Runyon Group Research Articles

Cross-reactions in cell mediated immunity induced by atypical mycobacteria.

Cross-reactivity in the delayed hypersensitivity response to mycobacteria of different Runyon groups was tested in Swiss white mice immunised with live mycobacteria. All the strains tested gave cross-reactions and, generally, slow growers gave stronger cross-reactions with other slow growers than with rapid growers and vice versa. Sonicates of cross-reacting mycobacteria were also tested for their ability to generate activated macrophages in mice immunised with Mycobacterium avium. All the mycobacterial sonicates generated activated macrophages but a sonicate of Salmonella typhi did not. The sonicate of M. tuberculosis H37Rv also generated activated macrophages, which indicates that there might be protective cross-reactions between M. tuberculosis and atypical mycobacteria.

Rapid and sensitive identification of Mycobacterium tuberculosis.

The fatty acid constituents of 14 species of Mycobacterium (14 isolates) and one isolate each of Corynebacterium xerosis, Nocardia asteroides, and Streptomyces albus were examined with the purpose of distinguishing Mycobacterium tuberculosis from other acid-fast bacilli. Combined thin-layer chromatography (TLC) of methyl mycolates and gas-liquid chromatography (GLC) of shorter-chain fatty acid esters provided an unequivocal identification of M. tuberculosis in a matter of 2 to 3 days. The methodology included rapid and simplified procedures for methanolysis and extraction of bacterial lipids with equally facilitated GLC and TLC analyses. These studies were performed with 0.5 to 1.0 mg of dry bacterial cells (approximately 2.5 X 10(7) CFU). When applied to 100 unknown cultures, the methodology with combined TLC-GLC correctly identified all 49 of the M. tuberculosis-Mycobacterium bovis cultures and a variety of other mycobacterium taxa. It was also interesting to note that 28 of 39 (72%) of the nontuberculous mycobacteria were correctly identified. An additional five species were tentatively identified as belonging to either of two species (Mycobacterium malmoense, Mycobacterium terrae), but in all cases, the two species belonged to the same Runyon group. All six nonmycobacterial species were differentiated from the mycobacteria studied.

Disease Due to Mycobacterium avium-intracellulare

Mycobacterium avium and M intracellulare are presently grouped together as the M avium-intracellulare complex. These closely related organisms belong to Runyon's group III nonchromogenic mycobacteria. The M avium-intracellulare organisms are increasingly significant pathogens in North America. Unlike M kansasii, the other most prominent nontuberculous Mycobacterium associated with human disease, infections with M avium-intracellulare are generally highly resistant to treatment. The reservoirs and routes of transmission are uncertain.

La flore bacterienne dans un reseau de distribution: Bacteria in distribution systems

Water quality upkeep in distribution systems is a major concern of drinking water suppliers. In the case of extended systems transporting water through several thousand kilometers of pipelines, the problem is not a simple one. This concern was, among others, at the root of the Water Authority for the Paris Suburbs' decision to modify treatment at the Méry-sur-Oise drinking water production plant (Damez, 1982). As can be seen in Fig. 1, the old line was upgraded by the addition of a raw water basin, two more ozonation stages, and activated carbon filtration. Furthermore, pre-chlorination is no longer used at the plant. A study to assess the impact of these changes on the quality of water in the system was launched prior to the switchover, in collaboration with researchers from the Pasteur Institute in Paris. It involved the enumeration and identification of the germs in analysed samples of river water, treated water and distributed water. Bacterial aftergrowth in the distribution system was significant with the old line, the mean value of the total germs at 20°C exceeding the limit value of European standards (100 germs ml −1). With the new line, bacterial aftergrowth has been limited to a great extent (Table 6). Removal of the biodegradable fraction of organics during treatment probably has a fundamental effect on this limitation of the aftergrowth (Brunet et al., 1981; Gaïd, 1981), which is achieved notwithstanding lower post-chlorination dosages. Moreover, chlorine is now in the form of free chlorine (Table 1). As regards organics, TOC removal has been only slightly increased by the new line (Table 7), but the permanganate consumption has been greatly reduced, from 1.5 to 0.5 mg −1 at the plant outlet. In river water, the germs are mainly Escherichía, Aeromonas hydrophila, Acinetobacter calcoaceticus, Alcaligenes faecalis, Yersinia enterocolitica were isolated in 6% of the samples. At the plant outlet, the samples are mostly uncontaminated in analysis conditions. In the distribution system, the species isolated are Aeromonas hydrophila and Alcaligenes faecalis. Enterobacteriaceae and Pseudomonodaceae also contaminated the samples, but with a very low frequency (5%) (Tables 3 and 4). As regards acid-fast bacteria, many species were isolated in river water ( M. fortuitum, M. chelonei, M. terrae and M. lactis). But no species were found in samples of 250 ml at the plant outlet. Still, 39 acid-fast bacteria were isolated from 126 samples of distributed water (Table 5). Most germs belong to Runyon's group 11 but were not identified (Runyon, 1959). The study is still under way. The onset of deterioration of bacteriological quality may be a criterion for selecting the regeneration cycle for activated carbon.

Isolation of mycobacteria from lymph nodes of pigs and their environment.

An attempt was made to isolate mycobacteria from the lymph nodes (LNs) of 459 pigs at four abattoirs (Obihiro, Tokachi-Shimizu, Fukagawa, and Kushiro). From 156 pigs selected at random at Tokachi-Shimizu highly contaminated with mycobacteria were isolated at 49% (29/59) in the submaxillary LN and 20% (13/65) in the mesenteric LN. The average isolation rates of mycobacteria from the lymph node in all the pigs, except those at Tokachi-Shimizu were as follows: 5.4% (16/294) from the submaxillary, 3.8% (11/293) from the pulmonary, and l.7% (5/298) from the mesenteric LN. When broken by the abattoir, they were 7% (7/100) at Fukagawa, 7.8% (8/102) at Kushiro, and 13.8% (14/101) at Obihiro. The average being 9.57% (29/303). It was found that 3 pigs harbored mycobacteria concurrently in two lymph nodes. A total of 76 strains of the mycobacterial isolates did not belonged to Mycobacterium tubercculosis or M. bovis, but a majority of them were so-called atypical mycobacteria. Of them, Runyon's Group III organisms were predominant and most of them identified as M. intracellulare serovars. M. avium serovar 1 was isolated from two pigs at Tokachi-Shimizu and Obihiro, respectively. This is the first isolation of M. avium in Hokkaido. From 202 environmental specimens collected, 17 strains of mycobacteria were isolated. It is known that the strains of serovars 4, 6, and 8 of M. intracellulare, which are potentially pathogenic for man and animals, have been isolated from soil, sewage and beddings. Isolation of M. intracellulare from soil, sewage and beddings is a new finding in Japan.

A SURVEY OF MYCOBACTERIOSIS OF FERAL PIGS IN THE NORTHERN TERRITORY

Seven hundred and fifty-one feral pigs from the subcoastal plains of the Northern Territory were examined. The sample population consisted of 52.4% females and 47.6% males. They ranged in age from newborn piglets to mature animals of over 72 months. Of the pigs examined 47.7% had macroscopic abscesses and of these 80.2% were probably caused by mycobacteria. Tissues from 193 pigs were examined bacteriologically and 93 strains of mycobacteria were isolated. These were typed as M. bovis (37 strains); M. avium serotype 2 (1); M. intracellulare serotypes 6 (2), 7 (3), 9 (1) and 18 (1); M. intracellulare double serotypes 6 + 12 (1), 8 + 12 (1), and 11 + 12 (1); M. intracellulare unclassified serotype (4); M. scrofulaceum serotype 41 (1); M. scrofulaceum unclassified serotype (7); M. gordonae (2); M. Kansasii (1); M. simiae (2); M. szulgai (2); M. vaccae (1); and M. xenopi (2). Additionally, 3 strains were unidentifiable members of the M. avium-M. intracellulare-M. scrofulaceum (MAIS) complex, one strain was a Runyon's group IV and 4 strains were typed as members of the genus Rhodococcus. Five strains were non-viable on subculture and 10 did not conform to any currently recognised species of mycobacteria. Of the 93 strains, 3 were isolated from tissue that did not contain macroscopic lesions, viz. M. simiae, Runyon's group IV and an unidentifiable member of the MAIS complex. It was concluded that the feral pig is probably an end host for both M. bovis and atypical mycobacteria and not a significant source of infection for cattle. M. bovis is not a significant cause of mortality in feral pigs but mycobacterioses are a significant cause of morbidity. With increasing age, the proportion of pigs having lesions increased whereas the proportion of lesions from which mycobacteria could be isolated decreased.

Possible monocyte killing defect in familial atypical mycobacteriosis

INFECTIONS in children caused by atypical mycobacteria general!y affect either skin as swimming pool granu!oma or superficial lymph nodes as cervical adenitis. Lincoln and Gilbert' identified only 12 published cases of disseminated atypical mycobacteriosis in children. O~f these, nine were caused by organisms belonging to Runyon group III (Mycobacterium inlracellulare/avium complex group). Since that review, only one additional report of disseminated infection in a child has appeared, which was also caused by a MIAC strain5 There has been only one previous report of disseminated atypical mycobacteriosis in more than one member of a family. :~ Previous studies assessing host defense mechanisms in a few of these patients have suggested that a deficiency of cell-mediatedimm unity may be an important factor allowing dissemination of the organisms. ~-'~ In addition, patients with microbicidal defects of phagocytic cells may be at increased risk for disseminated mycobacterial disease. 7 In this report an extensive immunologic evaluation in one of two affected siblings suggests the presence of a defect i n monocyte killing. Disseminated atypical tuberculosis should then alert the physician to the possibility of an immune deficiency. CASI~ REPORTS Patient 1. This 25-month-old Mexican-American girl was admitted to the Los Angeles County/USC Medical Center in

Occurrence and characterization of "avium-like" Mycobacteria isolated from animals in Sweden.

431 cultures of "avium-like" mycobacteria (ALM) were isolated from wild and domestic animals during 1974--76 at the National Veterinary Institute, Stockholm. Of these, 50 isolates from pigs were studied by growth-chromogenicity, pathogenicity, and biochemical tests. Furthermore, thin-layer chromathography was performed, and on some isolates serotyping. All 50 isolates belonged to Runyon's group III and were pathogenic for chicken; none was capable of splitting oleic acid from Tween 80. 47 gave tellurite reduction within a period of three days; one was arylsulphatase-positive after three days and a further four after 14 days. The biological and biochemical tests permit assignation of the 50 isolates to the M. avium-intracellulare complex. The lipid patterns of the isolates examined were analysed by thin-layer chromatography. Thirty-five of the isolates showed a lipid pattern similar to that of A 2 of the fowl reference strain; three belonged to lipid type A 1 and four to A 3. Eight could not be typed. Of 22 isolates, 14 could be assigned to M. avium serotypes.

Group III Atypical Mycobacterial Infections

We retrospectively and prospectively examined the clinical presentation, course of disease, and response to therapy in 63 patients whose mycobacterial isolates were identified as Runyon group III atypical mycobacteria. All of the patients except four had pulmonary disease similar to typical pulmonary tuberculosis. The others had scrofula or abxcess. There was a male predominance and whites were slightly more frequently infected in our series. Forty-six percent of the patients were from rural Oklahoma. Skin testing and chest roentgenography were important, but culture of the organism appeared to be the only productive method of distinguishing the atypical disease from typical tuberculosis. Eighty-one percent had an associated disease. The isolates showed a very high rate of primary drug resistance, and this was reflected in the difficulty in controlling the infection.

A Co-operative Numerical Analysis of Mycobacterium gastri, Mycobacterium kansasii and Mycobacterium marinum

A co-operative taxonomic study has been performed on slowly growing photochromogenic mycobacteria (Runyon Group I) and closely related organisms. Phenetic data on 54 strains, studied in seven laboratories, were collected and analysed by numerical taxonomic methods. Immunological properties and phage susceptibility patterns were analysed independently to establish correlation with numerical classification. Mycobacterium gastri, M. kansasii and M. marinum appeared as distinct well-defined clusters and the serological and phage data supported the resolution of these three species. A table of definitive properties is presented. Two strains each of M. simiae and M. asiaticum formed a loose cluster which was clearly separated from the previously mentioned three species; the small number of strains examined precluded the establishment of a list of definitive properties of these two species. It is concluded that the Runyon Groups, which provided a practical though arbitrary basis for establishment of a series of co-operative studies, have served their purpose and should now be supplanted by classification and nomenclature based on species.

Mycobacteriosis in mountain whitefish (Prosopium williamsoni) from the Yakima River, Washington.

Mycobacteriosis was found in mountain whitefish (Prosopium williamsoni) taken from the Yakima River near Richland, Washington in 1975 and 1976. The disease appeared to affect about 8% of the population sampled. Gross lesions were present in most visceral organs, but were most common in the kidney, liver and pyloric caeca. Microscopically, the lesions consisted of large numbers of macrophages containing numerous intracellular bacilli. An organism was isolated and has been tentatively classified as Mycobacterium sp., Runyon group III.

Pathogenicity for cattle of atypical mycobacteria isolated from feral pigs and cattle and the correlation of lesions with tuberculin sensitivity.

Two experiments involving the inoculation of cattle with atypical mycobacteria are described. In the first experiment groups of 5 cattle were inoculated either subcutaneously or into a mesenteric lymph node with a strain of M. scrofulaceum or M. intracellulare. Four weeks and 10 weeks after inoculation the cattle were tuberculin tested with bovine PPD, avian PPD and homologous PPDs. The pathological changes observed were similar within each group of cattle inoculated with the same strain of mycobacteria. A significant interaction was demonstrated between the strain and the route of inoculation. In the second experiment 17 cattle were similarly inoculated by either of the two routes with 1 of 6 strains of M. intracellulare, a strain of M. scrofulaceum or a strain of Runyon Group IV, all of which had been isolated from feral pigs, or a strain of M. intracellulare of bovine origin. Tuberculin tests were carried out after 4 weeks and 10 weeks. Only the isolate from a bovine lymph node produced a significant level of sensitivity to bovine PPD. Cultural isolation of the mycobacteria from autopsy material was not correlated with the presence of macroscopic lesions nor with sensitivity to bovine PPD. The response to bovine PPD of cattle infected with these atypical mycobacteria decreased between 48 h and 96 h after injection of the tuberculins. As the maximum difference in the response to bovine and avian tuberculins occurs at 72 h a comparative tuberculin test should be read at this time to eliminate non-specific reactors.

Atypical mycobacteria and the xenograft valve

During a 4 month interval, cultures taken at the time of porcine xenograft valve implantation grew a fastidious atypical mycobacterium, Mycobacterium chelonei (Runyon's groups IV), in eight of 20 patients. Initial growth occurred at 2 to 3 weeks in thioglycollate broth only; detailed biochemical and bacteriologic evaluation demonstrates altered catalase activity accounting for its initial slow growth. Only one patient has manifested clinical evidence of infection 5 months after mitral valve replacement. A large pericardial effusion required pericardiectomy. Pericardial tissue and fluid have grown M. chelonei with the same growth characteristics as the initial valve culture. It is recommended that manufacturers of xenograft valve bioprostheses maintain a quarantine of 6 weeks until cultures, smears of culture broth, and microscopic evaluation of aortic wall coupons are negative. Users of these prostheses should keep cultures for a minimum of 3 weeks. Should positive cultures result, antimicrobial therapy should be considered only with clinical evidence of infection.

In vitro susceptiiblity of Mycobacterium fortuitum and Mycobacterium chelonei to amikacin.

Infections due to Runyon group IV atypical mycobacteria, Mycobacterium fortuitum and Mycobacterium chelonei, in humans have been difficult to treat in the past because of the organisms' resistance to all of the conventional antimycobacterial drugs. Determinations of minimal inhibitory concentrations (MICs) by the agar dilution method suggest that amikacin may be useful in the treatment of infections due to M. fortuitum and M. chelonei. Further support for the efficacy of antimicrobial agents with good in vitro activity in the treatment of infections with M. fortuitum and M. chelonei will depend on correlation of future clinical experience with standardized data on the MICs for these organisms.

Tuberculin Reactions in Bulls And Boars Sensitized with Atypical Mycobacteria from Sawdust

Due to a planned export from a combined bull and boar station, more than 70 boars and 100 bulls were examined by tuberculin tests. Distinct reactions to avian tuberculin appeared in about half of the animals. Many of them also reacted to bovine tuberculin. For diagnostic purposes, many of the reactors were slaughtered, and samples from these and from the environment were examined bacteriologically. Strains of Mycobacterium avium were isolated from only 2 out of 14 reacting boars and from none of the 23 reacting bulls. No isolation of Mycobacterium bovis was made. However, atypical mycobacterial strains, classified as Runyon Group III and IV, were isolated from 3 boars, 2 bulls, 1 pigeon and from many samples of sawdust. The isolation of identical fast-growing mycobacterial strains from the sawdust used in the pens for the reacting boars and bulls, was especially remarkable. The strains differed enzymatically and biochemically from those isolated from other sources. This indicated a possible sensitization of the animals with similar mycobacterial strains. Possible cross-reactions to avian and bovine tuberculin were investigated in tuberculin assays with guniea pigs and pigs sensitized to one of the mycobacterial strains isolated. Distinct reactions to both avian and bovine tuberculin appeared in all the animals. From these results it was concluded that the tuberculin reactions in the boars and the bulls were not due to any tuberculous infection in the herd, but to a sensitization of the animals with atypical mycobacteria.

Cooperative Numerical Analysis of Rapidly Growing Mycobacteria: The Second Report

The second cooperative numerical taxonomic analysis of Runyon group IV rapidly growing mycobacteria was facilitated by combining the results of 12 individual participants, each performing one or more of the selected tests by his or her own choice of method. The 177 characters were coded for analysis and the matching matrix was generated. Eight of nine clusters which were defined in this investigation were of previously named species. The recognized clusters were as follows: Mycobacterium flavescens, M. vaccae, M. chitae, M. thermoresistibile, and M. smegmatis. One large cluster, designated “M. parafortuitum complex” was noted which contained strains representative of taxa earlier identified as M. parafortuitum, M. diernhoferi, M. aurum, M. neoaurum, and a cluster of strains of unnamed species. It is suggested that segregation of species or subspecies within this complex not be attempted at this time.

Antagonistic action of Streptococcus salivarius and Streptococcus faecalis to Mycobacterium tuberculosis.

Streptococcus salivarius and Streptococcus faecalis were found to inhibit the growth of Mycobacterium tuberculosis on Löwenstein-Jensen and Middlebrook 7H11 agars, but not on the latter medium when antibacterial drugs were added. S. faecalis was found to be more inhibitory than S. salivarius to 15 strains of M. tuberculosis. S. salivarius produced little or no inhibition of growth of Runyon group III organisms but was very antagonistic to Runyon group I mycobacteria.

The pathogenicity of mycobacterium fortuitum and mycobacterium chelonei in man: A report of seven cases

The clinical records of 7 patients referred to the National Jewish Hospital and Research Center over a 6-year period for evaluation of an abnormal chest x-ray and repeated sputum isolates of rapidly growing mycobacteria (Runyon's Group IV) were reviewed to determine the potential pathogenicity of these organisms. Mycobacterium fortuitum was isolated from 5 patients and Mycobacterium chelonei from 2. Haemoptysis, cough and weight loss were prominent in 6. Three had rheumatoid arthritis. Although two demonstrated cutaneous anergy, lymphocyte responsiveness to PHA was normal. PPD-F was not useful in skin testing or in the in vitro evaluation of lymphocyte function. Histologic examination of the lungs of 2 patients demonstrated caseating granulomata. One patient died of massive pulmonary haemorrhage soon after initiation of therapy. Multi-drug treatment regimens generally resulted in progressive sterilization of the sputum and improvement in the appearance of the chest x-ray. We conclude that some rapidly growing mycobacteria can cause potentially fatal cavitary lung disease and that intensive anti-tuberculosis therapy may successfully alter its course.

Studies on murine leprosy bacillus. X. Rapid growing mycobacteria isolated from experimental murine leprosy mice (author's transl)

In the course of primary cultivation, not only the supposed murine leprosy bacillus but also pigmented colonies of acid-fast bacilli were isolated from the tissues of mice in experimental murine leprosy. Of 612 specimens from the mice previously infected with the Hawaiian strain 11 yielded such colonies and 3 cultures were also obtained from among 306 specimens with the Keishicho strain.Tables 1 & 2 give the outlines of primary isolation of these cultures : source of materials, decontamination treatment, culture results, and their correlation on primary culture with the supposed murine leprosy bacilli. In these experiments the animals with the Hawaiian strain were killed during the period of 1 to 11 months after inoculation and the mice with the Keishicho strain were killed from 3 to 13 months after. These pigmented colonies, however, were obtained only from the mice of long standing, that is, from those which were sacrificed 6.5 months or more after inoculation. In the case of the mice with the Hawaiian strain, as indicated in Table 3, the tissues from which such colonies were isolated were the spleen, the liver and the lungs ; most often from the latter. With the Keishicho strain, however, they were isolated only from superficial lymph nodes and local site of the injection.All of the above isolates were found to belong to Runyon's Group IV organisms. Their colonies grown ogawa's egg and egg yolk media were uniformly S type and yellow-colored. Drug sensitivity test on 7 strains of them revealed that they were in general less susceptible to the anti-tuberculous drugs than the tubercle bacillus (Table 4). Virulence test on the same strains was made by inoculating mice intravenously with 0.1 mg amount of the bacilli. The animals were killed after a period of 1 to 6 months and evaluation was made according to gross involvement, spleen weight per gram of body weight, and the results of quantitative culture from the lungs and spleen. There was no gross evi-dence of disease, except that the spleen of a few animals at 1 month were found to be suggestive of being swollen. Table 5 shows that the bacilli of 4 strains disappeared from the tissues by 3 months after injection, while the ones of the other three survived there till 3 or 6 months later.

Numberical taxonomic analysis of some slowly growing mycobacteria using hypothetical median strain patterns.

SUMMARY: The reactions of 1498 isolates of slowly growing mycobacteria in 50 character states have been compared to those of 17 hypothetical median strain patterns. The establishment of intra- and intertaxon matching values for clusters so generated, allowed certain aggregates of taxa to be recognized for which a conventional n × n matching matrix might provide more clear cut taxonomic definition of species. Those species which are clearly distinct from the others include Mycobacterium tuberculosis, M. bovis, M. kansasii, M. flavescens, M. xenopi and M. gastri. Problems still exist regarding the precise separation of taxa within Runyon Groups II (M. scrofulaceum, M. gordonae and IIIB) and Group III (M. avium—M. intracellulare and M. terrae—M. nonchromogenicum—M. novum—M. triviale). These latter are discussed.