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Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis.

Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms). The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date. However, these mycobactins were unique in that they had more than one alkyl chain. The M. scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain. Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types. Each mycobactin could be subdivided further by the length of its R1 alkyl chain. No differences in the production of these novel mycobactin were observed among species. Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography. Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms. M. paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M. intracellulare M12. However, a sample of mycobactin J isolated by Merkal and McCullough (Curr. Microbiol. 7:333-335, 1982) from M. paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography. No reason for this difference could be evinced. Our findings indicate that there is a close taxonomic relationship between M. paratuberculosis and the MAIS complex.

Chemical Phenomena↗

Mycobacterium scrofulaceum infection in Erythrocebus patas monkeys.

Mycobacterium scrofulaceum was cultured from two of seven tuberculin reactors in a group of 12 Erythrocebus patas monkeys. One monkey reacted atypically to 0.1 ml of 2.5 mg veterinary tuberculin after having shown no reaction to four previous tests administered at 2-week intervals. The reaction consisted of edema with no induration or erythema at 24 hours and was completely dissipated at 36 hours. Responses to additional tests using veterinary tuberculin (2.5 mg) and Mycobacterium bovis purified protein derivative (1.1 mg) ranged from negative to slightly edematous. Radiographs were suggestive of pulmonary tuberculosis. Extensive granulomas were observed in the lungs, liver and spleen at necropsy. Mycobacterium scrofulaceum was isolated from lung and liver granulomas in this animal and from lung granulomas in another. The atypical skin hypersensitivity and organism isolation from multiple organs suggested Mycobacterium scrofulaceum as the etiologic agent in these animals. Reactions of six other Erythrocebus patas to veterinary tuberculin (2.5 mg) ranged from edematous to indurated.

Animals↗

Disseminated infection due to Mycobacterium scrofulaceum in an immunocompetent host.

Disseminated Mycobacterium scrofulaceum infection has rarely been reported, and the majority of infections have been associated with AIDS or other immunocompromising diseases. We describe a previously healthy man with M. scrofulaceum infection whose clinical manifestations included miliary lung lesions, mediastinal lymphadenitis, granulomatous hepatitis, osteomyelitis, subcutaneous abscesses, and probable renal involvement. M. scrofulaceum was isolated from multiple specimens of sputum, urine, and the abscesses. The patient was treated successfully with the combination of isoniazid, ethambutol, rifampin, and ofloxacin. To our knowledge, this is the first detailed description of an extensively disseminated infection due to M. scrofulaceum in an immunocompetent host since the advent of AIDS.

Adult↗

Plasmid-encoded copper resistance and precipitation by Mycobacterium scrofulaceum.

A copper-tolerant Mycobacterium scrofulaceum strain was able to remove copper from culture medium by sulfate-dependent precipitation as copper sulfide. Such precipitation of copper sulfide was not observed in a derivative that lacks a 173-kilobase plasmid. In addition, the plasmid-carrying strain has a sulfate-independent copper resistance mechanism.

Chemical Precipitation↗

Successful treatment of localized cutaneous infection caused by Mycobacterium scrofulaceum with clarithromycin.

Cutaneous infection arising from Mycobacterium scrofulaceum, a nontuberculous mycobacteria, has rarely been reported, and most of the reported infections were disseminated forms in patients with AIDS or other immunocompromising illness. We describe an occurrence of localized mycobacterial skin infection caused by M. scrofulaceum in a previously healthy child that manifested as a red nodule on the cheek. A biopsy specimen of the lesion demonstrated granulomatous infiltration in the dermis. M. scrofulaceum was isolated from culture of a tissue specimen. Polymerase chain reaction amplified specific fragments for M. scrofulaceum. The patient was treated successfully with clarithromycin as monotherapy for 6 months, leading to complete healing without recurrence during a follow-up period of 2 years.

Anti-Bacterial Agents↗

Factors influencing the chlorine susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.

Chlorine↗

[A case of Mycobacterium scrofulaceum lung infection occurring in old lung tuberculosis lesion].

A 68-year-old man was admitted because of a persistent productive cough of 6 weeks' duration and detection of acid-fast bacilli from sputum. Based on chest roentgenograms and isolation of Mycobacterium scrofulaceum from sputum, on admission, a diagnosis of Mycobacterium scrofulaceum lung infection was made. Although the organisms were resistant to 0.1 microgram/ml of INH, 2.5 micrograms/ml of EB and 10 micrograms/ml of RFP, sputum converted to negative by the use of INH (0.4 g/day), EB (0.5 g/day) and RFP (0.3 g/day) for 2 months. The incidence rate of nontuberculous lung mycobacteriosis seems to be increasing recently. This is partially considered to be due to an increase of various strains of nontuberculous mycobacteria in the environment. A striking advance of clinical medicine and changes in the constitution of human society and environment surrounding us produce an increase in the number of compromised hosts. The lung disease due to Mycobacterium scrofulaceum, a kind of opportunistic pathogen, is considered to be increasing in a wide variety of compromised hosts in the future.

Aged↗

Cloning, sequencing and expression in Escherichia coli of the gene for alpha antigen from Mycobacterium scrofulaceum.

The gene for the extracellular alpha antigen of Mycobacterium scrofulaceum (S-alpha) was cloned by using the alpha antigen gene fragments of Mycobacterium bovis BCG as probes. The complete nucleotide sequence was determined. The gene was expressed in Escherichia coli. The gene encodes 330 amino acids, including 40 amino acids for the signal peptide, followed by 290 amino acids for the mature protein. The deduced amino acid sequences were highly homologous to the alpha antigen of the species of other mycobacteria. Interestingly, the alpha antigens of MAIS complex (Mycobacterium avium-Mycobacterium intracellulare-M. scrofulaceum) were closely homologous even at the C-terminal regions which were variable among those of M. bovis BCG, Mycobacterium leprae and Mycobacterium kansasii.

Amino Acid Sequence↗

[Disseminated infection by Mycobacterium scrofulaceum: apropos of 3 cases].

BACKGROUND: Mycobacterium avium-M. Intracellulare complex and Mycobacterium kansasii cause most of nontuberculous mycobacteria infections in AIDS patients in Spain. Mycobacterium scrofulaceum is rarely isolated as a cause of disseminated disease and most of case have been reported before the AIDS occurrence. METHODS: Three clinical cases of patients with disseminated disease caused by M.scrofulaceum and AIDS are reviewed. RESULTS: Clinical presentation in all cases was similar to those of other disseminated mycobacterial infection, and was also linked to severe immunosuppression (CD4 cell count < 100 CD4/mm3). CONCLUSIONS: In our area, M. scrofulaceum is the second species of nontuberculous mycobacteria causing disseminated disease in AIDS patients. The most frequent species is M. avium-M. intracellulare complex. For diagnosis an AIDS patient complaining of febrile syndrome without foci and severe immunosuppression, is mandatory to obtain blood cultures and biopsy or adenopathy aspirates if present. To our knowledge, these are the first reports in our country.

AIDS-Related Opportunistic Infections↗

Production and characterization of monoclonal antibodies against specific serotypes of Mycobacterium avium and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex.

Serotype-specific and Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex (MAIS complex)-specific monoclonal antibodies (MAbs) were prepared. A series of MAbs were obtained, five specific for serotype 2, three specific for serotype 4, eight against a strain of serotype 19, two specific for the MAIS complex, and two against a common glycolipid shared by all the mycobacteria tested so far. The serotype-specific and the MAIS complex-specific MAbs reacted in immunofluorescence with intact mycobacteria and in enzyme-linked immunosorbent assay and immuno-thin-layer chromatography with lipid extracts of mycobacteria. The two MAbs against a common mycobacterial glycolipid reacted only in lipid enzyme-linked immunosorbent assay and immuno-thin-layer chromatography. All MAbs were directed against glycopeptidolipids (GPLs), except for four MAbs against proteins of serotype 19. The serotype- and MAIS complex-specific epitopes on GPLs are exposed on the mycobacterial cell wall, in contrast with the common mycobacterial glycolipid, which is probably located inside the cell wall. The serotype-specific MAbs reacted with native as well as deacetylated GPLs, in contrast with the MAIS complex-specific MAbs, which reacted only with native GPLs. The MAbs will be useful for the identification of MAIS complex and M. avium serotypes 2 and 4 and a strain of serotype 19, GPL analyses with immuno-thin-layer chromatography, and the localization of GPL epitopes in mycobacteria.

Animals↗

Disseminated Mycobacterium scrofulaceum infection: a potentially treatable complication of AIDS.

Disseminated Mycobacterium scrofulaceum infection has rarely been reported (only 8 cases to date), and no case of infection associated with AIDS has been reported in detail. We report a case of disseminated M. scrofulaceum infection in an AIDS patient that presented as chronic ulcerative and nodular skin lesions with probable cavitary lung involvement. We discuss reported cases of dissminated M. scrofulaceum infection and features of human immunodeficiency virus (HIV)-associated disease due to mycobacteria other than tuberculosis. Although our patient died before susceptibility testing could be completed, the M. scrofulaceum isolate was found to be susceptible to clarithromycin, ethambutol, and clofazimine. Physicians who evaluate skin lesions in HIV-infected persons should perform appropriate mycobacterial studies and search for disseminated disease. Drug susceptibility testing for mycobacteria other than tuberculosis is not yet standardized, but the broth dilution method, currently being studied in clinical trials of treatment for Mycobacterium avium complex, may be superior to older methods. After the possibility of Mycobacterium tuberculosis infection has been excluded, physicians should consider administering initial empirical therapy with two or more drugs, including a newer macrolide, to AIDS patients with disseminated mycobacterial disease.

AIDS-Related Opportunistic Infections↗

Variation within Mycobacterium scrofulaceum.

The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and investigate diversity within the species. Fifty-four cultures were identified as Myco. scrofulaceum by preliminary cultural and biochemical tests, thin-layer chromatography and double diffusion. These strains were examined by PCR based on the 65 kDa heat stress protein gene, followed by restriction enzyme analysis of the product with BstEII and HaeIII. This produced seven groups, most with fewer fragments than had been reported previously. The technique was a rapid and reliable method for studying variation within Myco. scrofulaceum but alone, was unable to discriminate between some of these variants and other genetically similar species. When PCR-RFLP results were combined with biochemical tests, the major groups appeared to relate to different disease situations and thus, may have some epidemiological value.

DNA, Bacterial↗

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.

DNA, Bacterial↗