Influence of pork consumption on human infection with Mycobacterium avian-intracellulare.

The hypothesis that eating pork from animals infected with organisms of the Mycobacterium avium-intracellular complex is a significant cause of human mycobacteriosis was examined by skin testing students with purified protein derivative from M. intracellulare to determine their sensitivity to M. avium-intracellulare. No significant differences in sensitivity could be demonstrated between those individuals who had never eaten pork and those who ate pork routinely. It was concluded that sensitivity to purified protein derivative from M. intracellulare in this population did not involve eating pork.     

Site-directed mutagenesis and virulence assessment of the katG gene of Mycobacterium intracellulare

Mycobacterial catalases have been suggested as acting as virulence factors by protecting intracellular mycobacteria from reactive oxidative metabolites produced by host phagocytes. Mycobacterium intracellulare , like many other mycobacteria, produces two proteins with catalase activity: a heat-stable catalase (KatE) and an inducible, heat-labile catalase peroxidase (KatG). The M. intracellulare katG gene was cloned, and a plasmid derivative with a 4 bp insertion in the katG coding sequence was constructed and used for site-directed mutagenesis of M. intracellulare 1403 (ATCC 35761). The resulting katG mutant was highly resistant to isoniazid (INH), showed an increased sensitivity to H₂O₂ and had lost peroxidase and heat-sensitive catalase activity but retained heat-stable catalase activity. The plasmid carrying the katG frameshift allele was also used for mutagenesis of the mouse virulent M. intracellulare isolate D673. After intravenous injection into BALB/c mice, D673 and the isogenic katG mutant showed the same growth kinetics in the spleen, liver and lungs of the infected mice. Our results demonstrate that the KatG catalase peroxidase mediates resistance to H₂O₂ and susceptibility to INH but is not an essential virulence factor for the survival and growth of M. intracellulare in the mouse.     

Expression profiles of low molecular mass heat shock proteins by Mycobacterium avium and Mycobacterium intracellulare

Closely related non-tuberculous mycobacterial species, Mycobacterium avium and Mycobacterium intracellulare, were compared for the profiles of their production of low molecular mass heat shock proteins at 45 degrees C, by performing polyacrylamide gel electrophoresis analysis of bacterial cell lysate proteins. All of the M. intracellulare but not M. avium strains potently increased the production of the 18-kDa heat shock protein, when cultured at 45 degrees C. Half of the M. intracellulare strains with high sensitivity to 45 degrees C produced not only the 18-kDa heat shock protein but also the 16-kDa heat shock protein at 45 degrees C. These findings indicate that M. avium and M. intracellulare differentially respond to 45 degrees C heat shock in terms of the production of low molecular mass heat shock proteins.     

An evaluation of the polyacrylamide gel electrophoresis fractionation method for the production of Mycobacterium tuberculosis skin test preparations. I. Production, physiochemical characterization and serological analyses

As a part of a cooperative inter-laboratory WHO supported project raw tuberculins were produced and purified protein derivative (PPD, 18.7 g protein) was prepared. Employing a multistage preparative polyacrylamide gel electrophoresis (PAGE) method the PPD was separated into four fractions corresponding to 15, 7, 4.75 and 3.5% gel concentrations. The PAGE procedure resulted in three lots of material--each representing 11 electrophoretic runs. Immunodiffusion analyses showed that the largest number of precipitinogens was found in the 15% fractions and that some precipitinogens cross-reacted with preparations of Mycobacterium bovis BCG, M. intracellulare, M. kansasii, M. smegmatis and M. vaccae.     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae

Abstract DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae , demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae , ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to posses DNA sequences homologous to the 18-kDa protein gene of M. leprae . RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare . The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae , and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae , based on the M. leprae 18-kDa protein gene.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Isolation of the Mycobacterium avium-M. intracellulare-M. scrofulaceum complex from tank water in Queensland, Australia.

Disease-associated serotypes of Mycobacterium intracellulare and M. avium have been isolated from 32 of 141 rainwater tanks situated in the basin of the Fitzroy River and its tributaries in central Queensland, 7 of 32 tanks situated in the hinterland of the coastal city of Rockhampton, and 2 of 32 tanks sampled repetitively in the southeastern Queensland city of Toowoomba. M. gordonae was also isolated from 23 of the river basin tanks, from 9 in the Rockhampton hinterland, and from 5 in the city of Toowoomba. One half of these isolates came from tanks which also yielded M. intracellulare. Mycobacteria of the M. terrae-M. triviale-M. nonchromogenicum complex were found in 7 tanks, usually in conjunction with M. intracellulare. The humans who consume the contaminated tank water are free of symptoms but have not been medically examined. It is suggested that mycobacteria adhering to dust particles disturbed by mechanical cultivation may be the source of contamination.     

Susceptibility of Mycobacterium avium and Mycobacterium intracellulare to various antibacterial drugs

Mycobacterium avium and M. intracellulare of human and natural sources, identified by the Gen-Probe Rapid Diagnostic System for M. avium Complex (MAC) were studied for susceptibility to eight different drugs. In the case of human isolates of MAC, the following was noted. M. avium showed nearly the same susceptibility to streptomycin, kanamycin, ethambutol, and clofazimine as was seen with M. intracellulare. M. avium was much more resistant to rifampicin and rifabutin than was M. intracellulare, and M. avium was more susceptible to quinolones such as ofloxacin and ciprofloxacin. Conversely, in the case of MAC from natural sources, there was no difference between the susceptibility of M. avium and M. intracellulare to these antibacterial agents.     

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.     

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Gene replacement through homologous recombination in Mycobacterium intracellulare.

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.     

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Isolation of tuberculin peptides from tuberculin purified protein derivative (PPD).

Tuberculin purified protein derivative (PPD) obtained from the filtrate of Mycobacterium tuberculosis was hydrolysed with proteinase, trypsin, or chymotrypsin. Each hydrolysate consisted of a tuberculin peptides mixture (TPM). From each TPM 16 fractions were obtained by ion-exchange chromatography on Dowex 50W-X8 but only one fraction was isolated from each of the 16 fractions which showed tuberculin activity in guinea pigs sensitized with M. bovis (bcg) or M. tuberculosis. This fraction was designated "purified tuberculin peptide" (PTP). The PTP fraction from the proteinase hydrolysate (PTP-proteinase) was rechromatographed on Dowex 1-X2 and two tuberculin peptide fractions having molecular weights of 3200 and 12,000 were isolated. The potency of these two fractions was assessed in guinea pigs sensitized with M. bovis (BCG) and with M. tuberculosis and they were approximately 4 to 7 times more potent than either the international standaCG and of at least equal potency to either PPD-S or Connaught PPD in guinea pigs sensitized with either M. kansasii, M. scrofulaceum, M. intracellulare, or M. avium whereas very little if any cross-reactivity was elicited by these two fractions. This lack of response indicates that either fraction could be used as an aid to differentiate between sensitization due to M. tuberculosis or M. bovis and sensitization attributed to other mycobacteria.     

Nucleotide sequence analysis and serologic characterization of the Mycobacterium intracellulare homologue of the Mycobacterium tuberculosis 19 kDa antigen

Disseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar-sized restriction fragments in M. tuberculosis and M. intracellular genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that M122 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species-specific diagnostic reagents.     

Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare

Mycobacterium avium and Mycobacterium intracellulare were grown in suspension and in biofilms, and their susceptibilities to chlorine were measured. M. avium and M. intracellulare readily adhered within 2 h, and numbers increased 10-fold in 30 days at room temperature in biofilms on both polystyrene flasks and glass beads. The chlorine resistance of M. avium and M. intracellulare cells grown and exposed to chlorine in biofilms was significantly higher than that of cells grown in suspension. Survival curves showed no evidence of a resistant, persisting population after 6 h of exposure to 1 mug chlorine/ml. The chlorine susceptibility of cells grown in biofilms and exposed in suspension (cells detached from bead surfaces) was also significantly higher than that of cells grown and exposed in suspension (planktonic cells), although it was lower than that of cells grown and exposed in biofilms. The higher resistance of the detached biofilm-grown cells was reversed upon their growth in suspension. There was a strong correlation between the chlorine susceptibility of cells of both M. avium and M. intracellulare and cell surface hydrophobicity measured by contact angle for both biofilm- and suspension-grown cells.     

Occurrence of Mycobacterium Other than Mycobacterium tuberculosis in the Oral Cavity and in Sputum

Mycobacteria were cultured from 9% of 424 paired mouthwash-induced sputum specimens. The majority of the organisms were not Mycobacterium tuberculosis. Sputum cultures contributed 59% of these isolations. M. intracellulare was the species most frequently isolated. The non-M. tuberculosis mycobacteria may constitute part of the oral flora of the general population and are not more prevalent in hospitalized patients. Approximately one-third more isolations were made from the patients furnishing two or three pairs of specimens as compared to those patients providing one pair of specimens each. From the paired specimens of 4 of 113 patients, two species of Mycobacterium were isolated. M. intracellulare was isolated from two of three samples of tap water and from the fingers of 1 of 52 patients.     

Identification of cytoplasmic membrane protein antigens of Mycobacterium avium, M. intracellulare, and M. scrofulaceum

The cytoplasmic membrane isolated from representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group contained approximately 20 proteins, as identified by SDS - polyacrylamide gel electrophoresis. One membrane protein predominated, comprising up to 50% of the total membrane protein. This major cytoplasmic membrane protein (MCMP) had a molecular weight of 31,000 and was surface accessible based on its susceptibility to proteinase digestion. The composition of the culture medium strongly influenced the amount of MCMP in the membrane fraction. Western blot analysis revealed that the MCMP and several other membrane proteins reacted with serum samples from patients infected with M. avium-intracellulare, M. tuberculosis, or other mycobacteria.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.