Electron impact and chemical ionization mass spectral analyses of methyl esters species of free mycolic acid fraction from Rhodococcus lentifragmentus

The free mycolic acid fraction from Rhodococcus lentifragmentus was derivatized to methyl esters and further fractionated into saturated (F-0), monounsaturated (F-1) and diunsaturated (F-2) species using argentation-TLC. Methyl esters fractions F-0, F-1 and F-2, accounting for approximately 7.4%, 53.1% and 39.5%, respectively, were analyzed by electron impact (EI) and chemical ionization (CI) mass spectrometries. According to EI-MS, peaks observed for M(+)-18, that were prominent compared to those representing M(+)-32 and M(+)-(18 + 32), indicated that the carbon chain size ranged from C-36 to C-48. The pyrolytic cleavage of methyl mycolates (R(2)-CHOH-CH(R(1))-COOCH3), following the McLafferty rearrangement released fragment ions corresponding to, (a) the alpha-subunit, representing the fatty acid methyl ester (R(1)-CH2-COOCH3), methyl hexadecanoate, methyl tetradecanoate and methyl dodecanoate in decreasing order of relative intensity of peaks, and (b) the beta-subunit, representing the meroaldehyde moiety (R(2)-CHO). The saturated meroaldehyde species exhibited peaks representing meroaldehyde minus 18 mass units in which R(2) ranged from C19H39 to C31H63. The monunsaturated species exhibited peaks representing the meroaldehyde in which R(2) ranged from C19H37 to C31H61; peaks corresponding to meroaldehyde minus 18 mass units appeared only in the most abundant components, C29H57CHO, C27H53CHO, C25H49CHO and C31H61CHO, in a decreasing order of relative abundance. The diunsaturated species exhibited peaks essentially corresponding to meroaldehyde in which R(2) corresponded to C31H59 and C29H55; the latter displayed a relative intensity that was about one-half compared to that of the former. Fractions F-0, F-1 and F-2 showed a more intense pyrolytic fragmentation under CI-MS in contrast to results found under EI-MS. Therefore, peaks representing the alpha-subunit and the beta-subunit were more prominent than the ones representing the fragmentation of the hydrocarbon chain. Moreover, the beta-subunit of saturated species exhibited peaks corresponding to meroaldehyde plus hydrogen, and no dehydration of the beta-subunit occurred in this case. In turn, the beta-subunit of monounsaturated and diunsaturated species showed peaks representing both the meroaldehyde plus hydrogen and its dehydration product plus hydrogen. Thus, the presence of unsaturation in the meroaldehyde subunit of methyl mycolate facilitates appearance of dehydration fragment ions under chemical ionization procedure.

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Thin-layer chromatography of mycobactins and mycolic acids for the identification of clinical mycobacteria

Forty seven strains of mycobacteria (35 strains isolated from clinical specimens and 12 reference strains) were analyzed for mycobactin and mycolate production by thin-layer chromatography (TLC). Different growth conditions had little or no effect on the production of individual mycobactins and the reproducibility of mycobactin Rf values. Mycolate profiles of isolated strains were compared with those of reference strains. Clinical isolates belonging to the same species showed the same profiles. The combined evaluation of mycobacterial products by TLC allowed the identification of pathogenic and opportunist cultivable mycobacteria. on routine examination, the analysis of mycobactin and mycolate production constitutes an adequate procedure for the characterization and identification of myobacteria.

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of Mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.

A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a β-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the α-branch of mycolic acids, followed by the joining of these two branches.

Identification of a gene involved in the biosynthesis of cyclopropanated mycolic acids in Mycobacterium tuberculosis.

Mycolic acids represent a major constituent of the mycobacterial cell wall complex, which provides the first line of defense against potentially lethal environmental conditions. Slow-growing pathogenic mycobacteria such as Mycobacterium tuberculosis modify their mycolic acids by cyclopropanation, whereas fast-growing saprophytic species such as Mycobacterium smegmatis do not, suggesting that this modification may be associated with an increase in oxidative stress experienced by the slow-growing species. We have demonstrated the transformation of the distal cis double bond in the major mycolic acid of M. smegmatis to a cis-cyclopropane ring upon introduction of cosmid DNA from M. tuberculosis. This activity was localized to a single gene (cma1) encoding a protein that was 34% identical to the cyclopropane fatty acid synthase from Escherichia coli. Adjacent regions of the DNA sequence encode open reading frames that display homology to other fatty acid biosynthetic enzymes, indicating that some of the genes required for mycolic acid biosynthesis may be clustered in this region. M. smegmatis overexpressing the cma1 gene product significantly resist killing by hydrogen peroxide, suggesting that this modification may be an important adaptation of slow-growing mycobacteria to oxidative stress.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Bone tuberculosis in Roman Period Pannonia (western Hungary)

The purpose of this study was to analyse a skeleton (adult female, 25-30 years) that presented evidence of tuberculous spondylitis. The skeleton, dated from the Roman Period (III-VI centuries), was excavated near the town of Győr, in western Hungary. The skeleton was examined by gross observation supplemented with mycolic acid and proteomic analyses using MALDI-TOF/TOF tandem mass spectrometry. The biomolecular analyses supported the morphological diagnosis.

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 (R-f) 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. (C) 2012 Elsevier Editora All rights reserved.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Isolation of Mycobacterium gordonae from poultry slaughterhouse water in São Paulo State, Brazil

Water samples (24 untreated water, 12 treated water and 24 served water) used in different stages of the slaughter process were examined to identify a possible source of pathogenic mycobacteria. The isolates were identified based on microscopy, morphological and biochemical features, mycolic acid analysis and molecular method - PCR-restriction-enzyme analysis. Eighteen mycobacterial strains were isolated from 60 water samples: 11 from untreated water, 5 from treated water and 2 from served water. All mycobacteria isolated were identified as Mycobacterium gordonae and showed the following PRA genotypes: III (27.8%), IV (38.9%) and V (33.3%).

Valorização do diagnóstico laboratorial, na identificação de Rhodococcus equi isolado do escarro de pacientes suspeitos de tuberculose

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Micobacteriose não tuberculosa pulmonar em hospital de referência no Estado do Pará: espécies mais frequentes, apresentação radiológica e evolução clínica

As micobactérias não tuberculosas (MNT) estão amplamente presentes no ambiente, tendo sido isoladas em águas naturais, sistemas de distribuição de água, solo e animais. Caracterizam-se pela presença de ácido micólico na parede celular. Em geral, são adquiridas através de inalação de gotículas de água contendo micobactérias. Podem causar formas variadas de doença como linfadenite, pulmonar, cutânea e disseminada. São patógenos oportunistas, com patogenicidade variável, que requerem defeitos na imunidade local ou sistêmica, congênitos ou adquiridos para causar doenças em humanos. Foram avaliados aspectos epidemiológicos, clínicos e radiológicos de 44 casos de micobacteriose não tuberculosa na forma pulmonar no Hospital João de Barros Barreto (HUJBB) através de estudo retrospectivo e foram tratados e acompanhados 21/44 (47,7%) pacientes durante um período de seis a dezessete meses através de estudo do tipo coorte prospectivo. Os dados mostraram um incremento de mais de 100% no número de casos a partir do ano de 2010 em relação aos anos anteriores no HUJBB. As micobactérias mais isoladas foram M. intracellulare (22,7%) e M. massiliense (20,5%). As condições mais frequentemente associadas à doença incluíram tratamento prévio para tuberculose (93,2%), bronquiectasias (59%), HIV (11,4%), asma (9,1%) e doença pulmonar obstrutiva crônica (9,1%). Não foram observadas diferenças nos aspectos radiológicos entre as espécies, exceto na análise das radiografias de tórax, onde atelectasias foram mais frequentes nos grupo M. massiliense do que no grupo de M. abscessus. A resposta ao tratamento de acordo com a análise das culturas para micobactérias mostrou que em 58,8% dos casos ocorreu negativação, persistência da positividade em 11,7% e positividade após negativação inicial em 11,7%. Durante o período de acompanhamento, a taxa de óbito foi de 17,7%. Os dados sugerem que a forma pulmonar da micobacteriose não tuberculosa tem se tornado uma doença com importância cada vez maior em nossa região. Adicionalmente, a resposta ao tratamento tem sido bastante satisfatória quando comparada à literatura. Entretanto, é necessário um seguimento desses pacientes por período mais prolongado para estabelecer o real desfecho da nossa abordagem terapêutica.

Fluidity of the lipid domain of cell wall from Mycobacterium chelonae.

The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. Hydrocarbon chains of much of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study, we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30 degrees C. Differential scanning calorimetry showed that much of the lipids underwent major thermal transitions between 30 degree C and 65 degrees C, that is at temperatures above the growth temperature, a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in the growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate labeled at different positions showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal, less fluid mycolic acid leaflet and an external, more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well-known intrinsic resistance of mycobacteria to such compounds.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.