Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.     

The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR.

The superoxide dismutase gene has been identified as a target in screening for the presence of mycobacteria on the genus level and differentiating relevant mycobacterial species from one another by PCR. Consensus primers deduced from known superoxide dismutase gene sequences allowed the amplification of DNAs from a variety of bacteria, fungi, and protozoa. Selected amplicons from Actinomyces viscosus, Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriticum, Mycobacterium avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, M. tuberculosis, M. xenopi, and Nocardia asteroides were subsequently cloned and sequenced. The alignment of those sequences facilitated the selection of primers targeting conserved regions present in myobacterial species but absent in nonmycobacterial species and thus allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific probes recognized 23 of the 28 mycobacterial species and did not cross-react with any of the 96 nonmycobacterial species tested. In addition, probes recognizing species-specific variable regions within the superoxide dismutase genes of M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, the M. tuberculosis complex, and M. xenopi were identified. All probes recognized only the species from which they were derived and did not cross-react with any other mycobacterial species or with any of the nonmycobacterial species tested. We conclude that the superoxide dismutase gene is a suitable target for amplifying mycobacteria by PCR on the genus level, confirming correct amplification by genus-specific probes, and differentiating relevant species from one another by a set of species-specific probes.     

Evolutionary bottlenecks in the agents of tuberculosis, leprosy, and paratuberculosis.

Parasitic mycobacteria cause important human and animal diseases including tuberculosis, leprosy, and paratuberculosis. Several methods demonstrate a high degree of sequence conservation in three parasitic mycobacterial species (Mycobacterium tuberculosis, M. leprae, and M. avium subspecies paratuberculosis). Each of these species has completely conserved deoxyribonucleic acid (DNA) sequence in an internal transcribed spacer. In contrast, several species of environmental mycobacteria (M. intracellulare, M. kansasii, M. gordonae, and M. scrofulaceum) have substantial strain-to-strain variation in this region. These data suggest that each of the parasitic species has gone through a recent evolutionary bottleneck. Comparisons of tandem-repeat DNA from ancient and modern mycobacterial strains may allow this hypothesis to be tested directly.     

Genetic diversity among Mycobacterium avium complex AccuProbe-positive isolates.

The partial 32-kDa-protein gene sequences of 22 Mycobacterium avium complex (MAC) clinical isolates that were positive by the AccuProbe MAC probe only (not by the M. avium or M. intracellulare probe) were determined. The obtained nucleotide sequences were compared with the published sequences for M. tuberculosis, M. avium, and M. intracellulare by a sequence analysis program. There was a wide range of genetic diversity among the strains studied. Most of them (16 of 22) had sequences similar but not identical to those of M. avium and M. intracellulare. These strains were considered to be true MAC strains. Five strains had sequences in the category of the novel MAIX sequence, which was very different from the sequences of other mycobacteria analyzed thus far. In addition to these strains, one isolate had a sequence that differed greatly from the reference sequences. These results support previous findings showing that the MAC probably contains several additional species. Our results also suggest that the MAC AccuProbe may react with strains that do not belong to the MAC.     

Identification of mycobacteria by PCR-based sequence determination of the 32-kilodalton protein gene.

In this study, a part of the nucleotide sequence of the mycobacterial 32-kDa protein gene was determined by PCR-based sequencing. A total of 24 mycobacterial strains, representing 10 species, were studied. Sequences of all tested members of the Mycobacterium tuberculosis complex were identical to each other and to the previously published sequence of M. tuberculosis H37Rv. The sequences of M. avium and M. intracellulare were different from each other. MAIX strains, identified with the Gen-Probe MAIX test, had sequences identical to each other but clearly different from those of M. avium and M. intracellulare. Each of the other mycobacterial species investigated, i.e., M. kansasii, M. gastri, M. gordonae, and M. malmoense, had a unique species-specific sequence. These results demonstrate that there is variation in the nucleotide sequence of the 32-kDa protein gene among different mycobacterial species. Thus, we propose that this gene can be used for PCR-based identification of mycobacteria.     

Identification and differentiation of Mycobacterium avium and M. intracellulare by PCR.

Known DNA sequences coding for the 16S rRNAs of 14 slowly growing Mycobacterium species were analyzed. Three sets of primers were synthesized: MAV and MIN, for M. avium and M. intracellulare, respectively, and MYCOB, for the slowly growing mycobacteria. Whole-cell DNAs of 14 reference species were extracted and amplified by PCR with the MYCOB, MAV, and MIN primers. The MYCOB primer amplified a 0.9-kb segment from the DNAs of all 14 species. The MAV and MIN primers each amplified one highly specific 1.3-kb segment from the homologous DNA, respectively. DNAs from each of 10 clinical isolates of M. avium and M. intracellulare identified by conventional methods were amplified with the MYCOB as well as the MAV and MIN primers; 9 of 10 isolates of each species were identified with their respective primers. One isolate of M. intracellulare was subsequently found to have been mislabeled. One isolate designated M. avium reacted only with the MYCOB primer. The hypervariable region of this strain was shown by DNA sequence analysis to be distinct from all known 16S rRNA sequences of Mycobacterium spp. Our data indicate that the currently identified M. avium-M. intracellulare complex includes strains genetically diverse from M. avium and M. intracellulare.     

Evaluation of the LiPA MYCOBACTERIA assay for identification of mycobacterial species from BACTEC 12B bottles

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacterium species and differentiates M. tuberculosis complex, M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonae group, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive for M. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive for Mycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.     

Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.

Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. kansasii, M. marinum, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonei, M. haemophilum, and M. paratuberculosis). On the basis of the dnaJ gene sequences, we developed dot blot hybridization analysis with species-specific oligonucleotide probes for the M. tuberculosis complex. M. avium, M. intracellulare, and M. kansaii, allowing a rapid identification of these species following polymerase chain reaction for the dnaJ gene. We conclude that polymerase chain reaction with the genus-specific primer that amplifies the dnaJ genes and subsequent dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and nontuberculous mycobacterial infections.     

Sequence analysis and amplification by polymerase chain reaction of a cloned DNA fragment for identification of Mycobacterium tuberculosis.

Analysis of the 1,016-base-pair sequence of a putative probe for identification of Mycobacterium tuberculosis revealed two almost identical fragments of 507 and 509 bases. From this sequence two pairs of primers were synthesized (MtbAB and MtbCD), ranging from 18 to 22 nucleotides, for use in polymerase chain reactions (PCRs) with DNA from six reference strains of M. tuberculosis, as well as type strains of M. bovis, M. bovis BCG, M. kansasii, M. avium, M. intracellulare, and M. scrofulaceum. Although there was amplification of DNA from all mycobacterial strains included in the study, when used as probes, a predominant band, fragment CD from M. tuberculosis H37Rv DNA, proved to be more specific for strains of M. tuberculosis than the original probe, pMTb4, was. Amplified fragments from as little as 1 fg of DNA (equivalent to one-fifth of an organism) could be resolved on ethidium bromide-stained gels loaded with a 1/10 volume of PCR. Furthermore, it was possible to amplify specific DNA sequences from frozen M. tuberculosis H37Rv organisms which were thawed prior to PCR.     

A simple and rapid method for the detection and identification of mycobacteria using mycobactin.

A system was developed for the identification of mycobacteria such as Mycobacterium tuberculosis and M. avium, by thin layer chromatography of 55Fe-labelled mycobactin. Approximately 2 x 10(3) mycobacteria were detected within 24 h and little operator time or skill was required. M. avium, M. intracellulare and M. scrofulaceum were found to have lower requirements for iron than other mycobacteria and this may influence their growth in host organisms.     

DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory.

A method incorporating DNA amplification and reverse dot blot hybridization for the detection and identification of mycobacteria to the species level is described. The amplification procedure allowed for the incorporation of digoxigenin-labeled UTP, which was detected by chemiluminescence, removing the need for radioactivity. Using a set of primers and probes from the gene for the 65-kDa heat shock protein of mycobacteria, previously reported in the literature, the reverse dot blot method correctly identified 12 of the 12 M. tuberculosis isolates and 45 of the 50 M. avium complex isolates. Two of the nonhybridizing M. avium complex isolates were reidentified as M. xenopi. The other three nonhybridizing M. avium complex isolates, which were identified as M. intracellulare, hybridized with the probe for M. tuberculosis, as did two ATCC strains of M. intracellulare. The amplified DNA of M. intracellulare was sequenced, and the sequence was compared with the sequence from M. tuberculosis. The sequence for M. avium differed from M. tuberculosis by 5 of 20 bases. The sequence for M. intracellulare differed from M. tuberculosis by 2 of 20 bases, but this difference did not result in sufficient thermal instability to affect hybridization. The use of chemiluminescence allowed as few as 10(2) CFU to be detected. The format of the assay is readily applicable for implementation in the clinical laboratory.     

Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.     

Use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients.

The presence of mycobacteria in blood culture fluids (BACTEC) of AIDS patients with positive growth indices (GIs, > 20 U) was investigated by using a multiplex PCR to detect and identify members of the genus Mycobacterium, M. avium, M. intracellulare, and M. tuberculosis. Three different methods of extracting mycobacterial DNA from blood culture fluid were compared for use with the multiplex PCR. Mycobacterial cells were pelleted from a small aliquot of blood culture fluid by centrifugation, and the DNA was extracted from cells by heat lysis or a sodium iodide-isopropanol or a phenol-chloroform method. DNAs of different sizes were amplified from a region of the MPB70 gene of M. tuberculosis (372 bp) and from a region of the 16S rRNA gene of members of the genus Mycobacterium (1,030 bp), M. intracellulare (850 bp), or M. avium (180 bp) as a multiplex PCR in a single tube. The amplified DNA products were detected by agarose gel electrophoresis and ethidium bromide staining in all 41 (100%) positive cultures after sodium iodide-isopropanol extraction, in 18 (44%) after heat lysis, and in 5 (12%) after phenol-chloroform extraction. Of the 41 positive cultures, 38 were identified as M. avium and 2 were identified as M. intracellulare by both routine methods and multiplex PCR. The remaining mycobacterium was identified as M. intracellulare by routine methods and as M. avium by the multiplex PCR. Another six blood cultures that were negative for the presence of acid-fast bacilli after Ziehl-Neelson staining were also negative by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of Mycobacterium tuberculosis and Mycobacterium avium by non-radioactive probes: evaluation of the Snap Syngene system]

We evaluated an alkaline phosphatase-labeled oligonucleotide probe for the rapid identification of Mycobacterium tuberculosis and mycobacteria belonging to the M avium and M intracellulare complex (MAIS). Sixty-two strains of mycobacteria and eight strains belonging to related genera were studied. All M tuberculosis strains hybridized with the tuberculosis probe. All M avium and M intracellulare gave a strong signal with their probes. However the 3 M xenopi strains tested hybridized with all probes for MAIS complex.     

Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n = 18) or M. intracellulare (n = 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.     

Cross-Reactive Immune Responses Against Mycobacterium bovis BCG in Mice Infected with Non-Tuberculous Mycobacteria Belonging to the MAIS-Group

Two bacillus Calmette–Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare , M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum . Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-γ production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum -infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis .     

Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare.

The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR-amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex.     

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples.

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

Comparative evaluation of PCR and commercial DNA probes for detection and identification to species level of Mycobacterium avium and Mycobacterium intracellulare.

Selective amplification of a 187-bp fragment within the DT6 sequence using the AV6 and AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of Mycobacterium intracellulare using the IN38 and IN41 primers was performed for 69 clinical isolates identified as M. avium complex by conventional methods. The results were compared in parallel with results with commercial M. avium and M. intracellulare probes. A positive response to either of the two PCRs or M. avium-M. intracellulare AccuProbes constituted positive detection as M. avium complex; this cumulative detection limit was 94.2% for PCR, compared with 90% for AccuProbe. Concordance, on the other hand, was considered an identical species identification using either DT1 PCR and the M. intracellulare probe or DT6 and DT1 PCRs are inexpensive and at least equally sensitive, in-house options to the AccuProbe system for species identification of M. avium and M. intracellulare.     

Superoxide dismutase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.

Superoxide dismutase (EC 1.15.1.1) (SOD) activity has been detected in crude cell extracts of representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group. Polyacrylamide gel electrophoresis demonstrated a single SOD activity band for each of the MAIS strains, though there were differences in mobility. All M. avium and M. intracellulare and two of five M. scrofulaceum strains demonstrated a single activity band of identical mobility (Rf = 0.83), while the SOD activity band for the three remaining M. scrofulaceum strains migrated farther (Rf = 0.85). The differences in mobility correlated with differences in sensitivity to NaN3 and H2O2. The SOD activities of the majority of the MAIS strains which displayed the slower-migrating activity band were inhibited 22 to 81% after 15 min of exposure to 5 mM H2O2, suggesting that both iron and manganese may be present in a single enzyme. The SOD activities of the three M. scrofulaceum strains which had the faster-migrating activity band were inhibited 100% after only 5 min of exposure to 5 mM H2O2 and exhibited greater sensitivity to 5 and 10 mM NaN3, characteristics of an iron-containing SOD. A concentration of 1 mM KCN did not cause inhibition of enzyme activity in any of the MAIS strains tested. Extracellular SOD activity was detected in four of six MAIS strains and was shown to be identical in mobility to the SOD activity of the crude extracts.