High-catalase strains of Mycobacterium kansasii isolated from water in Texas.

Isolation techniques with membrane-filtered potable water samples resulted in the isolation of potentially pathogenic high-catalase strains of Mycobacterium kansasii from 8 of 19 representative outlets in a small central Texas town. Mycobacterium gordonae was isolated from all samples, and Mycobacterium fortuitum was isolated from two samples. Data on chlorine levels are presented along with a possible explanation for the unusually high numbers of mycobacteria in these potable water samples. Findings suggest that water is a source of M. kansasii and may be an important link in the epidemiological picture of the disease.     

Niacin-positive Mycobacterium kansasii isolated from immunocompromised patients.

Niacin-positive Mycobacterium kansasii was isolated from three patients, two with respiratory infections and one with a perirectal abscess. The isolates were phenotypically similar to other strains of M. kansasii, differing only in their ability to produce niacin. This phenotype has been reported only twice in the literature, during the 1960s.     

Phenol-Soluble Antigens from Mycobacterium kansasii, Mycobacterium gastri, and Mycobacterium marinum

Many of the demonstrable antigens derived from mycobacteria are common to members of different species. Agglutination tests have yielded the most specific characterizations. An antigen which may be associated with the specific agglutination is soluble in phenol and can be extracted and separated from other antigens by use of this solvent. Phenol-soluble antigens have been extracted from representative cultures of Mycobacterium kansasii, M. gastri, and M. marinum. Most representatives of each of these species yielded an antigen which was characteristic of the species but was distinct from antigen derived from the other two species.     

Identification of Mycobacterium kansasii by DNA hybridization.

A DNA probe specific for Mycobacterium kansasii was obtained from a plasmid clone library of EcoRI-digested genomic DNA. The probe specifically identified culture-confirmed isolates of M. kansasii and isolates in cultures of environmental water samples. In an attempt to distinguish between isolates of M. kansasii, we used two methods to demonstrate restriction fragment length polymorphisms in the genomic DNA. Both of these methods failed to detect any differences between the isolates. These isolates included the type strain TMC 1201, environmental isolates, and clinical isolates from Australia and the Solomon Islands. This result suggests that the genome of M. kansasii is highly conserved and that genetic divergence within this species in insignificant.     

First report of isolation of Mycobacterium novocastrense from water supplies

We herein present the first documented report associated with the isolation of Mycobacterium novocastrense from environment. The identification and characterization of four unrelated isolates, one from the surface water and the other three from hospital water, were achieved by various conventional and molecular tests including a genus‐specific PCR for Mycobacterium based on 65‐kDa heat shock protein (hsp) gene and 16S rDNA sequencing. Our findings might shed further light on the natural habitat of this rare Mycobacterium.     

Genotypic characterization of five subspecies of Mycobacterium kansasii.

Different molecular typing methods including restriction fragment length polymorphism (RFLP) analysis with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PCR restriction analysis of the hsp-65 gene (PRA) were applied to clinical and water isolates of Mycobacterium kansasii. RFLP with the MPTR probe, PRA, PFGE, and AFLP analysis revealed five homogeneous clusters which appeared to be subspecies. RFLP with the MPTR probe and PRA gave patterns specific for each cluster, whereas PFGE and AFLP analysis gave polymorphic patterns. IS1652 was present in two of the five clusters and provided polymorphic patterns for one cluster only. The two IS1652-positive clusters were Accuprobe negative (Accuprobe test; Gen-Probe Inc.), and only two other clusters were Accuprobe positive. A PCR test based on the detection of a species-specific fragment (M. Yang, B.C. Ross, and B. Dwyer, J. Clin. Microbiol. 31:2769-2772, 1993) was positive for all M. kansasii strains. This PCR test is an accurate, rapid, and specific M. kansasii identification test. No subspecies was particularly more virulent, because all clusters contained clinical strains, from AIDS patients and non-AIDS patients, and environmental strains.     

Tentative evidence of AIDS-associated biotype of Mycobacterium kansasii.

Previous studies revealed heterogeneous behavior within the species Mycobacterium kansasii against commercially available DNA probes (Accuprobe M. kansasii culture identification test; Gen-Probe); several isolates, conventionally identified as M. kansasii, failed in fact to hybridize. Looking for a possible association with phenotypic features, we tested a fully characterized panel of 69 clinical isolates of M. kansasii (19 of which were Accuprobe negative) with a semiquantitative micromethod which tests for 19 enzymatic activities (Api Zym; BioMérieux). The strains were from 25 hospitals in 18 Italian towns; 20 isolates came from human immunodeficiency virus type 1-positive patients who fulfilled the Centers for Disease Control criteria for AIDS diagnosis. On the basis of the whole set of phenotypic traits, our strains clustered in two groups, allowing the differentiation of biotypes within the species. There was a perfect association between biotype 2 and hybridization failures with Accuprobe and a very significant association between this novel biotype 2 and AIDS status, which suggests that it differs in virulence.     

Molecular analysis of Mycobacterium kansasii isolates from the United States

We studied the population genetics of Mycobacterium kansasii isolates from the United States by PCR restriction enzyme analysis (PRA) of the 441-bp Telenti fragment of the hsp-65 gene and pulsed-field gel electrophoresis (PFGE) of genomic DNA with the restriction endonucleases AseI, DraI, and XbaI, and we compared the patterns to those previously reported from France and Japan. By PRA, 78 of 81 clinical isolates (96%) from the United States belonged to subspecies I. With PFGE, 28 AseI patterns, 32 DraI patterns, and 35 XbaI patterns were produced. PFGE showed marked clonality of the U.S. isolates, with differences between genotypes involving only one or two bands. Isolates within Texas showed lower pattern diversity than those from different states. With DraI, 31 of 71 isolates (44%) had the same common PFGE pattern, which matched the predominant pattern in France (pattern Ia), determined by Picardeau et al. (M. Picardeau, G. Prod'hom, L. Raskine, M. P. LePennec, and V. Vincent, J. Clin. Microbiol. 35:25-32, 1997), and in Japan (type M), determined by Iinuma et al. (Y. Iinuma, S. Ichiyama, Y. Hasegawa, K. Shimokata, S. Kawahara, and T. Matsushima, J. Clin. Microbiol. 35:596-599, 1997). With AseI, 42% of isolates produced a common pattern indistinguishable from the common pattern seen in French isolates (Ia) and with only one band difference from the common pattern (type M) in Japan. This study demonstrates that subspecies I is the predominant subspecies of M. kansasii among clinical isolates in the United States, as it is in Europe and Japan, and that genotype I is highly clonal worldwide, with the same major genotype responsible for human infection. The fact that a single clone of M. kansasii is responsible for most cases of human disease suggests that specific virulence factors may be associated with this specific genotype.     

Lysogeny associated with mucoid variation in Mycobacterium kansasii.

Ten of 200 strains of Mycobacterium kansasii were found to produce very mucoid growth on L?wenstein-Jensen medium. By electronmicroscopy these 10 strains were found to be lysogenic, whereas no phage was observed in cultures of 30 non-mucoid strains. The cultural and biochemical properties of the lysogenic strains are compared with those of non-lysogenic strains, and the morphology of the phages is described.     

Identification of a genetically distinct subspecies of Mycobacterium kansasii.

To assess the usefulness of a specific DNA probe for Mycobacterium kansasii, 105 isolates from Australia, Belgium, Japan, South Africa, and Switzerland were collected and analyzed. Twenty of these isolates were probe negative, of which 18 were from Belgium and Switzerland. Analysis of all isolates by Southern blot hybridization indicated a lack of variability among probe-positive isolates, while probe-negative isolates were clearly distinct and showed greater diversity. Sequence analysis of the 250 nucleotides at the 5' end of the 16S rRNA gene revealed that 19 of the 20 probe-negative isolates had a sequence different from that of M. kansasii. A total of five nucleotide differences were present in a cluster consisting of two nucleotide deletions and three nucleotide substitutions. These results suggest the existence of a genetic subspecies of M. kansasii.     

Assessment of morphology for rapid presumptive identification of Mycobacterium tuberculosis and Mycobacterium kansasii

Mycobacterium tuberculosis often exhibits serpentine cording when grown in liquid medium, whereas Mycobacterium kansasii can be larger and cross-barred. We assessed the use of these morphologic characteristics as a cost-effective method for rapid presumptive identification of isolates from BACTEC bottles. Without specific training, using the Kinyoun acid-fast stain, definitive cording was found in 237 of 373 specimens positive for M. tuberculosis (64%) and cross-barring was recognized within 63 of 76 (83%) of the specimens positive for M. kansasii, giving sensitivities specificities, positive predictive values, and negative predictive values of 63.5, 96, 92, and 79%, respectively, for M. tuberculosis and 83, 95, 59, and 98%, respectively, for M. kansasii. With training and experience, these results improved to 74.5, 98, 96, and 84% and 93, 98, 79, and 98%, respectively. The major improvements were in distinguishing the pseudocording, or loose aggregation of Mycobacterium avium complex from M. tuberculosis and the long beaded forms of Mycobacterium gordonae from M. kansasii. Mycobacterium asiaticum and Mycobacterium szulgai, which rarely occur, are genetically related to M. kansasii and morphologically difficult to distinguish. In defined circumstances, serpentine cording and cross-barring can be used for rapid presumptive identification of M. tuberculosis and M. kansasii, respectively, and as guides for initial probe selection to reduce costs.     

Immune responsiveness in mice heavily infected with Mycobacterium kansasii.

Growth of Mycobacterium kansasii TMC 1203 in B6D2 F1 hybrid mice was associated with increased splenic cellular proliferation, hyperplasia and the generation of non-specific antibacterial resistance. Both responses were dose dependent; the larger the inoculum, the more rapid and extensive the cellular response. However, such mice were still unable to reduce the mycobacterial load within the tissues, apparently because of their inherent resistance to inactivation by immunologically activated macrophages. On the other hand, mice infected with the non-persistent strain of M. kansasii 1214 exhibited only a transient increase in non-specific (anti-listeria) resistance which rapidly declined as the number of viable mycobacteria within the spleen fell below an arbitrary threshold level. Mice infected with either M. kansasii 1203 or 1214 could be immunized with sheep red blood cells (SRBCs), an unrelated T cell-dependent antigen. The humoral (PFC) response was not affected by the mycobacterial load within the spleen. However, the delayed footpad swelling reaction was severely depressed. The latter could be restored merely by increasing the size of the intravenous sensitizing inoculum 100-fold. The present study indicates that mice chronically infected with M. kansasii are not severely immunosuppressed (as had been inferred from earlier in vitro lymphoproliferation studies) but are fully capable of responding to appropriate in vivo stimuli.     

Infection with Mycobacterium kansasii and efficacy of vaccination against tuberculosis.

The purpose of the present study was to examine further the recent hypothesis that subcutaneous infection with Mycobacterium kansasii resulted in the generation of a cell-mediated hypersensitivity reaction ('Koch' reaction) which could, it was argued, subsequently interfere with the generation of acquired immunity following vaccination of the animal with BCG. The results of the present study were unable to confirm this hypothesis in that they show, firstly, that subcutaneous M. kansasii infection was associated with the development of substantial Arthus-like reactivity which masked the detection of any subsequent delayed response, and that furthermore, attempts to adoptively transfer this form of delayed reaction by means of passive transfer of cells were unsuccessful. Furthermore, the results show that, despite the presence of the M. kansasii infection, BCG-vaccinated animals were fully resistant to subsequent aerosol-delivered challenge with virulent M. tuberculosis.     

Systemic Mycobacterium kansasii infection and regulation of the alloantigenic response.

Specific-pathogen-free B6D2 F1 hybrid mice were infected intravenously with 10(7) to 10(8) viable Mycobacterium kansasii cells. The growth of the five test strains in vivo was correlated with the level of delayed hypersensitivity to a cytoplasmic protein antigen injected into the footpad. M. kansasii TMC no. 1201 and 1203 gave rise to persisting systemic infections with an early delayed hypersensitivity response (day 7) followed by a profound anergy to the cytoplasmic protein antigen injections. Strains 1204, 1214, and 1217 declined in viability relatively rapidly and failed to induce detectable levels of delayed hypersensitivity. Spleens harvested from mice infected 20 to 30 days earlier with 10(8) M. kansasii 1203 cells contained a T-cell subpopulation capable of suppressing mixed lymphocyte reactions between normal B6D2 and C3H(He) cells. On the other hand, splenic T-cells taken from M. kansasii 1214-infected mice enhanced, rather than suppressed, the indicator mixed lymphocyte reactions. The kinetics of stimulator-suppressor T-cell production within the spleens of the heavily infected mice differed as the two contrasting M. kansasii infections progressed. Such cellular interactions could well be responsible for the observed persistence of the systemic M. kansasii 1203 infection.     

Adoptive transfer of acquired resistance to Mycobacterium kansasii by T cells harvested from chronically-infected mice.

Growth of Mycobacterium kansasii in intravenously infected mice ceases when the spleen cells express an enhanced non-specific resistance to a secondary challenge. Mice inoculated with 10(6) CFU M. kanasii 1203 develop a population of splenic T cells which are able to transfer protection passively to sublethally-irradiated syngeneic recipients when challenged with M. kansasii. Although the T-cell activated macrophages were unable to eliminate the mycobacteria from the spleen, they were able to prevent further growth of the organism in vivo. When mice which lack T cells (congenitally athymic, or 'nude' mice) were infected with M. kansasii, the cellular defences were unable to halt the progressive growth of the challenge organisms within the tissues. When normal mice were inoculated with large numbers of viable M. kansasii 1203 (up to 5 X 10(7) CFU), the activated macrophages within the spleen were capable of limiting the further growth of the bacterial population in vivo, but with no T-cell response capable of adoptively immunizing naive recipients against a secondary M. kansasii challenge. Thus, it seems likely that M. kansasii can induce the formation of activated macrophages by two separate mechanisms: one is a T-cell dependent process which occurs in mice inoculated with moderate doses (10(6) CFU) of M. kansasii, while the other is T-cell independent and occurs when a large infectious inoculum is employed.     

Evaluation of a modified single-enzyme amplified-fragment length polymorphism technique for fingerprinting and differentiating of Mycobacterium kansasii type I isolates

The usefulness of single-enzyme amplified-fragment length polymorphism (AFLP) analysis for the subtyping of Mycobacterium kansasii type I isolates was evaluated. This simplified technique classified 253 type I strains into 12 distinct clusters. The discriminating power of this technique was high, and the technique easily distinguished between the epidemiologically unrelated control strains and our clinical isolates. Overall, the technique was relatively rapid and technically simple, yet it gave reproducible and discriminatory results. This technique provides a powerful typing tool which may be helpful in solving many questions concerning the reservoirs, pathogenicities, and modes of transmission of these isolates.     

Cloning and expression of the gene for the cross-reactive alpha antigen of Mycobacterium kansasii.

The gene for the extracellular alpha antigen of Mycobacterium kansasii was cloned by using the alpha-antigen gene fragments of M. bovis BCG as probes. Gene analysis revealed that this gene encodes 325 amino acid residues, including 40 amino acids for the signal peptide, followed by 285 amino acids for the mature protein. A comparison of the nucleotide sequences of the genes isolated from these two mycobacterial species showed that the levels of DNA and amino acid homology were 84.8 and 89.1%, respectively. The hydropathy profiles were also compared, and two highly changed hydrophilic regions were observed, which might account for the antigenic diversity of this antigen or its acquirement of antigenic specificity.     

Microscopic morphology in smears prepared from MGIT broth medium for rapid presumptive identification of Mycobacterium tuberculosis complex,Mycobacterium avium complex and Mycobacterium kansasii

Mycobacterium species has a specific morphology when grown in liquid medium. Mycobacterium tuberculosis complex (MTB) often exhibits serpentine cording, which is different from the dot and cross-barring morphology observed in Mycobacterium avium complex (MAC) and Mycobacterium kansasii (MK), respectively. These characteristic morphologies can be used as a cost-effective method for rapid, presumptive identification of mycobacterial isolates cultured from the MGIT 960 system. By using Kinyoun acid-fast stain, serpentine cording was found in 840 of 904 (92.1%) samples positive for MTB; dot or loose aggregation was observed in 112 of 136 (82.3%) samples positive for MAC; and the cross-barring, ladder-like, morphology was observed in 45 of 56 (80.5%) samples positive for MK. The sensitivity and specificity were 92.9% and 96.4% for MTB; 82.4% and 94.5% for MAC; and 80.4% and 94.6% for MK, respectively. Using growth rate selection to exclude rapid growers, the positive and negative predictive values were 98% and 87.6% for MTB; 78.3% and 98% for MAC; and 78.9% and 99.1% for MK, respectively. Twenty-eight (93.3%) of 30 strains with ball morphology were rapid growers. Microscopic morphology can be used for rapid, presumptive identification of M. tuberculosis complex, M. kansasii, and M. avium complex and act as a guide for appropriate selection of initial probes to reduce costs.     

Use of Gen-Probe AccuProbes to identify Mycobacterium avium complex, Mycobacterium tuberculosis complex, Mycobacterium kansasii, and Mycobacterium gordonae directly from BACTEC TB broth cultures.

To evaluate the utility of Gen-Probe AccuProbes for the identification of mycobacteria directly from BACTEC TB 12B vials containing acid-fast bacilli, culture results for 11,375 clinical specimens other than blood received from 1 January 1992 to 30 September 1993 were reviewed retrospectively. During this period, a total of 359 of 11,375 BACTEC vials were positive for acid-fast bacilli and were evaluated for mycobacteria with one or more probes: 224 were probed for Mycobacterium tuberculosis complex, 253 were probed for Mycobacterium avium complex, 64 were probed for Mycobacterium kansasii, and 77 were probed for Mycobacterium gordonae. After initial testing with the probes, 75 vials were positive for M. tuberculosis complex, 99 were positive for M. avium complex, 11 were positive for M. kansasii, and 55 were positive for M. gordonae. Repeat testing of vials that were initially probe negative or testing of colonies from subcultures of these vials identified an additional 11 M. tuberculosis, 27 M. avium complex, 1 M. kansasii, and 9 M. gordonae that were not detected on initial screening. On the basis of these data, the percentage of organisms identified directly from the BACTEC TB 12B vials upon initial screening with each of the four AccuProbes was 87.2% for M. tuberculosis complex, 78.6% for M. avium complex, 91.7% for M. kansasii, and 85.9% for M. gordonae.     

Modified culture technique for Corynebacterium diphtheriae isolation from desiccated swabs.

Corynebacterium diphtheriae was isolated from pyoderma and ulcerative skin lesions with a modified delayed culture procedure as late as 9 weeks after field collection of silica gel-desiccated swabs. Biotypes gravis and mitis were identified. Most isolates were nontoxigenic. Todd-Hewitt broth enrichment enhanced recovery of C. diphtheriae by 70%.