogénica se le aplicó el estudio de los patrones de las fracciones de ácidos micólicos. Los resultados fueron: 9 Mycobacterium avium, 8 Mycobacterium fortuitum, 4 Mycobacterium flavescens, 4 Mycobacterium smegmatis, 3 Mycobacterium marinum, 4 Mycobacterium gastri, 2 Mycobacterium gordonae, 2 Mycobacterium chelonae, 1 Mycobacterium xenopi, 1 Mycobacterium phlei, 1 Mycobacterium triviale, y 1 Mycobacterium malmoense. Sólo 5 de estas cepas estaban asociadas a cuadros clínicos: 2 Mycobacterium avium (micobacteriosis diseminada y renal respectivamente), 1 Mycobacterium gordonae (lesiones en piel), 1 Mycobacterium fortuitum (linfadenitis submaxilar), 1 Mycobacterium malmoense (linfadenitis submaxilar). Las especies más frecuentemente aisladas fueron M. avium y M. fortuitum acorde con lo revisado en la literatura. La aplicación simultánea de las técnicas convencionales y el estudio de las fracciones de ácidos micólicos ha permitido obtener resultados más confiables por lo que recomendamos su aplicación en estos estudios.

Non tuberculosis mycobacteria are opportunist pathogens whose frequency in human infections has increased after the appearance of the Acquired Immunodefficiency Syndrome (AIDS). In this work we studied 40 strains isolated from patients infected by the Human Immunodefficiency Virus and isolates with pathogenic significance were further analyzed for diagnostic confirmation by the method that studies mycolic acid fractions. After identification, the results were: 9 Mycobacterium avium, 8 Mycobacterium fortuitum, 4 Mycobacterium flavescens, 4 Mycobacterium smegmatis, 3 Mycobacterium marinum, 4 Mycobacterium gastri, 2 Mycobacterium gordonae, 2 Mycobacterium chelonae, 1 Mycobacterium xenopi, 1 Mycobacterium phlei, 1 Mycobacterium triviale, and 1 Mycobacterium malmoense. Only five of these strains were associated to clinical symptoms: 2 Mycobacterium avium (disseminated and renal mycobacteriosis respectively), 1 Mycobacterium gordonae (skin lesions), 1 Mycobacterium fortuitum (submaxilar lymphoadenitis), and 1 Mycobacterium malmoense (submaxilar lymphoadenitis). The species most frequently isolated were: M. avium and M. fortuitum, in agreement with a bibliographic revision. The simultaneous application of conventional techniques and the study of mycolic acid allowed us to obtain more trustworthy results.