Evaluation of needle trap micro-extraction and solid-phase micro-extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 µm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L-1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.

Nucleotide-binding oligomerization domain-2 (NOD2) regulates type-1 cytokine responses to Mycobacterium avium but is not required for host control of infection.

Nucleotide-binding oligomerization domain-2 (NOD2) is an innate immune receptor that recognizes peptidoglycan-derived muramyl dipeptide from intracellular bacteria and triggers proinflammatory signals. In this study, we sought to evaluate the role played by this receptor during early and late stages of infection with Mycobacterium avium in mice. We demonstrated that NOD2 knockout (KO) animals were able to control M. avium infection similarly to wild-type mice at all time points studied, even though IL-12 and TNF-α production was impaired in NOD2-deficient macrophages. At 100 days following infection with this bacterium, but not at 30 days post-infection, NOD2-deficient mice showed significantly diminished production of IFN-γ, as confirmed by reduced accumulation of IFN-γ and IL-12 mRNA in the spleens of KO mice. Additionally, a reduction in the size and in the number of lymphocytes/granulocytes of hepatic granulomas from NOD2 KO animals was observed only during late time points of M. avium infection. Taken together, these data demonstrate that NOD2 regulates type-1 cytokine responses to M. avium but is not required for the control of infection with this bacterium in vivo.

Infection and immunity on a chip: a compartmentalised microfluidic platform to monitor immune cell behaviour in real time.

Cells respond to their environments and self-organise into multicellular assemblies with dedicated functions. The migratory and homing response of cells to soluble ligands can be studied by using different techniques, but for real time studies of complex multicellular self-organisation, novel and simpler systems are required. We fabricated a flexible open access microsystem and tested the design by studying cell recruitment from an immune cell reservoir towards an infectious compartment. The two compartments were connected by a network of bifurcated microchannels allowing diffusion of signalling molecules and migration of cells. Bacterial filters were incorporated in the design to prevent bacteria and activated cells from entering the network, permitting migration only from the recruitment reservoir. The fabricated microsystem allows real-time continuous monitoring of cellular decision-making based on biologically produced gradients of cytokines and chemokines. It is a valuable tool for studying cellular migration and self-organisation in relation to infections, autoimmunity, cancer, stem cell homing, and tissue and wound repair.

Síndrome de Lady Windermere: afectación del lóbulo medio y língula por Mycobacterium avium complex / Lady Windermere syndrome: involvement of the middle lobe and lingula by Mycobacterium avium complex

No disponible

CCL20 is overexpressed in Mycobacterium tuberculosis-infected monocytes and inhibits the production of reactive oxygen species (ROS).

CCL20 is a chemokine that attracts immature dendritic cells. We show that monocytes, cells characteristic of the innate immune response, infected with Mycobacterium tuberculosis express the CCL20 gene at a much higher level than the same cells infected with non-tuberculous mycobacteria. Interferon (IFN)-γ, a fundamental cytokine in the immune response to tuberculosis, strongly inhibits both the transcription and the translation of CCL20. We have also confirmed that dendritic cells are a suitable host for mycobacteria proliferation, although CCL20 does not seem to influence their intracellular multiplication rate. The chemokine, however, down-regulates the characteristic production of reactive oxygen species (ROS) induced by M. tuberculosis in monocytes, which may affect the activity of the cells. Apoptosis mediated by the mycobacteria, possibly ROS-dependent, was also inhibited by CCL20.

Measurement of phagocytosis and of the phagosomal environment in polymorphonuclear phagocytes by flow cytometry.

Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies.

Optimization of a rapid viability assay for Mycobacterium avium subsp. paratuberculosis by using alamarBlue.

A microtiter alamarBlue assay was adapted and optimized for Mycobacterium avium subsp. paratuberculosis. Using cell concentrations ranging from 10(4) to 10(8) CFU/ml, a minimum incubation time to indicate viability was obtained after 24 h. Rifampin (rifampicin) was used to demonstrate that this method has applications for high-throughput screening against M. avium subsp. paratuberculosis.

Similaridades clinicopatológicas entre paratuberculosis y enfermedad de crohn: ¿posible vínculo zoonótico? / Clinical and pathological similarities between paratuberculosis and crohn’s disease: ¿a possible zoonotic link?

EL artículo presenta a la luz de la literatura científica actual la asociación del Mycobacterium avium subespecie paratuberculosis con la enfermedad de Crohn, las principales similaridades clinicopatológicas. La revisión se limitó a las publicaciones contenidas en el programa HINARI de la WHO, en especial de Elservier Science, Bioline Internacional, Blackwell Publishing, BMJ Publishing, Lippincott Williams & Wilkins, Nature Publishing, PubMed, Springer Science y publicaciones de la Asociación Internacional para estudio de Paratuberculosis. Evidencias significativas apoyan un posible vinculo zoonótico entre las enfermedades. El aislamiento del Mycobacterium avium subespecie paratuberculosis en pacientes con enfermedad de Crohn desde muestras de intestino, leche, sangre periférica y nódulos linfáticos; de igual manera la respuesta inmune especifica a algunos antígenos de Mycobacterium en pacientes con la enfermedad; aunque estos hallazgos tienen cada uno sus propias controversias. La evidencia actualmente disponible no es suficiente para validar o negar al Mycobacterium avium subespecie paratuberculosis como agente causal de por lo menos algunos casos de enfermedad de Crohn. Los estudios son inconclusos en aceptar un vinculo zoonótico dada la naturaleza multifactorial de esta enfermedad. Se requieren estudios para determinar sí Mycobacterium es un agente espectador ó patogénico; paralelamente realizar estudios epidemiológicos a gran escala que permitan analizar la distribución geográfica y temporal de ambas enfermedades.

Effects of isoniazid on viability, cell morphologies and acid fastness properties of Mycobacterium avium NCTC 8559 during the growth cycle.

Our previous study demonstrated that the effects of isoniazid (INH) on Mycobacterium tuberculosis at the cellular level varied according to the growth phases. In this study, the variations in the INH action on M. avium strain NCTC 8559 are reported. M. avium cells grown on Middlebrook 7H10 agar were harvested at different stages of their growth cycle, exposed to the minimum inhibitory concentration of INH, stained with acid-fast staining for morphological changes and acid fastness properties, and the number of colonies were evaluated for viability studies. The study demonstrated that M. avium NCTC 8559 cells at the initial and fragmentation stages of the growth cycle were most susceptible to INH.

Modulation of cellular phosphatidylinositol 3-phosphate levels in primary macrophages affects heat-killed but not viable Mycobacterium avium's transport through the phagosome maturation process.

Most disease causing mycobacteria are intramacrophage pathogens which replicate within nonacidified phagosomes that can interact with the early endosomal network but fail to mature to a phagolysosome. The mycobacterial phagosome retain some proteins required for fusion with endocytic vesicles including Rab5 but lack others such as early endosomal autoantigen 1 (EEA1). As the membrane lipid phosphatidylinositol 3-phosphate (PtdIns-3-P) is required for EEA1 membrane association and phagosome maturation, it may be a potential target of pathogenic mycobacteria. To test this hypothesis, macrophage cellular levels of PtdIns-3-P were altered by retroviral introduction of the type III Phosphoinositide 3-Kinase (VPS34) and the PtdIns-3-P phosphatase myotubularin 1 (MTM1). By utilizing the PtdIns-3-P-specific probes FYVE and PX coupled to EGFP (EGFP-2-FYVE and EGFP-PX, respectively), the expression of PtdIns-3-P on the mycobacterial phagosome was addressed. All phagosomes containing viable Mycobacterium avium stained positive for EGFP-2-FYVE and EGFP-PX despite obvious differences in PtdIns-3-P concentrations in cells expressing MTM1 or VPS34. Altering PtdIns-3-P cellular concentrations did not affect trafficking of live bacilli. However, a significant increase in the transport of killed bacilli to a late endosomal/lysosomal compartment was observed in VPS34-compared to MTM1-transduced macrophages. Therefore, although overexpression of PdtIns-3-P in macrophages can facilitate phagosome maturation, its effect on phagosomes containing viable M. avium was negligible.

Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex and Mycobacterium kansasii.

Mycobacterium species has a specific morphology when grown in liquid medium. Mycobacterium tuberculosis complex (MTB) often exhibits serpentine cording, which is different from the dot and cross-barring morphology observed in Mycobacterium avium complex (MAC) and Mycobacterium kansasii (MK), respectively. These characteristic morphologies can be used as a cost-effective method for rapid, presumptive identification of mycobacterial isolates cultured from the MGIT 960 system. By using Kinyoun acid-fast stain, serpentine cording was found in 840 of 904 (92.1%) samples positive for MTB; dot or loose aggregation was observed in 112 of 136 (82.3%) samples positive for MAC; and the cross-barring, ladder-like, morphology was observed in 45 of 56 (80.5%) samples positive for MK. The sensitivity and specificity were 92.9% and 96.4% for MTB; 82.4% and 94.5% for MAC; and 80.4% and 94.6% for MK, respectively. Using growth rate selection to exclude rapid growers, the positive and negative predictive values were 98% and 87.6% for MTB; 78.3% and 98% for MAC; and 78.9% and 99.1% for MK, respectively. Twenty-eight (93.3%) of 30 strains with ball morphology were rapid growers. Microscopic morphology can be used for rapid, presumptive identification of M. tuberculosis complex, M. kansasii, and M. avium complex and act as a guide for appropriate selection of initial probes to reduce costs.

Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates.

BACKGROUND: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. RESULTS: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. CONCLUSIONS: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Profiles of the mRNA expression by macrophages infected with mycobacterium leprae and mycobacterium avium complex

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (M phi s) in terms of up-regulation of M phi expression of proinflammatory and immunosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated M phi mRNA expression IL-12, TNF-alpha, IL-10, and transformating growth factor-beta (TGF-beta), except that M. leprae-infected M phi s showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected M phi s with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that M phi s co-infected with MAC and M. leprae showed the Il-12, TNF-alpha and IL-10 mRNA expression in an intermediate mode of those of M phi s infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected M phi s may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal M phi s in terms of the activation of signal transduction pathways for expression of some kinds of immunoregulatory cytokines and immunoprotective enzymes.

Activation of the mitogen-activated protein kinase signaling pathway is instrumental in determining the ability of Mycobacterium avium to grow in murine macrophages.

Of the two common morphotypes of Mycobacterium avium, designated smooth transparent (SmT) or smooth opaque (SmO), the SmO morphotype is avirulent, whereas the SmT morphotype is virulent. The role of the host macrophage in determining these different virulence phenotypes was analyzed using an in vitro model of macrophage infection. Initial studies confirmed previous reports of the increased ability of the SmT bacteria to grow in macrophages; this increased virulence correlated with reduced induction of inflammatory cytokines. Examination of the response of the mitogen-activated protein kinase (MAPK) pathway following infection with either morphotype revealed that all three members of the MAPK pathway were activated. Pharmacologic inhibition of either the extracellular signal-regulated kinase (ERK) or p38(MAPK) pathways resulted in distinct consequences for the growth of the two morphotypes. In particular, inhibition of the p38(MAPK) resulted in attenuated growth of the SmT morphotype, which correlated with reduced PGE(2) production. Inhibition of cyclooxygenase 2 by indomethacin also inhibited growth of SmT, substantiating the role for PGE(2) in promoting the growth of SmT. In contrast, SmO induction of the ERK pathway was increased compared with the SmT morphotype, and inhibition of ERK resulted in decreased TNF-alpha synthesis and enhanced SmO growth. Pharmacologic inhibitors of the MAPK pathway were present for only the first 4 h of infection and yet had consequences for bacterial growth at 7 days. Therefore, the data suggest that induction of the MAPK pathway during uptake of bacteria is instrumental in determining the eventual fate of the bacteria.

Mycobacterium avium interaction with macrophages and intestinal epithelial cells.

Mycobacterium avium is an environmental microorganism that is adapted to live both in the environment (mainly in water and soil) and in bird, fish and mammal hosts. In humans, M. avium infection is seen in patients with some sort of immunosuppression, such as patients with chronic lung disease, and Acquired Immunodeficiency Syndrome. More recently, other populations were shown to be at risk to develop M. avium disease. For the majority of time, humans acquire M. avium through the intestinal tract where the bacterium comes in contact with and translocates the intestinal mucosa. M. avium possesses a unique manner to interact with the intestinal mucosa, and, following invasion, can enter and survive within macrophages and monocytes. Although in vitro entry seems to be dependent on binding to the complement receptor, this finding has not been observed in vivo where the bacterium appears to enter macrophages by alternative mechanisms. The bacterium appears to trigger little inflammatory response, and is able to adapt itself to different environments in the host.

Lipid domains of mycobacteria studied with fluorescent molecular probes.

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non-tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non-covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl-arabinogalactan-peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high-resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.

Search for the molecular basis of morphological variation in Mycobacterium avium.

Isolates of Mycobacterium avium exhibit three different colonial variations: smooth domed (SmD), smooth transparent (SmT), and rough (Rg). Because the discrimination between morphotypes is founded on morphological rather than molecular principles and because of the absence of consensus over the relevance of morphology to pathogenesis and drug sensitivity, a comparative study at the protein level was undertaken. By direct immunization of BALB/c mice with the soluble sonicate of one of the morphotypes of M. avium serovar 2, eight monoclonal antibodies (MAbs) were identified, of which one was M. avium specific. Cross immunization of syngeneic mice with serum-absorbed antigens allowed the generation of 15 further MAbs; 11 were M. avium or M. avium complex specific, but none of them was morphotype specific. Subcellular fractions analyzed by electrophoresis showed similar profiles, with the exception of a cytosolic protein with a relative molecular mass of ca. 66 kDa (protein SmT 66), which was most highly expressed in SmT variants of M. avium serotypes 2 and 4. Because a well-known, ubiquitous stress-heat shock protein (hsp65) has a similar molecular mass, protein SmT 66 was compared with hsp65. Western blot (immunoblot) analyses using several cross-reacting MAbs and N-terminal amino acid sequencing established that this protein was not the ubiquitous stress protein. Thus, SmT 66 is the first product to be described which might be associated with the SmT morphotype.

The electron-transparent zone in phagocytized Mycobacterium avium and other mycobacteria: formation, persistence and role in bacterial survival.

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages.

Mycobacterium avium is a cause of nontuberculous chronic granulomatous infections which is attracting increased attention as a frequent opportunistic pathogen in acquired immunodeficiency syndrome. Some important aspects of its human pathogenicity were investigated by using cultured human macrophages infected with it. The uptake and replication of various strains of M. avium in the macrophages could be measured by CFU counts of the bacteria in samples of lysed, sonicated macrophages. Microscopic counts of acid-fast bacilli were not useful because the bacteria multiplying in the macrophages were usually not acid fast. Electron microscopy showed the intracellular bacilli to multiply by transverse fission, to be surrounded in individual vacuoles by a broad electronlucent zone, and to have thinner cell walls than extracellularly grown M. avium. Fifteen strains, including examples of serovars 1, 2, 4, 8, and 9, were studied for uptake and rate of replication in cultured macrophages from three normal subjects. The strains were isolates from patients with nontuberculous granulomatous infection, acquired immunodeficiency syndrome, or unrelated problems, or they were laboratory reference cultures. There were no differences among them in phagocytosis, but there were differences in intracellular replication. Laboratory strains tended to be avirulent, that is, they did not replicate in the macrophages. Patient isolates usually were virulent and could be compared for virulence by intracellular replication rates. Virulence correlated with flat, transparent bacterial colony morphology on nutrient agar but not with serovar or kind of patient from whom the bacteria were isolated. However, among strains of transparent colony morphology there were wide differences in virulence. A virulent bacilli generally produced domed, opalescent colonies on nutrient agar. A virulent bacilli predominated in populations of M. avium conditioned to growth in bacteriologic culture medium. Bacilli of virulent colony morphology predominated in populations passaged through cultured macrophages. The model described here presents a new approach to the investigation of the pathogenicity of M. avium for human subjects and may be more patient relevant than animal models.