Seventeen strains of mycobacteria, recovered from six armadillos experimentally infected with Mycobacterium leprae, were examined in ten different laboratories. This collaborative study included use of conventional bacteriological tests, lipid analyses, determination of mycobactins and peptidoglycans, characterization by Py-MS, and immunological, metabolic, pathological and DNA studies. These armadillo-derived mycobacteria (ADM) formed five homogeneous groups (numbered ADM 1 to 5) on the basis of phenetic analyses. However, DNA studies revealed only four homogeneous groups since group ADM 1 and one of the two strains in group ADM 3 showed a high level of DNA relatedness. The phenetic and DNA studies confirmed that the ADM strains differed from all other known mycobacteria. Cultural, biochemical, metabolic and pathogenic properties as well as DNA-DNA hybridizations clearly differentiated these ADM from M. leprae.

Genome sizes of 22 species of mycobacteria have been calculated. Thirteen of these genome sizes are based on results from re-examinations. Base ratios of some of the strains have been determined.

M. avium and M. intracellulare are difficult to distinguish by means of biochemical tests and by means of numerical taxonomy. DNA-DNA hybridization confirms that these species are different but indicates that some serovars of M. intracellulare actually belong to the species M.avium, viz.: serovars 4, 5, 6 and 8. This corresponds to results obtained with sensitin tests on guinea-pigs. The status of strains belonging to serovar 9 is uncertain. The two strains investigated in this study were closely related to, but not identical with M. scrofulaceum serovar 41.

The DNA-DNA homology percentages obtained in this study indicate M. smegmatis, M. phlei, M. fortuitum, M. peregrinum, M. senegalense, M. farcinogenes, M. chelonei, M. parafortuitum, M. vaccae, M. diernhoferi and M. aurum are separate species. Taxonomically there is no justification for the designation M. fortuitum complex, and M. peregrinum should be revived as an independent species. The type strains of M. parafortuitum, M. vaccae, M. aurum and the non-valid M. diernhoferi are so distinct from each other that they should not be combined.

This study is a contribution to the elucidation of the taxonomic relationships among Mycobacterium simiae serovars 1 and 2, other photochromogenic, mycobacterial species and the non-photochromogenic species M. avium and M. intracellulare. DNA-DNA homology percentages and comparative reciprocal intradermal sensitin reactions in guinea-pigs have confirmed that M. habana is a subjective synonym of M. simiae serovar 1 and that M. asiaticum is a separate species. The two strains of M. simiae serovar 2 studied were distinct from each other and both were distinct from M. simiae serovar 1. M. simiae was so different from M. avium and even from M. intracellulare serovars 12, 14, 16 and 19, that there is no basis for including M. simiae in the so-called M. avium-intracellulare complex. Neither did M. simiae bear any close relation to the photochromogenic species M. kansasii and M. marinum. A fairly close negative correlation was observed between the DNA-DNA homology percentages and the specificity differences of the sensitins from the same pairs of strains. This is per se a proof of the veracity of the two taxonomic methods used in the study.

Mycobacterium phlei PN-bb, induced in the parental strain PN by gamma-radiation and ethyl methanesulfonate, should be reclassified as Rhodococcus bronchialis PN-bb. The reclassification is based on the results of lipid analysis, DNA-DNA hybridization and several biochemical tests, performed in comparison with the parental strain PN, earlier reclassified as R. bronchialis, and R, bronchialis (N654).

Study of lipid and DNA, biochemical tests and phage typing performed on the strain PA previously labelled Mycobacterium phlei, lead to the conclusion that this strain belongs to the species M. smegmatis. Parallel studies performed on strain PN, isolated from a culture of strain PA, as well as DNA homology percentage of the two strains, do not support the assumption that strain PN could have resulted from a mutation of strain PA Strain PN produces mycolic acids similar to those found in Rhodococcus bronchialis; the few biological tests applied quite agree with such a classification.

DNA-DNA hybridization is a reliable method for determining the phylogenetic relationship between bacterial strains The hybridization kinetics for DNA from different slowly-growing mycobacteria were measured optically in a spectrophotometer. The results indicate that M. tuberculosis and M. bovis belong to one species. M. avium and M. intracellulare are two species, but some serotypes, now designated M. intracellulare, actually belong to M. avium.

The homology percentages between DNA from M. smegmatis ATCC 607 and DNA from nine various species of mycobacteria have been determined. DNA-DNA hybridization was measured in a spectrophotometer. The technique, calculation of results and the uncertainty of the method are described.

The genome size and the base ratio of deoxyribonucleic acid (DNA) from 15 different species of mycobacteria have been determined. The molecular weight of the sheared DNA specimens has been estimated.

An account is given of isolation of DNA from mycobacteria. Mechanical disruption was carried out in a French pressure cell press, followed by isolation of DNA by means of Marmur's method. This DNA contained considerable amounts of polysaccharides which, however, could be removed by precipitation with cetyltrimethylammonium bromide (CTAB). The degree of purity obtained and the GC contents are given.

Although phage-typing of some pathogenic microbes-e.g., Escherichia coli and the genera Salmonella, Staphylococcus, and Streptomyces-is in routine use, little progress has yet been made in the phage-typing of mycobacteria, chiefly because mycobacteriophages possess a polyvalence that prevents the reliable identification of individual species of mycobacterium. Furthermore, laboratories cannot compare their results because they employ different methods and culture media and also because the phages used cannot be accurately identified. It has been shown that internationally reproducible results can be obtained for M. tuberculosis by selecting suitable mycobacteriophages and sensitive culture media, and by using a standardized technique of phage-typing. Under these conditions, phage lysis may be utilized to classify strains of M. tuberculosis.

SUMMARY: A co-operative numerical taxonomic analysis of rapidly growing mycobacteria of Runyon's group IV is reported. There was no limitation on the number, nature, or method of performance of the test characters contributed by each of the 12 participants. Initially 415 test characters were coded for analysis; deletion of irrelevant and repetitious data resulted in a final 195 characters used to generate the matching matrix. All nine major clusters defined in the study were of named species; however, three of these were noted to contain two or more species and the reduction of some of these to synonymy with prior epithets is discussed. Recognized clusters were: Mycobacterium smegmatis, M. phlei, M. vaccae (including M. para-fortuitum), M. diernhoferi, M. flavescens, the rhodochrous taxon, M. thamnopheos, M. fortuitum, M. chelonei. Results of the pooled information are compared with those of the individual participants. Immunological results generally correlated well with numerical analyses.

A description is given of a rapidly-growing mycobacterium (designated C.U.) which produces bacteriophages spontaneously. The strain was isolated from an abscess that occurred 2 months after a BCG vaccination. The phage content was maintained after subculture. Since only some of the colonies produced phages and the content of phages could be eliminated after growth in antiserum against the phage (designated BK5) it is concluded that' C.U. is a pseudolysogenic mycobacterium. No phage-mediated changes in the characteristics of the bacterium were: demonstrated. The literature and various forms of pseudo-lysogeny are reported.

Non-photochromogenic mycobacteria belonging in serotype Davis deviate from the typical pattern of M. avium. The divergencies in the in vitro experiments occurred especially in connection with drug resistance, effect of temperature, and catalase test. In the in vivo examinations, there were divergencies in all the animal studies. The Davis strains are unstable in subcultures on Lowenstein-Jensen and 7H-10 agar media and pure cultures of smooth, transparent colonies (“light”) were dominated by opaque variants (“dark”) after 7–8 passages. It is probable that the variants are the chief reason for the inhomogeneity within the Davis group. On the basis of virulence experiments, the strains were divided into three groups. The most virulent (Group A, virulent for hens, rabbits, guinea pigs) gave growth of “light” colonies and the attenuated (Group C) “dark” colonies. However, the correlation between the two characteristics is not absolute, since both types of colonies were represented in pure culture in Group B (virulent for hens and guinea pigs, attenuated for rabbits), i.e. also the strains which give growth of “dark” colonies may possess virulence for the classical experimental animal, the hen. In the in vitro experiments, the attenuated strains (Group C) were more sensitive to streptomycin, viomycin, rifampicin, and penicillin than the strains in the other groups. This greater sensitivity of the variants must be taken into consideration when selecting test strains for assay of the effect of new drugs or drug combinations.

Non‐photochromogenic mycobacteria belonging in serotype Davis deviate from the typical pattern of M. avium. The divergencies in the in vitro experiments occurred especially in connection with drug resistance, effect of temperature, and catalase test. In the in vivo examinations, there were divergencies in all the animal studies. The Davis strains are unstable in subcultures on Löwenstein‐Jensen and 7H‐10 agar media and pure cultures of smooth, transparent colonies (“light”) were dominated by opaque variants (“dark”) after 7–8 passages. It is probable that the variants are the chief reason for the inhomogeneity within the Davis group. On the basis of virulence experiments, the strains were divided into three groups. The most virulent (Group A, virulent for hens, rabbits, guinea pigs) gave growth of “light” colonies and the attenuated (Group C) “dark” colonies. However, the correlation between the two characteristics is not absolute, since both types of colonies were represented in pure culture in Group B (virulent for hens and guinea pigs, attenuated for rabbits), i.e. also the strains which give growth of “dark” colonies may possess virulence for the classical experimental animal, the hen. In the in vitro experiments, the attenuated strains (Group C) were more sensitive to streptomycin, viomycin, rifampicin, and penicillin than the strains in the other groups. This greater sensitivity of the variants must be taken into consideration when selecting test strains for assay of the effect of new drugs or drug combinations.

Acta Pathologica Microbiologica ScandinavicaVolume 76, Issue 3 p. 464-474 SUBDIVISION OF M. TUBERCULOSIS BY MEANS OF BACTERIOPHAGES With Special Reference to Epidemiological Studies Inga Baess, Inga Baess Tuberculosis Department, Statens Seruminstitut, Copenhagen, Denmark.Search for more papers by this author Inga Baess, Inga Baess Tuberculosis Department, Statens Seruminstitut, Copenhagen, Denmark.Search for more papers by this author First published: September 1969 https://doi.org/10.1111/j.1699-0463.1969.tb03276.xCitations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume76, Issue3September 1969Pages 464-474 RelatedInformation