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Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis) and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium.

A search has been made for corynebacterial phospholipase D, "ovis toxin," a sphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among a wide variety of corynebacteria. Phospholipase D activity has been found in strains exhibiting the biochemical properties characteristic of Corynebacterium pseudotuberculosis or of Corynebacterium ulcerans and in no other species of Corynebacterium. Methods for the assay of phospholipase D as a sphingomyelinase and methods for screening for phospholipase D in the presence of Corynebacterium equi on washed sheep blood agar are discussed.

Corynebacterium↗

Identification of some charcoal-black-pigmented CDC fermentative coryneform group 4 isolates as Rothia dentocariosa and some as Corynebacterium aurimucosum: proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum.

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum.

Centers for Disease Control and Prevention, U.S.↗

Corynebacterium ulcerans and Corynebacterium pseudotuberculosis responses to DNA probes derived from corynephage beta and Corynebacterium diphtheriae.

Strains of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (Corynebacterium ovis) were examined for the production of diphtheria toxin. A majority of C. ulcerans strains (25 of 37) and 1 C. pseudotuberculosis strain (1 of 14) gave a positive Elek test for diphtheria toxin, and for all strains but 1, production of diphtheria toxin was inhibited at the same level of Fe2+ as was the Corynebacterium diphtheriae control. All Elek-positive cultures as well as two Elek-negative isolates of C. ulcerans gave a positive signal when hybridized with a DNA probe unambiguous for the diphtheria toxin gene (tox) under conditions of high stringency. The majority of probe-positive C. ulcerans strains contained three or more DNA restriction fragments that hybridized with converting corynephage beta, suggesting that in C. ulcerans as in C. diphtheriae there may be a relationship between toxinogeny and carriage of beta-related phage. Selected strains of C. diphtheriae, C. ulcerans, and C. pseudotuberculosis were examined for DNA homology by a semiquantitative technique. There was very little homology between C. diphtheriae and members of the other two species. Strains of C. ulcerans and C. pseudotuberculosis, although more closely related, appeared to belong to distinct species as well.

Bacteriophages↗

Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2.

The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described. Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated. Transfections were carried out with DNA from corynephage 782, a member of the beta family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin-resistant strain of C. diphtheriae and carries the resistance gene. Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA. Transfection frequencies were in the range of 3-8 X 10(3) plaque-forming units/micrograms of phage DNA, and transformation frequencies were in the range of 0.2-150 colony-forming units/micrograms of plasmid DNA. Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin. Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram-negative bacteria without the intervention of genetic engineering. pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains. The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin.

Bacteriophages↗

In vitro activity of newer antibiotics against Corynebacterium jeikeium, Corynebacterium amycolatum and Corynebacterium urealyticum.

The in vitro activity of ciprofloxacin, erythromycin, telithromycin, teicoplanin, linezolid and quinupristin-dalfopristin was tested against human derived pathogenic corynebacteria. The MICs of these antibiotics were measured using the agar dilution method against 31 strains of Corynebacterium jeikeium, 58 Corynebacterium amycolatum (including 33 multidrug-resistant strains) and 64 Corynebacterium urealyticum clinical strains. A high resistance rate to ciprofloxacin and erythromycin was found in the three species. Telithromycin was much more active than erythromycin (MIC(90) of erythromycin >or=128 mg/l for all three species; MIC(90) of telithromycin: 4 mg/l for C. jeikeium, 64 mg/l for C. amycolatum and 1 mg/l for C. urealyticum). There were no teicoplanin-resistant (MIC(90) 1, 0.5 and 1 mg/l, respectively) or linezolid-resistant strains (MIC(90) 1, 0.2 and 0.5 mg/l, respectively). Quinupristin-dalfopristin was active against most strains with an activity similar to linezolid, but three C. jeikeium and one C. amycolatum showed MICs >or=4 mg/l. Telithromycin showed much better activity against corynebacteria than older macrolides. Synercid and linezolid were active against most isolates tested, including multidrug resistant strains.

Acetamides↗

Adhesion of Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis to bovine urinary bladder epithelial cells of various ages and levels of differentiation.

The adhesion of Corynebacterium renale, Corynebacterium pilosum, and Corynebacterium cystitidis to various epithelial cell layers of bovine urinary bladders was examined. Adhesion was most efficient to the urinary sediment epithelial cells and the superficial cells immediately before shedding, followed by the remaining superficial cells and intermediate cells in this order, and least efficient to the deeper intermediate and basal cells. Incubation of the intermediate cells for 6 h increased the number of bacteria that adhered to these cells.

Age Factors↗

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP site of gamma-corynephage.

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.

Attachment Sites, Microbiological↗

Survival of Corynebacterium renale, Corynebacterium pilosum and Corynebacterium cystitidis in soil.

Survival of the causative agents of bovine pyelonephritis, Corynebacterium renale, C. pilosum and C. cystitidis, was examined at 30 degrees C in autoclaved soil. In the soil from a paddock, C. renale and C. cystitidis survived for 56 and 63 days, respectively, and C. pilosum for a longer period of at least 210 days. In soil from a pasture, sand from an athletic field and sea sand, the survival of these bacteria was of shorter duration.

Animals↗

Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese.

Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).

Biomass↗

[DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)].

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.

Base Sequence↗

Taxonomy of Corynebacterium diphtheriae and related taxa, with recognition of Corynebacterium ulcerans sp. nov. nom. rev.

Levels of genomic DNA relatedness were determined using a S1 nuclease procedure for reference bacteria identified as biotypes of Corynebacterium diphtheriae, biovars of Corynebacterium pseudotuberculosis, and 'Corynebacterium ulcerans'. These results showed that the three species are separate taxa at the genomospecies level whereas biotypes and biovars are closely related genomically within each species. Phylogenetic analyses of small-subunit rDNA sequences revealed that 'Corynebacterium ulcerans' forms a tight cluster with Corynebacterium pseudotuberculosis within the robust branch that groups all Corynebacterium sequenced to date. Therefore, we propose that the species incertae sedis 'C. ulcerans' should be conclusively recognized as a distinct species within the genus Corynebacterium with strain CCUG 2708 = NCTC 7910 as type strain. This species is characterized by urease production and fermentation of glycogen.

Base Composition↗

Corynebacterium aurimucosum sp. nov. and emended description of Corynebacterium minutissimum Collins and Jones (1983).

Two coryneform bacteria isolated from human clinical specimens were 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 sensu stricto. Comparative 16S rRNA gene sequence analysis showed that the two strains are genealogically highly related (99.8% sequence similarity) and constitute a new subline within the genus Corynebacterium, with Corynebacterium minutissimum as their nearest phylogenetic neighbours (98.8% sequence similarity). However, DNA-DNA hybridization experiments demonstrated unambiguously that the isolates are genealogically distinct from Corynebacterium minutissimum (42% homology). Biochemical testing indicated that the two isolates were hardly differentiated from Corynebacterium minutissimum. Based on both phenotypic and phylogenetic evidence it is proposed that these isolates be classified as a new species, Corynebacterium aurimucosum sp. nov. The type strain of Corynebacterium aurimucosum is represented by strain IMMIB D-1488T (= DSM 44532T = NRRL B-24143T).

Bacterial Typing Techniques↗

Corynebacterium testudinoris sp. nov., from a tortoise, and Corynebacterium felinum sp. nov., from a Scottish wild cat.

Two unknown gram-positive, rod-shaped bacteria isolated from a tortoise and a Scottish wild cat were subjected to a polyphasic taxonomic analysis. Chemical analysis revealed the presence of straight-chain and monounsaturated fatty acids and short-chain mycolic acids in the two isolates consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing confirmed that the unknown isolates were members of the genus Corynebacterium, with the two organisms displaying greater than 3% sequence divergence from each other and from established species of the genus. The unknown Corynebacterium isolates were readily distinguished from each other and from all recognized species of the genus by biochemical tests. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown organisms from a tortoise and a cat be classified in the genus Corynebacterium as Corynebacterium testudinoris sp. nov. and Corynebacterium felinum sp. nov., respectively. The respective type strains of C. testudinoris and C. felinum are CCUG 41823T and CCUG 39943T.

Animals↗