Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques.

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

Corynebacterium sputi sp. nov., isolated from the sputum of a patient with pneumonia.

A coryneform bacterium isolated from the sputum of a patient with pneumonia was characterized by phenotypic and molecular taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short chain mycolic acids consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing studies confirmed this assignment, with the organism forming a hitherto unknown subline within the genus associated with a subcluster containing Corynebacterium hansenii, Corynebacterium freneyi, Corynebacterium xerosis, Corynebacterium amycolatum and Corynebacterium sphenisci. Sequence divergence values of >2.7 % from established corynebacterial species suggested that the new isolate represented a novel species. This was also supported by the results of the biochemical tests. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown bacterium be classified as a novel species of the genus Corynebacterium, Corynebacterium sputi sp. nov. (type strain IMMIB L-999(T)=DSM 45148(T)=CCUG 55795(T)).

Corynebacterium hansenii sp. nov., an alpha-glucosidase-negative bacterium related to Corynebacterium xerosis.

A novel strain, C-138(T), belonging to the genus Corynebacterium was isolated from a severe thigh liposarcoma infection and its differentiation from Corynebacterium xerosis and Corynebacterium freneyi is described. Analysis of 16S rRNA gene sequences, rpoB sequences and the PCR profile of the 16S-23S spacer regions was not conclusive enough to differentiate strain C-138(T) from C. xerosis and C. freneyi. However, according to DNA-DNA hybridization data, strain C-138(T) constitutes a member of a distinct novel species. It can be differentiated from strains of C. xerosis and C. freneyi by colony morphology, the absence of alpha-glucosidase and some biochemical characteristics such as glucose fermentation at 42 degrees C and carbon assimilation substrates. The name Corynebacterium hansenii sp. nov. is proposed for this novel species; the type strain is C-138(T) (=CIP 108444(T)=CCUG 53252(T)).

Lipid composition of leprosy-derived corynebacteria, a distinct group of corynebacteria, and of a reference Corynebacterium.

Leprosy-derived corynebacteria (LDC) are diphtheroid organisms isolated from leprosy patients and previously characterized by DNA and cell wall analysis. Three groups of LDC components of taxonomic value, glycolipids, and phospholipids and cell-wall-bound lipids were analyzed in comparison with those of a reference strain C. hoffmannii (CH). The main CH glycolipid, "cord factor" (trehalose dimycolate), was missing from LDC. Among phospholipids, phosphatidylinositol and phosphatidylglycerol had lowered proportions in LDC, as compared to CH, whereas phosphatidylethanolamine and cardiolipin were absent from both microorganisms. Bound lipids in acidic extracts of delipidated LDC yielded arabinose corynomycolate in lesser quantity with respect to CH. Alkaline hydrolysis of whole cells released fatty acids and mycolic acids, which were analyzed by gas chromatography/mass spectrometry. Reference CH, grown in the absence of serum, yielded C16:0 and C18:1 (major) and C18:0 (minor) fatty acids, as well as C32, C34, and C36 corynomycolic acids. All these components, particularly mycolates, had lowered proportions when this organism was grown in the presence of serum. Dominant LDC components were, in addition to C16:0, C18:0, and CI8:u fatty acids, cholesterol from serum. Very low concentrations of corynomycolic acids with a high degree of unsaturation were found in these organisms, suggesting a dependence of lipid metabolism on growth conditions. The presence in LDC of tuberculostearic acid (C19r:0), a mycobacterial component found in some pathogenic corynebacteria, was carefully explored: Traces of C19r:0 were found in LDC 19 grown in the presence of delipidated serum, but not in LDC 15 nor in C. hoffmannii. Present data, in conjunction with previous studies on DNA and mycolic acids, disclose basic differences in the composition of LDC and conventional corynebacteria.

Size and homology of the genomes of leprosy-derived corynebacteria, Mycobacterium leprae, and other corynebacteria and mycobacteria.

The genomes of Mycobacterium leprae and leprosy-derived corynebacteria (LDC), which have a similar base composition of guanine + cytosine 56 mol %, have been compared with those of reference bacteria of the CMN group (genera Corynebacterium, Mycobacterium, Nocardia). Genome sizes of three LDC strains were (1.2-2.5) x 10(6) base pairs. DNA from four of seven LDC strains examined had homology levels greater than 60%. Two other strains had a homology of 40% when compared with the CMN strains and one strain was distinctly different. The DNA from all seven LDC strains gave 0.3-18% hybridisation with that of M. leprae, 5-16% with reference corynebacteria, 5-12% with M. bovis, and 2-8% with Nocardia caviae. The small size of the LDC genome and its unrelatedness to those of M. leprae and organisms of the CMN group shows the uniqueness of LDC.

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.

Description of Corynebacterium tuberculostearicum sp. nov., a leprosy-derived Corynebacterium.

Leprosy-derived corynebacteria (LDC) have been extensively studied over the past decade. A composite of their biological properties (cell morphology, staining reactions, cellular inclusions and guanine-plus-cytosine content of their deoxyribonucleic acid; 16 strains studied) and their chemical structures (peptidoglycan type, major cell wall polysaccharide, major glycolipid as well as characteristic mycolic acids) appears to define them as members of the genus Corynebacterium. In relation to other corynebacteria found in humans, including "JK corynebacteria", they seem to be distinct. They are here named Corynebacterium tuberculostearicum sp. nov. because they produce a 10-methyloctadecanoic (tuberculostearic) acid (8 strains studied). This and some of their other attributes are considered in relation to properties of leprosy bacilli and Mycobacterium leprae.

Chemical identification of some cell-wall components of microorganisms isolated from human leprosy lesions.

The cell walls of 24 coryneform non-acid-fast, Gram-positive organisms isolated from human leprosy lesion, were hydrolysed and analysed. Four known chemical markers of different high polymer components of the walls of microorganisms of the CMN (Corynebacterium, Mycobacterium, Nocardia) group were detected in whole cells and cell wall hydrolysates of the coryneform bacteria analyzed. These markers were: meso-diaminopimelic acid (peptidoglycan), arabinose and galactose (arabinogalactan), and mycolic acids. In addition, mycolic acids proved to be of the corynomycolic type, as shown by thin layer chromatography analysis. The conclusion was drawn that these coryneform strains independently isolated from patients of different countries, represent a homogeneous group within the genus Corynebacterium. This inference is supported by a parallel work showing that the guanine-plus-cytosine content of the DNA of these coryneform strains falls within the range of values characteristic of true corynebacteria pathogenic for animals.

Immunological relatedness of ribosomes from mycobacteria, nocardiae and corynebacteria, and microorganisms in leprosy lesions.

Serological relatedness of ribosomes from microorganisms of the Mycobacterium, Nocardia, and Corynebacterium genera has been analyzed by the microplate immunodiffusion technique. Mycobacterium and Nocardia proved homogeneous and closely related taxa, whereas Corynebacterium was found to be a heterogeneous phylum connected by remote links to the others. The taxonomic position of "diphtheroid microorganisms" (non-acid-fast, gram-positive bacteria morphologically similar to corynebactria), which were found together with Mycobacterium leprae in human leprosy lesions, was also investigated. Ribosomes of diphtheroid bacteria strongly cross-reacted with antisera against several mycobacteria and nocardiae but not against corynebacteria. Moreover, ribosomes from independently isolated diphtheroid strains proved serologically related and yielded strong cross-reactions with antisera against M. leprae as well as with sera from leprosy patients. Hence, diphtheroid microorganisms represent a homogeneous group immunologically related to mycobacteria in general and more specifically to M. leprae.

Chemical characterization of organisms isolated from leprosy patients.

CHEMICAL ANALYSES OF THE CELL WALLS OF ORGANISMS ISOLATED IN VARIOUS PARTS OF THE WORLD FROM CASES OF LEPROMATOUS AND TUBERCULOID LEPROSY MAKE POSSIBLE THEIR ASSIGNMENT TO ONE OF THE THREE GENERA: Corynebacterium, Mycobacterium, or Propionibacterium. One, bacterium 22M, remains unassigned. The combined chemical and enzymatic properties attributed to leprosy bacilli freshly harvested from lepromata are found collectively, but not individually, in these three genera.