Mycobacterium avium-intracellulare contamination of mammalian cell cultures.

Mycobacterium avium contamination has been described as a putative contaminant of nonphagocytic mammalian cells. Screening of numerous cultured nonphagocytic mammalian cell lines revealed the presence of intracellular bacteria that were identified as M. avium-intracellulare. An extensive and critical analysis of the origin of infection, of cure protocols, and of biological manifestations in M. avium-infected cells is presented. As no tremendous visible alteration of turbidity or pH of cell culture media, and no morphological change occurred in most M. avium-infected cell cultures, detection of an infection by these bacteria is rather difficult. Recommendations are given for treatment of irreplaceable cultures and prevention of mycobacterial contamination in a tissue culture facility.

The ability to form biofilm influences Mycobacterium avium invasion and translocation of bronchial epithelial cells.

Organisms of the Mycobacterium avium complex (MAC) are widely distributed in the environment, form biofilms in water pipes and potable water tanks, and cause chronic lung infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Pathological studies in patients with pulmonary MAC infection revealed granulomatous inflammation around bronchi and bronchioles. BEAS-2B human bronchial epithelial cell line was used to study MAC invasion. MAC strain A5 entered polarized BEAS-2B cells with an efficiency of 0.1 +/- 0.03% in 2 h and 11.3 +/- 4.0% in 24 h. In contrast, biofilm-deficient transposon mutants 5G4, 6H9 and 9B5 showed impaired invasion. Bacteria exposed to BEAS-2B cells for 24 h had greater ability to invade BEAS-2B cells compared with bacteria incubated in broth. M. avium had no impact on the monolayer transmembrane resistance. Scanning electron microscopy showed that MAC A5 forms aggregates on the surface of BEAS-2B cell monolayers, and transmission electron microscopy evidenced MAC within vacuoles in BEAS-2B cells. Cells infected with the 5G4 mutant, however, showed significantly fewer bacteria and no aggregates on the cell surface. Mutants had impaired ability to cause infection in mice, as well. The ability to form biofilm appeared to be associated with the invasiveness of MAC A5.

Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis-like mechanism and survives in a compartment that differs from that with extracellular phenotype.

Mycobacterium avium uptake by human macrophages differs between the phenotypes of bacterium grown in laboratory media (extracellular growth, EG) and bacterium grown within macrophages (intracellular growth, IG). Studies in vivo have confirmed that, when spreading, pathogenic mycobacteria enter macrophages by a complement receptor 3-independent pathway, in contrast to mycobacteria uptake in vitro. M. avium, grown in macrophages (IG) for 3 or more days, invade fresh macrophages by a macropinocytosis-like mechanism, in contrast to bacteria grown in media (EG), confirmed by the inhibitory effect of wortmannin, an inhibitor of phosphoinoside-3-kinase, on the uptake of IG, but not EG, by macrophages. The IG phenotype was seen in vacuoles with lower pH than those inhabited by the EG phenotype. Incubation of macrophages with bafilomycin A1, an inhibitor of vacuole acidification, decreased the viability of intracellular IG, but not EG, phenotype, suggesting the importance of an acidic environment for the regulation of IG genes. In addition, the percentage of vacuoles that incorporate and retain LAMP-1 is smaller with EG than with IG bacteria. The formation of M. avium macropinosomes was also shown to be independent of microtubules. These data suggest that uptake of extracellular fluid is part of M. avium IG phenotype uptake by macrophages, and that the IG phenotype inhabits a slightly different vacuole than that of EG.

[Mycobacterium avium-intracellulare complex: phenotypic and genotypic markers and the molecular basis for interspecies transmission]. / Le complexe Mycobacterium avium-intracellulare: marqueurs phénotypiques et génotypiques et les bases moléculaires de la transmission inter-espèces.

The Mycobacterium avium complex (MAC) comprises a heterogeneous group of slowly-growing mycobacteria that are pathogenic for both humans and animals. Two genetically distinct species within MAC are M. avium, which tends to infect HIV-infected patients, and M. intracellulare more common among immunocompetent individuals. Contrary to M. intracellulare which relates to a single species, M. avium is separated into three subspecies; M. avium subsp. avium, a major opportunistic pathogen leading to a disseminated disease among terminal AIDS patients; M. avium subsp. paratuberculosis, causing Johne's disease among ruminants and implicated in Crohn's disease among humans; and M. avium subsp. silvaticum, a pathogen affecting birds that may cause chronic enteritis among calves but has not yet been associated with human disease. With the exception of mycobactin-dependent growth of M. paratuberculosis, most of the biochemical and cultural tests cannot discriminate among the three subspecies of M. avium. However, recently developed molecular methods and fingerprinting of strains using insertion sequences allows not only to distinguish among them but also further to explore the polymorphism of human and animal isolates. Numerous studies have underlined the probable role of various ecological niches (water, dust, soil, pigs, poultry and ruminants etc.) as a possible source of contamination for AIDS patients. This paper reviews the phenotypic and genotypic markers and epidemiology of M. avium complex organisms and current knowledge of the molecular basis of of inter-species transmission.

Ultrastructural study of Mycobacterium avium infection of HT-29 human intestinal epithelial cells.

Mycobacterium avium is a common pathogen in AIDS patients and, in a large percentage of those patients, M. avium infection appears to be acquired via the gastrointestinal tract. M. avium is able to bind to and enter human and murine intestinal epithelial cells in vitro and in vivo. The invasion by and intracellular fate of M. avium in the HT-29 intestinal epithelial cell line was examined in an ultrastructural study. Bacterial contact with polarised cells was observed 10-15 min after monolayer infection and in polarised monolayers this always occurred in areas lacking microvilli. Contact with HT-29 cells did not appear to take place in a preferential area on the bacterial cell. Following invasion, M. avium was encountered within vacuoles containing either single or multiple bacteria; the latter evolved to contain only an individual bacterium. Vacuoles containing more than one bacterium were seen early in the infection and eventually underwent segmentation, with each bacterium occupying a vacuole. No bacteria were observed outside vacuoles up to 5 days after infection.

Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls.

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-micron-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.

Growth within macrophages increases the efficiency of Mycobacterium avium in invading other macrophages by a complement receptor-independent pathway.

Infections caused by organisms of the Mycobacterium avium complex occur in approximately 50 to 60% of patients with AIDS. M. avium is an intracellular pathogen that survives and multiplies within mononuclear phagocytes. In this study, we investigated the uptake of M. avium grown within macrophages (intracellular growth M. avium [IG]) by a second macrophage compared with M. avium cultured in broth (extracellular growth M. avium [EG]). The results showed that IG was six- to eightfold more efficient than EG in entering macrophages. In addition, while an anti-CR3 antibody was able to inhibit approximately 60% of EG uptake by macrophages, it failed to inhibit the entry of IG. In contrast to EG, IG uptake into macrophages was significantly inhibited in the presence of anti-beta1-integrin and anti-transferrin receptor antibodies. Entry into macrophages by alternate receptors was associated with resistance to tumor necrosis factor alpha (TNF-alpha) stimulation. While stimulation with TNF-alpha resulted in inhibition of the growth of EG, it was not associated with inhibition of intracellular growth of IG. Investigation of the reason why M. avium is able to sense the changes in the intracellular environment triggering a change to the invasive phenotype suggests a direct relationship with macrophage apoptosis. These results suggest that intracellular growth is associated with novel mechanisms of M. avium uptake of macrophages and that those mechanisms appear to offer advantages to the bacteria in escaping the host defense.

[Utility of the direct culture examination (Ziehl-Neelsen) using the Bactec system for the presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi, and Mycobacterium kansasii]. / Valor del examen directo (Ziehl-Neelsen) del cultivo en sistema Bactec para la identificación presuntiva de: Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi y Mycobacterium kansasii.

BACKGROUND: The usefulness of the microscopic examination of Bactec 12B and 13A growth medium as a method for the possible identification of M. tuberculosis complex, M. avium complex, M. xenopi, and M. kansasii was performed out to guide the selection of different genetic identification probes and, in the case of M. xenopi, the selection of the temperature of subcultures incubation. METHODS: Upon detection of an index of growth greater than 100 in Bactec tubes, staining was performed by the Ziehl-Neelsen technique. On the basis of the morphology observed, the possible identification was performed by genetic probes. Subcultures were used for definitive identification. RESULTS: Three hundred forty-four positive samples were studied by radiometric technique. A total of 190 strains were identified as M. tuberculosis, 88 strains as M. avium-intracellulare (MAI), 33 strains as M. xenopi, 14 strains as M. kansasii and 19 strains were identified as: M. gordonae (10), unpigmented rapid growth microbacteria (7), and M. simiae (2). Sensitivity, specificity, positive predictive value, and negative predictive value were 97.9%, 95.4%, 96.4%, and 97.3%, respectively for M. tuberculosis complex, 84.0%, 99.2%, 97.3% 94.7% for M. avium complex; 63.6%, 98.3%, 80.7%, 96.2% for M. xenopi; 35.7%, 98.1%, 45.5% 97.2% for M. kansasii. CONCLUSIONS: The morphology of M. tuberculosis complex examined in the radiometric system in useful to differentiate this species from other microbacteria (MOTT), allowing the selection of specific probe used. Within the MOTT, M. avium complex also has morphological characteristics which are useful for its differentiation, the morphology usually described for the remaining species was frequently not observed.

Pneumocystis carinii and Mycobacterium avium-intracellulare infection of the choroid.

It has been hypothesized that coinfection with mycobacteria occurs in patients with Pneumocystis carinii choroiditis, but cases demonstrating ocular infection by both organisms have not been reported. This study reports the case of a patient with P. carinii choroiditis who was treated with intravenous trimethoprim and sulfamethoxazole, followed by intravenous trimethoprim and dapsone. The choroidal lesions failed to resolve despite 6 weeks of treatment, and the patient died from massive pulmonary infection caused by P. carinii, Mycobacterium avium-intracellulare, and cytomegalovirus infections. Ocular histologic and electron microscopic examinations revealed choroidal infection by both P. carinii and M. avium-intracellulare. Serum levels of sulfamethoxazole were below the recommended therapeutic range for treating P. carinii infection during the first week of therapy, but adequate drug levels were subsequently obtained. Failure of choroidal lesions of P. carinii to resolve in some cases may suggest insufficient antimicrobial levels in the blood or raise the possibility of coexistent M. avium-intracellulare or other opportunistic infection.

Effect of Tween 80 on the growth of Mycobacterium avium complex.

The effect of Tween 80 on the growth of Mycobacterium avium complex (MAC) in liquid culture condition was investigated. Observation of the colony-forming units (CFU) and the morphology of MAC with transmission and scanning electron microscopy showed that Tween 80 at 0.05% in the medium (ca. 0.5 mg/ml) had bacteriostatic action and caused cell elongation. Tween 80 at 0.5% or more in the medium (ca. 5 mg/ml) reduced the quantity of MAC glycolipids and also led to false positive or positive results in biochemical tests for mycobacterial identification using nitrate reductase, urease, or arylsulfatase. To determine whether or not surfactants could reduce the MAC permeability barrier, the minimal inhibitory concentration (MIC) of antituberculosis drugs on MAC was determined in liquid medium with or without several kinds of surfactants including Tween 80. Five surfactants including Tween 80 increased the activity of antituberculosis drugs to MAC. These findings suggest that Tween 80 acts directly on the cell wall of MAC.

Ability of smooth and rough variants of Mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides.

A spontaneous rough (Rg) mutant of M. avium ATCC 15769 was mutagenized using N-methyl-N-nitro-nitrosoguanidine (MNNG). Out of 54 clones initially chosen on the basis of their morphological appearance on the 7H11 agar, seven Rg clones were selected on the basis of their response to current biochemical tests, and were later characterized for their cell wall amphiphilic contents (analysis of loosely-bound surface lipids for mycosides, peptidolipids, phospholipids, and mycolic acids by thin layer chromatography, and fatty acids by gas chromatography), and ability to grow intracellularly inside J-774 macrophages. A parallel study was also performed on a previously reported Rg mutant of M. intracellulare serovar 20 (W.W. Barrow and P.J. Brennan, J. Bact. 150 (1982) 381-384) which lacked the ability to synthesize mycosides C (MYC-). The results obtained were compared to parental smooth (Sm) colony-types of the respective M. avium-intracellulare strains. Our results showed that neither the ninhydrin-reacting lipids (probably peptidolipids) nor the glycopeptidolipids (mycosides C) were primary factors responsible for the intracellular survival and multiplication of these bacteria. Ultrastructural studies showed that although the polysaccharide-rich outer wall layer (POL) in case of MYC- Rg strain was not uniformly distributed and contained blebs, these bacteria formed the characteristic electron-transparent zone (ETZ) upon phagocytes by macrophages. Furthermore, the multiplication of MYC- Rg strain inside macrophages did not result in intracellular selection of MYC+ bacteria, nor in Rg to Sm transition.

Effects of surface-active agents on drug susceptibility levels and ultrastructure of Mycobacterium avium complex organisms isolated from AIDS patients.

The multiple-drug-resistance property of Mycobacterium avium complex (MAC) is mainly attributed to a cell envelope permeability barrier. MAC treated with subinhibitory levels of dimethyl sulfoxide (DMSO) and ethylenediaminetetraacetic acid (disodium salt) (EDTA) did not have altered minimum inhibitory concentration (MIC) levels or show ultrastructural changes; the effect of sodium dodecyl sulfate (SDS) was variable. With SDS, the visualization of the nucleoid and ribosomes decreased, and amorphous electron-dense material accumulated near the structurally altered cytoplasmic membrane and cell wall. Use of 0.005% Tween-80 resulted in a 2-4-fold reduction in MIC in the case of rifampicin, ansamycin (LM 427), cephapirin, and ciprofloxacin. Tween-80 treated cells were swollen, and deposits of low electron-density accumulated in the cytoplasm; distortions in the outer-cell integuments were observed. These findings are consistent with the idea that Tween-80 increases cell-envelope permeability, thereby enhancing drug penetrability and reducing MIC levels. Because of the action of Tween-80, its use in drug-susceptibility media or diluent fluids should be avoided.