Mycobacterium abscessus is one of the most important nontuberculous mycobacteria that cause lung diseases. In vitro infection models developed to analyze the immune response are frequently based on ...the addition of mycobacteria to mononuclear cells or neutrophils from peripheral blood. An important requirement of these assays is that most cells phagocytose mycobacteria, only accomplished by using large multiplicities of infection (1 or more bacteria per cell) which may not adequately reflect the inhalation of a few mycobacteria by the host. We propose modifications that try to mimic some of the conditions in which immune cells deal with mycobacteria. For the preparation of the inoculum mycobacteria are grown in solid media followed by preparation to a single cell suspension. Multiplicities of infection (number of bacteria per cell) are below 0.01. Serum-free cellular media is used to allow the growth of M. abscessus. After several days of incubation Bacterial Colonies in Cellular Culture (BCCC) develop, which are enumerated directly under an inverted microscope. These colonies may represent biofilm formation during chronic infections.
•Low multiplicity of infection (below 0.01 bacteria per cell) reflects more realistically conditions encountered by immune cells in the lungs.•The surface of mycobacteria prepared for infection assays that are grown in solid media are less affected than that of mycobacteria grown in liquid media with detergents.•Colony formation in the infected cells may reflect the aggregation and biofilm formation in the lungs during chronic infection.
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Nontuberculous mycobacteria (NTM) are environmental and ubiquitous, but only a few species are associated with disease, often presented as nodular/bronchiectatic or cavitary pulmonary forms. ...Bronchiectasis, airways dilatations characterized by chronic productive cough, is the main presentation of NTM pulmonary disease. The current Cole's vicious circle model for bronchiectasis proposes that it progresses from a damaging insult, such as pneumonia, that affects the respiratory epithelium and compromises mucociliary clearance mechanisms, allowing microorganisms to colonize the airways. An important bronchiectasis risk factor is primary ciliary dyskinesia, but other ciliopathies, such as those associated with connective tissue diseases, also seem to facilitate bronchiectasis, as may occur in Lady Windermere syndrome, caused by
infection. Inhaled NTM may become part of the lung microbiome. If the dose is too large, they may grow excessively as a biofilm and lead to disease. The incidence of NTM pulmonary disease has increased in the last two decades, which may have influenced the parallel increase in bronchiectasis incidence. We propose that ciliary dyskinesia is the main promoter of bronchiectasis, and that the bacteria most frequently involved are NTM. Restoration of ciliary function and impairment of mycobacterial biofilm formation may provide effective therapeutic alternatives to antibiotics.
Bronchiectasis is considered a consequence of the neutrophilic inflammatory response to infection. Mycobacterial infections, mainly from the Mycobacterium avium complex and M. abscessus, have been ...inextricably linked to bronchiectasis development. The most important pathogen that infect patients with bronchiectasis is Pseudomonas aeruginosa, associated with an increased risk of death. Patients with bronchiectasis are often co-infected with P. aeruginosa and M. avium complex, and it was studied whether they interacted in immune cell cultures. Peripheral blood mononuclear cells from healthy volunteers were infected overnight with clinical isolates of mycobacteria, 18 h later co-infected with P. aeruginosa and Pseudomonas multiplication was quantified. Inoculated P. aeruginosa multiply faster when cells were previously infected in vitro with M. avium complex or M. tuberculosis, but not with M. kansasii or M. gordonae, mycobacteria not regularly isolated from patients with bronchiectasis. The interaction between mycobacteria and P. aeruginosa also takes place in the absence of cells, but to a lower degree. Growth of Staphylococcus aureus, less frequently co-isolated with mycobacteria, was not affected by previous infection with mycobacteria. Surprisingly, multiplication of P. aeruginosa in neutrophil cultures did not vary in the presence of mycobacteria. Nevertheless, co-infection of mycobacteria and P. aeruginosa induced the production of IL-1β, a mediator of neutrophilic inflammation. P. aeruginosa stimulation by mycobacteria provides evidence for explaining their common clinical association. Strategies to control mycobacteria may be useful to impair P. aeruginosa colonization.
•M. avium complex and M. tuberculosis co-infected cells promote P. aeruginosa growth.•Peripheral blood mononuclear cells, but not neutrophils, promote growth.•Less pathogenic mycobacteria (e.g. M. gordonae) do not promote growth.•Co-infected cells produce IL-1β, mediator of neutrophilic inflammation.•Observed mycobacteria - P. aeruginosa interaction may have clinical relevance.
Cutaneous fungal infections can result in disastrous episodes if improperly diagnosed and treated, especially in immunosuppressed patients. Although dermatopathologists are highly familiar with some ...filamentous fungi – such as Aspergillus and Zygomycetes – they are not so aware of other less common species. We report a case of ocular infection by Scedosporium apiospermum that started as conjunctivitis and resulted in Phthisis bulbi and subsequent exeresis of the left eye. We describe some of the main morphological features of the fungus as well as the important morphological clues for the differential diagnosis with some similar species, such as Aspergillus, Scopulariopsis, Fusarium, Paecilomyces and Zygomycetes.
Highlights • Blood antimycobacterial activity is normal in individuals with type 2 diabetes. • Phagocytosis is inhibited in neutrophils from individuals with diabetes. • Higher production of TNFα in ...blood from diabetes patients infected with M. tuberculosis.
The increasing worldwide incidence of mycobacteriosis and the need to achieve improved clinical management makes nontuberculous mycobacteria (NTM) genotyping a useful tool. However, because of ...technical difficulties, medium size microbiology laboratories do not attempt to compare the genetic patterns that each of their isolates present. We have aimed to optimize a genotyping method with a reduced hands-on experimental time and that requires few technical resources. A strategy based on the Amplified Fragment Length Polymorphism (AFLP) methodology was developed using two rare-cutters enzymes (
Sac
I and
Bgl
II). One out of seven primers was sequentially used in each amplification reaction that was analyzed by agarose gel electrophoresis. This approach makes it possible the timely genotyping of a moderate number of strains and its characterization without the need of image analysis software. We have genotyped 28
Mycobacterium intracellulare
and 4 M
. abscessus
. Clinical researchers are encouraged to routinely genotype their NTM isolates.
In vitro analysis of mycobacterial pathogenicity or host susceptibility has traditionally relied on the infection of macrophages, the target cell of mycobacteria, despite difficulties reproducing ...their antimycobacterial activity. We have employed alternative models, namely whole blood and leukocytes in plasma, from QuantiFERON negative individuals, and performed infections with the pathogenic M. tuberculosis, the less pathogenic M. avium, M. kansasii and M. chelonae and the occasionally pathogenic M. gordonae and M. bovis. The anticoagulant used in blood extraction, heparin or EDTA, had a major influence in the outcome of the infection. Thus, while in the heparinized models a similar number of bacteria were enumerated in the inoculum and after seven days, in the presence of EDTA a killing effect was observed, despite the inhibitory effect of EDTA on cellular functions like the production of cytokines or reactive oxygen species (ROS). A special case was the rapidly growing mycobacteria M. chelonae, that multiplied in heparinized models but was eliminated in models with EDTA. We verified that EDTA is not responsible for the bactericidal effect, but acts as a bacteriostatic agent. Further work will determine whether blood derived models are a better alternative to the classical macrophage.
Part of the susceptibility to tuberculosis has a genetic basis, which is clear in primary immunodeficiencies, but is less evident in apparently immunocompetent subjects. Immune responses were ...analysed in blood samples from tuberculosis patients and their healthy first-degree relatives who were infected in vitro with mycobacteria (either Mycobacterium tuberculosis or M. bovis BCG). The antimicrobial activity against M. tuberculosis in blood from relatives was significantly lower than that observed in healthy controls. Tuberculosis patients exhibited a higher number of neutrophils, and monocyte phagocytosis was inhibited in both relatives and tuberculosis patients. A remarkable finding was that the production of reactive oxygen species by infected neutrophils was higher in relatives than in healthy controls. A higher production of TNFα in infected blood from relatives was also observed. These results may indicate that relatives display a stronger inflammatory response and that their immune response to M. tuberculosis is different from those of unrelated controls. First-degree relatives may represent a highly informative group for the analysis of tuberculosis susceptibility.
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