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The main characteristics of Mycoplasma ulcerans are its remarkably distinct drug sensitivity pattern and its rather poor enzymatic activity. M. microti is clearly distinguishable from M. bovis and M. tuberculosis. Some of the M. buruli strains appear to be identical with M. ulcerans, Whereas others show similarity either to M. microti or to M. tuberculosis. It seems that M. buruli is not a legitimate species.

The suitability of phage-impregnated paper discs for the phage typing of mycobacteria was studied. The relevance of the routine test dilution, the volume of the phage used, the mode of incubation, and the effect of prolonged storage of phage-impregnated paper discs were considered. By using paper discs, each impregnated with one of 5 different mycobacteriophages (BG1, BK1, G37, CRI-3 and LG) that lyse Mycobacterium smegmatis 607B, it was determined that 100 x the routine test dilution in a volume of at least 20 microliter was required for phage lysis. Soaked and dried paper discs produced larger areas of lysis than those with 20-microliter volumes. Soaked discs were found to be stable even after storage for 8 weeks at 4 degrees C.

Rapid methods for the differentiation of Mycobacterium tuberculosis/M. bovis (TB complex) from other mycobacteria (MOTT bacilli) were developed and evaluated in a three-phase study. In the first phase, techniques for identification of Mycobacterium species were developed by using radiometric technology and BACTEC Middlebrook 7H12 liquid medium. Based on 14CO2 evolution, characteristic growth patterns were established for 13 commonly encountered mycobacterial species. Mycobacteria belonging to the TB complex were differentiated from other mycobacteria by cellular morphology and rate of 14CO2 evolution. For further differentiation, radiometric tests for niacin production and inhibition by Q-nitro-alpha-acetyl amino-beta-hydroxy-propiophenone (NAP) were developed. In the second phase, 100 coded specimens on Lowenstein-Jensen medium were identified as members of the TB complex, MOTT bacilli, bacteria other than mycobacteria, or "no viable organisms" within 3 to 12 (average 6.4) days of receipt from the Centers for Disease Control. Isolation and identification of mycobacteria from 20 simulated sputum specimens were carried out in phase III. Out of 20 sputum specimens, 16 contained culturable mycobacteria, and all of the positives were detected by the BACTEC method in an average of 7.3 days. The positive mycobacterial cultures were isolated and identified as TB complex or MOTT bacilli in an average of 12.8 days. The radiometric NAP test was found to be highly sensitive and specific for a rapid identification of TB complex, whereas the radiometric niacin test was found to have some inherent problems. Radiometric BACTEC and conventional methodologies were in complete agreement in Phase II as well as in Phase III.

A total of 339 strains of the Mycobacterium avium-intracellulare-scrofulaceum complex were phage typed using our previously described technique and 11 typing phages. These included 235 strains of human origin obtained from state health laboratories in Virginia, Georgia, Florida, and Arkansas, 26 strains isolated from persons with AIDS, 38 strains isolated from animals, and 40 environmental isolates. A phage-typing scheme was developed that denotes sensitivity to 8 primary typing phages: the JF group (JF1, JF2, JF3, and JF4), phage D302, and the AN group (AN3, AN9, and AN1-8). The 3 auxiliary phages (VC3, VA6, and D32) define subgroups of the strains sensitive to the AN phages. A total of 99 strains were sensitive to at least 1 phage. Of 31 serotype 1 or 2 strains from animals, 13 were sensitive to AN phages but resistant to JF phages. In contrast, 7/33 serotype 4 or 8 strains from animals or from persons with AIDS were sensitive to JF phages but not to the AN phages. Of the clinical isolates not associated with AIDS, 78/235 were phage sensitive. These strains could be divided roughly into 4 groups: sensitive to AN phages, sensitive to JF phages, sensitive to phage D302, and sensitive to multiple phages. Only 1 of the environmental isolates was phage sensitive. The results indicate that phage typing can subdivide this heterogeneous group of organisms and is a useful tool for epidemiological studies.

Based on conventional taxonomic analyses, as well as molecular level studies on DNA and catalases, the tuberculosis complex (M. tuberculosis, M. microti, M. bovis, and M. africanum) appears to consist of a single species. This species does not occur normally free in nature, but depends on host-to-host transmission for its continued existence. Members of the complex are sufficiently distinct genetically from known free-living mycobacteria so that it is unlikely that they would evolve from the free-living species at a significant frequency in nature, although that is presumably how they arose in the first place. Therefore, eradication of tuberculosis seems to be a realistic hope. The technical means now exist for control of the disease; some improvements in diagnostic technology can be expected to accelerate the control efforts. At a basic science level, more information is needed about the ability of M. tuberculosis to survive in a latent state in a host, only to revive years later, if the control of tuberculosis is to progress to eradication.

Cultures of the Mycobacterium avium-intracellulare-scrofulaceum (MAIS) complex, isolated from patients who lived throughout the state of Georgia, were studied using the serotyping scheme of Schaefer. One hundred four (77 per cent) of the 135 isolates tested could be classified into 16 serotypes. The rest were not typable. Fifty-eight per cent of all the typable strains were serotypes 1, 14, 16, and 42. Results of the serotyping were recorded, mapped, and analyzed by county of residence. Most frequent isolations of these mycobacteria were from patients who resided in the coastal plain of Georgia. This finding correlates with the mycobacterial skin test studies of Edwards and associates, who found that the largest percentage of reactors to PPD-Battey resided in the costal plain. The disparate distributions of certain serotypes suggest the possible importance of regional factors that may affect the serotypes of mycobacteria indigenous to an area. The data do not suggest that chicken or swine reservoirs are highly significant sources of human mycobacteria.

Strains of the species Mycobacterium simiae give a positive niacin test. On the basis of their cultural and biochemical characteristics and by seroagglutination they can be classified into 2 subspecies. (1) The strains of serotype M. simiae 1 hydrolyze urea regularly and nicotinamide and pyrazinamide irregularly. They are photochromogenic after prolonged exposure to light. (2) The strains of serotype M. simiae 2 hydrolyze urea only. Three of 4 strains are scotochromogenic; the fourth has a pale pink pigment. Two of these strains possess alpha- and beta-esterase activity. The other two are negative in this test. Strains of M. habana are culturally and biochemically identical with the serotype M. simiae 1 and show the same serologic specificity as M. simiae 1. M. habana sera, after having been absorbed by M. simiae 1, retain a small amount of agglutinins specific for M. habana. We believe that the M. habana strains belong to the species M. simiae and are closely related to the serotype M. simiae 1.

The Tween opacity test can be used to differentiate (1) Mycobacterium flavescens from Mycobacterium gordonae and Mycobacterium szulgai, and (2) nonphotochromogenic strains of Mycobacterium kansaii from strains of the Mycobacterium terrae complex.

The heat-stable (70degrees C) acid phosphatase test performed by the method of Kind and King is a simple method for differentiating Mycobacterium kansasii, M. marinum, M. gastri, M. nonchromogenicum, and M. triviale from other slowly growing mycobacteria, and M. fortuitum from other rapidly growing acid-fast bacilli.