[Conditionally pathogenic mycobacteria in the environment and their effect on living organisms]. / Výskyt podmienecne patogénnych mykobaktérií v prostredí a ich vplyv na zivý organizmus.

Since 1968 when bovine tuberculosis was eliminated in the Czech Republic, the epidemiological situation of bovine tuberculosis has been stabilized. At present the incidence of the disease in men and animals caused by conditionally pathogenic mycobacteria is worldwide increasing. In human population, especially people with impaired immunity are affected. In farm animals infections caused by conditionally pathogenic mycobacteria may often result in complications in intravital and postmortal diagnosis of bovine and avian tuberculosis. Those infections are then often incorrectly diagnosed which could have a great negative impact on health, economy and breeding. Therefore the objective of the study was to summarize data from literature and our own experience concerning the occurrence of atypical mycobacteria in environment. The study is divided into 5 summarizing chapters, supplemented with 13 Tables.

The tuberculin test.

Tuberculin tests in general use today rely on the response to intradermal injections of tuberculin with assessment of the injection site for swelling at 72 hours post injection. Estimates of the sensitivity of tuberculin tests range from 68-95% while specificity is estimated to be 96-99%. The sensitivity of the test is affected by the potency and dose of tuberculin administered, the interval post-infection, desensitisation, deliberate interference, post-partum immunosuppression and observer variation. Specificity is influenced by sensitisation as a result of exposure to M. avium, M. paratuberculosis and environmental mycobacteria and by skin tuberculosis.

Mycobacterium hiberniae sp. nov.

Strains of a new type of slowly growing scotochromogenic, rose-pink-pigmented mycobacterium were isolated repeatedly from sphagnum vegetation, true moss, and soil in Ireland. These strains grew at 22, 31, and 37 degrees C but not at 45 degrees C and possessed acid phosphatase and arylsulfatase activities. They reduced nitrate, tolerated 0.1% NaNO2, did not split amides, and were resistant to most of the antituberculous drugs tested, except ethambutol. They did not form acid from glucose and mannose. Their internal phenetic similarity was 97.08% +/- 2.07%. The whole mycolate pattern confirmed the homogeneity of the taxa sharing similar mycolate types with several other mycobacterial species. However, on the basis of the nature of the major pyrolysis esters, the taxon appeared unique. The phylogenetic analysis based on evolutionary distance values revealed that the strains belong to a new species of slowly growing mycobacteria. The DNA-DNA hybridization values confirmed that these strains differ significantly from Mycobacterium nonchromogenicum, M. terrae, M. triviale, and M. thermoresistibile. The strains produced a unique rose-pink pigment and were nonpathogenic for mice, guinea pigs, and rabbits, but they provoked a nonspecific hypersensitivity reaction to bovine tuberculin in guinea pigs and cattle. Hence, they are considered a member of a new species of nonpathogenic slowly growing mycobacteria, for which the name Mycobacterium hiberniae is proposed. Strain Hi 11 is the type strain, a culture of which has been deposited in the American Type Culture Collection as strain ATCC 49874.

Isolation of Mycobacterium simiae from the environment.

An examination of 18 sphagnum samples collected in two different biotopes of the coastal region of southeastern Madagascar revealed an unexpectedly high positivity for mycobacteria (83.3%). The concentration of alcohol acid-fast bacilli reached a high level of 10(5) and 10(6)/g, respectively, compared with the sphagnum biotopes in moderate climates. Besides the habitat-specific mycobacterial species in sphagnum vegetation, like M. sphagni, M. gordonae and M. madagascariense, potentially pathogenic species, like M. avium, M. scrofulaceum and M. xenopi and M. marinum, were found. Furthermore, pathogenic M. simiae was found in sphagnum vegetation of Madagascar, first time isolated in the environment until now. It should be considered as a potential source of infection for human and animals.

Phylogeny of rapidly growing members of the genus Mycobacterium.

The 16S rRNAs from nine rapidly growing Mycobacterium species were partially sequenced by using the dideoxynucleotide-terminated, primer extension method with cDNA generated by reverse transcriptase. The sequences were aligned with 47 16S rRNA or DNA sequences that represented 30 previously described and 5 undescribed species of the genus Mycobacterium, and a dendrogram was constructed by using equally weighted distance values. Our results confirmed the phylogenetic separation of the rapidly and slowly growing mycobacteria and showed that the majority of the slowly growing members of the genus represent the most recently evolved organisms. The 24 strains which represented 21 rapidly growing species constituted several sublines, which were defined by the following taxa: (i) Mycobacterium neoaurum and M. diernhoferi, (ii) M. gadium, (iii) the M. chubuense cluster, (iv) the M. fortuitum cluster, (v) M. kommossense, (vi) M. sphagni, (vii) M. fallax and M. chitae, (viii) M. aurum and M. vaccae, (ix) the M. flavescens cluster, and (x) M. chelonae subsp. abscessus. Our phylogenetic analysis confirmed the validity of the phenotypically defined species mentioned above, but our conclusions disagree with most of the conclusions about intrageneric relationships derived from numerical phenetic analyses.

Mycobacterium cookii sp. nov.

Strains of a new type of slowly growing scotochromogenic mycobacterium were isolated repeatedly from sphagnum vegetation and surface water of moors in New Zealand. These strains grew at 31 and 22 degrees C but not at 37 degrees C and possessed catalase, acid phosphatase, and arylsulfatase activities. They did not split amides, and most of them were susceptible to antituberculotic drugs. Furthermore, they did not tolerate 0.1% NaOH2 and 0.2% picric acid and did not grow on compounds used as single carbon sources and single nitrogen and carbon sources. The internal similarity of the strains as determined by numerical taxonomy methods was 96.6% +/- 3.09%. The whole-mycolate pattern is unique in that it has not been found previously in 23 species of slowly growing mycobacteria. Evaluation of long-reverse-transcriptase-generated stretches of the primary structure of the 16S rRNA confirmed that these organisms belong to the genus Mycobacterium. The phylogenetic position of these bacteria is unique; they are situated between slowly growing pathogenic and rapidly growing saprophytic species. The strains are not pathogenic for mice, guinea pigs, and rabbits, but they provoke a nonspecific hypersensitivity reaction to bovine tuberculin. Hence, they are considered members of a new species of nonpathogenic, slowly growing mycobacteria, for which the name Mycobacterium cookii is proposed. Strain NZ2 is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 49103.

Acid-fast bacilli found in sphagnum vegetation of coastal Norway containing Mycobacterium leprae-specific phenolic glycolipid-I.

In the grey layer of sphagnum vegetation originating from former leprosy-endemic regions of coastal Norway, acid-fast bacilli (AFB) containing Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) on the surface have been found. These AFB survived in foot pads of nude mice with multiplication but without swelling. This contrasts to experimental leprosy with clinically derived M. leprae where swelling and unlimited multiplication takes place. The naturally occurring AFB may be of a lower pathogenicity than M. leprae obtained from clinical cases. The possibility of M. leprae surviving in sphagnum vegetation was assessed by inoculation of clinically derived M. leprae into the grey layer of the sphagnum. It multiplied more than tenfold and retained its pathogenicity in nude mice for 16 weeks, the duration of the experiment. The lack of pathogenicity of sphagnum-derived, M. leprae-like mycobacteria may be relevant to the decline of leprosy in Norway.

Evidence for phagosome-lysosome fusion in Mycobacterium leprae-infected murine Schwann cells.

Murine Schwann cells were infected with viable armadillo-derived Mycobacterium leprae in vitro, and the lysosomal marker enzyme, acid phosphatase, was stained by the Gomori reaction. Electron microscopic analysis revealed that Schwann cells infected with M. leprae possess acid phosphatase and that lysosomes fuse with infected phagosomes.

The primary structure of the 16SrRNA of Mycobacterium leprae: its use in phylogeny and development of DNA probes.

Two long stretches of the 16S from Mycobacterium leprae were sequenced using reverses transcriptase and the chain termination technique. Homology values were calculated for 11 cultivable mycobacteria and a phylogenetic tree constructed from evolutionary distance values (Knuc). Slow and fast growing mycobacteria used in this study form a taxonomic unit but were phylogenetically well separated. It could be confirmed that M. leprae is a true member of the slowly growing pathogenic mycobacteria branching off intermediate to other members of this subgroup. Comparison of the 16 rRNA primary structures reveals that the nucleotide sequence of M. leprae contains regions of sufficient variation to serve as potential target sites for DNA probes. Here we describe the designation of a DNA oligonucleotide and its use in dot blot hybridization experiments were it was directed against bulk RNA isolated from several mycobacteria.

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand.

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation H-Group, which differed from any known mycobacterial species. Mycobacteria of this H-group had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. H-Group mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing H-Group mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.

Molecular-genetic evidence for the relationship of Mycobacterium leprae to slow-growing pathogenic mycobacteria.

A total of 1170 nucleotides of the 16S rRNA from Mycobacterium leprae were compared to the homologous regions of M. tuberculosis, M. bovis Vallée, M. avium, M. scrofulaceum, M. phlei, M. fortuitum and one representative each of the genera Corynebacterium, Nocardia, and Rhodococcus. Homology values were calculated and a phylogenetic tree was constructed from the evolutionary distance values. Despite differences in DNA G + C content and genome size, M. leprae is a true member of the slow-growing pathogenic mycobacteria, branching off intermediate to the other members of this subgroup. Slow- and fast-growing mycobacteria are phylogenetically well separated but constitute an individual branch of the actinomycetes proper. Significant structural variation of certain regions of the 16S rRNA may allow construction of M. leprae-specific probes used for rapid identification.

The simultaneous inoculation of Mycobacterium leprae and M. intracellulare into nude mice: development of cutaneous leproma and accelleration of foot pad swelling.

Organisms of the non-pathogenic Mycobacterium intracellulare serotype 19 Darden enhanced the pathogenicity of M. leprae when inoculated together into the foot pads of nude mice. This supporting effect could be demonstrated by an accelleration of foot pad swelling, beginning 4 months after inoculation and by the development of cutaneous leproma on dorsal and lateral body sites within 6 months after inoculation. These leproma increased in number and size during the 9 months they were under observation and demonstrated micromorphological characteristics similar to those of human leprosy.