M u r~i a ,~ SpainUrealytic strains of coryneform bacteria that are designated Corynebacterium group D2 and are isolated from human urine are a cause of urinary tract infections. Cell wall and lipid analyses confirmed that these organisms are members of the genus Corynebacterium but can be separated from other species in the genus on the basis of DNA base composition and DNA-DNA hybridization values. Biochemically, strains in this taxon can be distinguished from other Corynebacterium spp. by their failure to produce acid from carbohydrates, by their failure to reduce nitrates, and by their ability to hydrolyze urea. We regard these bacteria as a new species of the genus Corynebacterium and propose the name Corynebacterium urealyticum. The type strain is strain NCTC 12011 (= ATCC 43042).The identification system for medically important coryneform bacteria devised by the Centers for Disease Control, Atlanta, Ga., designates one group of urealytic, nonfermentative bacteria as Corynebacterium group D2 and suggests that these organisms may be nitrate-negative variants of Corynebacterium pseudodiphtheriticum (9). This group has tentatively been referred to as Corynebacterium urealyticum (23), but this name has not been validly published previously. Strains identified as C. urealyticum have been associated with cases of encrusted cystitis and other urinary tract infections in hospital patients (20), where they have been highly resistant to antibiotics (22). The natural habitat of these strains is human skin (24), but they readily colonize urinary tracts (22); strains have also been isolated from blood (14, 17) and wounds (28). C. urealyticum is relatively inactive in conventional tests apart from its ability to hydrolyze urea rapidly. Colonially and microscopically, this species resembles Corynebacterium jeikeium, with which it shares a common habitat and resistance to antibiotics (4). However, the latter species is consistently nonurealytic and acidifies glucose and maltose. Evidence from lipid analysis (7, 21), from pyrolysis-mass spectrometry (8), and from rRNA gene restriction fragment polymorphism studies (26) indicates that there are clear differences between the two species. Chemotaxonomic evidence that group D2 is related to the genus Corynebacterium has been provided by Herrera-Alcarez et al. ("), who determined that the cell wall type is chemotype IV, with mycolic acids having chain lengths of 26 to 36 carbon atoms, and that the isoprenoid quinone MK-9(H2) is present.In this study, we estimated the DNA base compositions of Corynebacterium strains and levels of DNA-DNA relatedness between strains of C. urealyticum and other Corynebacterium species; we concluded that this taxon merits status as a new species in the genus Corynebacterium.* Corresponding author. MATERIALS AND METHODSBacterial strains and culture. The strains used in this study are listed in Table 1. Strains identified as C. urealyticum were isolated in Madrid, Spain, from the urine (strains A516/89, A524190, A532/90, A548/90, and NCTC 12011T [T =...

Nocardia brasiliensis is a Gram-positive facultative intracellular bacterium frequently isolated from human actinomycetoma. However, the pathogenesis of this infection remains unknown. Here, we used a model of bacterial delipidation with benzine to investigate the role of N. brasiliensis cell wall-associated lipids in experimental actinomycetoma. Delipidation of N. brasiliensis with benzine resulted in complete abolition of actinomycetoma without affecting bacterial viability. Chemical analyses revealed that trehalose dimycolate and an unidentified hydrophobic compound were the principal compounds extracted from N. brasiliensis with benzine. By electron microscopy, the extracted lipids were found to be located in the outermost membrane layer of the N. brasiliensis cell wall. They also appeared to confer acid-fastness. In vitro, the extractable lipids from the N. brasiliensis cell wall induced the production of the proinflammatory cytokines interleukin-1␤ (IL-1␤), IL-6, and CCL-2 in macrophages. The N. brasiliensis cell wall extractable lipids inhibited important macrophage microbicidal effects, such as tumor necrosis factor alpha (TNF-␣) and nitric oxide (NO) production, phagocytosis, bacterial killing, and major histocompatibility complex class II (MHC-II) expression in response to gamma interferon (IFN-␥). In dendritic cells (DCs), N. brasiliensis cell wall-associated extractable lipids suppressed MHC-II, CD80, and CD40 expression while inducing tumor growth factor ␤ (TGF-␤) production. Immunization with delipidated N. brasiliensis induced partial protection preventing actinomycetoma. These findings suggest that N. brasiliensis cell wall-associated lipids are important for actinomycetoma development by inducing inflammation and modulating the responses of macrophages and DCs to N. brasiliensis.

No abstract

No abstract

A trehalose-containing glycolipid was detected in several strains of Mycobacteriutn fortuitum and characterized as 2,3-di-O-acyltrehaIose (DAT) by constituents of the molecule were identified as a mixture of straight-chain (14-18 carbon atoms) and methyl-branched-chain (17-21 carbon atoms) fatty acyl groups. DAT was further fractionated by reverse phase TLC into four fractions that were designated DAT-I-DAT-IV. DAT-I contained 70-75 O/ O straightchain acyl substituents (hexadecanoyl and octadecanoyl predominating) and 25-30 O/ O 2-methyl branched substituents (mainly 2-methyl octadecadienoyl). DAT-II was composed of a mixture in which the acyl groups were almost exclusively 2-methyl branched, with 2-methyl octadecadienoyl and 2-methyl octadecen-2-oyl predominating. DAT-Ill, which was the major isolated fraction, consisted of compounds in which the ratio linear to branched acyl groups varied between 0.8 to 09,2-methyl octadecen-2-oy1, hexadecanoyl and octadecanoyl being the most abundant. Finally, DAT-IV comprised a mixture of DAT molecules containing mostly 2-methyl octadecadienoyl, 2-methyl octadecen-2-oy1, 2-methyl eicosadienoyl and 2-methyl eicosen-2-oyl groups.combined NMR spectroscopy, IR spectroscopy, GLC and GLC-MS. Lipid

Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of 'M. habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-ll and GPGIII; group B contained GPGI, GPGII', GPGll and GPGIII; group C contained GPGII', GPL-II and GPGIII; group D did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of a=, a'-and ketomycolates.Partial structural analysis by M S carried out in strains 'M. habana' TMC 5135 and M. simiae ATCC 25275l revealed that a-and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and a'-mycolates from 58 to 67 carbon atoms. The a-and ketomycolates belonged to several structural series, and minor variations were found between the two strains examined. The data obtained justified the synonymy between 'M. habana' and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of 'M. habana' can be defined by the presence of GPGll and GPGIII, a finding that could be useful in the quality control of potential vaccine strains.

The fatty acids and mycolic acids of 16 clinical isolates of Mycobacterium malmoense were studied by gas chromatography and thin-layer chromatography. All strains contained 2-methyleicosanoic and 2,4,6-trimethyltetracosanoic acids and alpha-, alpha'-, and keto-mycolic acids. The reported findings suggest that lipid analysis is a very useful approach in the species identification of M. malmoense.

The addition of D-arabinose, D-galactose, D-glucosamine, or D-mannose to the growth medium of Mycobacterium smegmatis suppressed the inhibitory effects of ethambutol both on acetate labeling of cell wall-linked mycolic acids and on the increase in the delipidated cell dry weight. The addition of D-glucose or D-fructose had no effect. It is proposed that ethambutol inhibits an early step of glucose conversion into the monosaccharides used for the biosynthesis of structurally and biologically important cell wall polysaccharides: arabinogalactan, arabinomannan, and peptidoglycan.Ethambutol (EMB) is an efficient antituberculosis molecule used in combination with other drugs (3). Moreover, there is renewed interest in EMB because of its activity against opportunistic pathogens, like Mycobacterium avium (4), which are resistant to conventional antituberculosis drugs and because of its ability to increase the susceptibility of this pathogen to other antimycobacterial drugs (5,7,13).The mode of action of EMB has not yet been elucidated (for reviews, see references 7 and 16). The early inhibitory effects described for EMB concerned the synthesis of phospholipids (1, 8), the transfer of mycolic acids to cell walllinked arabinogalactan (14), and the incorporation of labeled glucose into the arabinose-containing polysaccharides of the cell wall (15).Inhibition of arabinogalactan synthesis would explain the inhibition of its acylation by mycolic acids, but the corresponding experiments have been performed with intact cells (14) and could be a secondary effect resulting from inhibition of some central metabolism.Inhibition The effect of EMB on the arabinogalactan content of the cell wall was determined for cells that were delipidated with chloroform-methanol and then extracted with 70% ethanol to eliminate arabinomannan and mannan, which are not covalently linked to the wall (6). Cell residues were hydrolyzed (with 1 M trifluoroacetic acid at 110°C for 2 h), and the galactose content was determined by gas-liquid chromatography, with erythritol being added to the hydrolysis medium as a standard for quantification.The cell-free system that incorporates acetate into mycolic acids was prepared and used as described previously (12) by isolating at the tops of centrifuge tubes the fluffy layer of cells from an exponential-growth-phase culture disrupted in a French pressure cell. The protein content of the fluffy layer was determined by the Lowry procedure as described previously (12). The assay medium did not contain magnesium, an ion known to reduce the activity of EMB. To test the drug effect, the cell-free system was preincubated for 30 min in the presence of 25 ,ug of EMB per ml (5 x the MIC), and then [14C]acetate was added for 90 min of incubation. Lipids were extracted as indicated above; lipids and cell residues were saponified, and total acids were isolated. Aliquots were counted, and total acids were analyzed as methyl esters by thin-layer chromatography (the solvent was CH2Cl2); radioactivity was determined on thin-layer...