Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Direct stimulation of superoxide output in neutrophils by lipid A of Escherichia coli.

Two different results have been published in regard to the superoxide-stimulating activity of lipopolysaccharide or Lipid A in neutrophils: first, a direct stimulation after a lag time of about 30-60 sec and second, the inactivity of Lipid A if applied alone, being able only to "prime" the cells for a second challenge during a longer incubation period. In order to achieve clarity regarding these two different opinions, we asked the questions whether: (a) Lipid A is able to stimulate PMN directly, i.e. without a preincubation and a second stimulus; (b) fMLP and Lipid A show a synergistic effect; (c) a preincubation ("priming") of the PMN with Lipid A really increases the superoxide output after a second challenge. We observed (a) a direct stimulation of the chemiluminescence with Lipid A without an additional second challenge, accompanied by a seemingly unimodal kinetics of the superoxide output, i.e. mainly the second phase of the usually bimodal kinetics has been stimulated. As for question (b), a clearly detectable synergism between Lipid A and fMLP could be measured. Regarding question (c), a preincubation ("priming") with Lipid A was of no beneficial effect; the chemiluminescence count could be equally well increased without a "priming" compound.

Enhancement by cimetidine of chemotactic peptide-stimulated ATP release and chemiluminescence in human neutrophils.

As determined by luciferase-luciferin, we recently found that the H2-blocker CIM considerably increased the ATP release from fMLP-stimulated PMN. This observation correlates well with our previous [1] regarding the enhancement of superoxide output (chemiluminescence) in in human neutrophils. by CIM plus fMLP. In order to compare the ATP release from PMN of different donors, a standard procedure has been developed consisting of the determination of the ATP present initially in the cell suspension (without stimulation), ATP release after stimulation with fMLP, and ATP release in the presence of CIM plus fMLP. The whole ATP content per neutrophil was determined after ultrasonication of the cells as well. The mean value of the initially present ATP was 0.45 x 10(-17) mol/cell in the suspension. Stimulation with fMLP plus CIM yielded within 5-10 minutes considerably higher ATP amounts than fMLP alone. The corresponding and statistically significantly different mean values were 2.46 x 10(-17) mol/PMN (s.d. = 1.047) and 1.38 x 10(-17) mol/PMN (s.d. = 0.55), respectively. The whole ATP per neutrophil was found to be 1.22 x 10(-15) mol (mean; s.d. = 0.60) and thus, the stimulation with CIM plus fMLP released about 2.0 per cent, with fMLP alone about 1.0 per cent of the whole ATP. CIM without fMLP did not enhanced the ATP release during the reaction time applied. On the other hand, fMLP-stimulated, lucigenin-amplified chemiluminescence determinations were carried out in the presence of CIM as well; contrarily to our previous method, CIM was dissolved in PBS without DMSO, because DMSO inhibited the chemiluminescence slightly.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of cimetidine and histamine with superoxide generated in a cell-free system and in neutrophils.

We investigated the influence of CIM and HIS on the SUP and CH in the SUP generating cell-free xanthine oxidase system and in human neutrophils. As measured by CCR or CH, we found that HIS inhibited SUP output in both systems. The HIS concentration required for a measurable inhibition was somewhat higher in the xanthine oxidase system than in neutrophils. CIM increased CH in neutrophils significantly. A similar effect of CIM was not observed in the xanthine oxidase system. CIM antagonized in neutrophils the effect of HIS. However, a five or ten times higher molar concentration of CIM than of HIS was necessary to produce this effect. The antagonistic effect of CIM was not found in the xanthine oxidase system within the concentrations applied. In addition to an interaction of HIS with neutrophils on the cellular level, we suggest a superoxidase scavenging effect caused by HIS. At the present no suggestions can be made regarding the observed SUP output stimulating activity of CIM.

Histamine (HIS) degradation in microorganisms: imidazole ring splitting and formation of imidazolyl ethanol (IMET) and imidazolyl acetic acid (IMAC) in mycobacteria.

Mycobacterium smegmatis SN 46, isolated from a fatal human oesophagus infection, is able to degrade HIS by imidazole ring splitting. In this respect, SN 46 is unique under the bacteria strains tested. Some other mycobacteria (M. diernhoferi, M. fortuitum, M. chelonei etc.) oxidize HIS to IMET and IMAC without further change. Cell-free extracts of SN 46 transform HIS to IMET and IMAC, while ring splitting enzymes are not detectable. IMAC and IMET were identified by combined gas-liquid chromatography and mass-spectrometry. At present, the possibility of HIS degradation by SN 46 via imidazolyl acetaldehyde (according to the pathway in vertebrates) can not be excluded.

[Nicotinamidase and the so-called pyrazinamidase in mycobacteria; the simultaneous occurrence of both activites (author's transl)]. / Nikotinamidase und die sogenannte Pyrazinamidase in Mycobakterien; gekoppeltes Auftreten der beiden Enzymaktivitäten.

Nicotin- and the so-called pyrazinamidase (in the following: "pyrazinamidase") have been found in strains of four mycobacteria species, M. fortuitum, M. gastri, M. bovis and M. microti. These findings are in contradiction to those summarized in Bergey's Manual of Determinative Bacteriology (1974). The reason for the discrepancies is that the original method (Bönicke, 1961) for amidase determination has not taken the following aspects into consideration: a) The inducibility of the nicotin- and "pyrazinamidase" (example: M. fortuitum); b) The temperature sensitivity of these enzymes (M. gastri); c) The light sensitivity of nicotinamidase (in photochromogenic M. gastri strains); d) The optimal substrate concentration which must be at least 4 mM instead of 0,8 mm. The following consequences can be drawn for the taxonomy and biochemistry of the tested organisms: e) The species status of M. gastri should be annuled. The main difference between M. gastri and M. kansasii consists only of the non-agglutinability of M. gastri by anti-M. kansasii serum. "Pyrazinamidase" and also nitrate reductase (Tarnok et al., in press) are positive in strains of both species; f) M. bovis possesses nicotin- and "pyrazinamidase" as M. tuberculosis too. Thus, these two species are more closely related than suggested earlier; g) Till now, no Mycobacterium has been found showing nicotinamidase without "pyrazinamidase" activity (or vice versa). It seems to be very probable that nicotinamidase, an enzyme of low substrate specificity, is able to hydrolyze several compounds with a nicotinamide-like structure such as pyrazinamide. Thus, we suggest the annulment of the term pyrazinamidase or the employment of quotation marks ("pyrazinamidase") to show the fictitious value of this designation.

Biochemical background of some enzymatic tests used for the differentiation of mycobacteria.

The break-down of benzamide, acetamide, malonamide and allantoin in M. smegmatis was investigated. It has been stated that the uptake of liberated NH3 into the cells, favoured by the presence of an organic acid, occasionally results in a negative NH3 determination. This difficulty can be overcome by an increase of the substrate concentration from 0.8 up to 4 mM. All antoinase activity in mycobacteria can be demonstrated only by an NH3 determination, when all the enzymes necessary for the complete break-down of allantoin are present. Bacteria containing allantoinase but not urease will be negative in this test. Using high amide concentrations (4 mM) some doubtful results concerning the degradation of acetamide, benzamide, nicotinamide and pyrazinamide can be eliminated as could be demonstrated for different strains of mycobacteria.

[Studies of Nocardia pellegrino SN 5108 pigment mutants: reasons for differences in pigmentation (author's transl)]. / Untersuchungen über Pigmentmutanten von Nocardia pellegrino SN 5108; Ursache der verschiedenartigen Pigmentierung

Yellow and white mutants of the strains Nocardia pellegrino SN 5108 R have been isolated. Regarding their morphological and physiological properties, the mutants are identical with the wild type bacteria with the exception of their pigmentation and lipid composition. However, the pigment composition (number, Rf-values and spectra of the pigment components) of the yellow mutant is identical with that of the wild type; as a consequence, the modified pigmentation of the yellow mutant cannot be explained by an altered pigment synthesis. The wild type cells and the mutant SN 5108 G contain three main pigment components designated as I, II and III. Components II and III posses a marked indicator character and show a bathochromic shift in solutions of pH 12 or higher. Components II and III contain functional groups which are able to react with acetic acid yielding acetylated products; after acetylating, no bathochromic shift in alkali occurs. Intact cells of the wild type retain their orange-red pigmentation in buffer solution with a pH-value of 12 or higher. Cells of the yellow mutant, however, change the yellow color immediately after the alkali treatment to orange-red; this new color is identical with that of the wild type and can be changed to yellow by placing the cells into 1 N HCl. Regarding these facts it seems to be very probable that the functional groups of the pigment components II and III are differently bound in the wild type and mutant cells. In the mutant, they are accessible to OH- ions yielding a bathochromic shift while in the wild type cells, OH- ions are unable to provoke this shift. It seems to be also probable that different lipids in the two strains are responsible for the binding of the pigments. So far known, this is the first observation about the occurence of pigment mutants with an altered pigment binding site in the cells.