Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.     

Mycolic acid analysis in Nocardia species. The mycolic acid compositions of Nocardia asteroides, N. farcinica, and N. nova.

Mycolic acids from twelve Nocardia species were analyzed for structure using capillary gas chromatography and mass spectrometry. This high-resolution procedure permitted good separation of the trimethylsilyl (TMS) ether derivatives of mycolic acid methyl ester according to the total number of carbon and double bonds. The profiles of the mycolic acid molecular species were used as models to illustrate the difference in the structures of each species, even in the case of N. asteroides complex; N. asteroides, N. farcinica and N. nova. Although N. asteroides and N. farcinica had similar lengths of carbon skeleton, i.e., 51.9-53.7 was the average carbon number (Av.Nc.), they had different compositions of unsaturated acids. Mycolic acids from N. asteroides were composed of abundant saturated acids and less than 1% tetraenoic acids; mycolic acids from N. farcinica were composed of unsaturated acids, which were composed of abundant dienoic acids, 2-12% of tetraenoic acids and a trace of pentaenoic acids. In contrast, Av.Nc. of mycolic acids from N. nova were 55.7-56.3, which were relatively longer than those from N. asteroides or N. farcinica. Regarding the characteristics of the structure of alpha-branch, major components were C16:0 and C18:0 for N. asteroides 23206T, and C16:0 and C14:0 for N. farcinica 23157T, respectively. The presence of monounsaturated alpha-branch (C18:1 and C16:1) was characteristic of N. nova.     

Nocardia asteroides in the Soil of Kuwait

A pilot study was undertaken to determine the occurrence and distribution of pathogenic nocardiae in Kuwaiti soil. A total of 102 soil samples collected from two localities were investigated by the paraffin bait technique. Nocardia asteroides was the only species isolated from 42 (41%) soil samples. None of the isolates fulfilled the criteria required for identification of N. farcinica or N. nova. Thirty one (73.8%) isolates showed equivalent growth at 45 °C and 35 °C, 17 (40.4%) isolates utilized acetamide for carbon and nitrogen requirements and 3 (7.1%) isolates showed delayed arylsulphatase activity. Only a solitary isolate was resistant to cefamandole. Soil samples originating from the Kuwait University Campus Shuwaikh, which were rich in humus/organic matter, were more productive for N. asteroides (67%) than the samples which were devoid of it but were mixed with crude oil (39%). Sand samples that lacked organic matter and crude oil samples were least productive of N. asteroides. These preliminary findings do not suggest that massive oil contamination of soil in the Ahmadi oil field area during the Gulf war promoted the natural occurrence of N. asteroides. However, isolation of N. asteroides in as many as 41% of the soil sample is a significant observation warranting further epidemiologic studies including its possible role in the operation desert storm sickness syndrome. This is the first report on the natural occurrence of N. asteroides in Kuwait. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of streptomycin on lipid composition with particular reference to cyclic depsipeptide biosynthesis in Serratia marcescens and other micro-organisms

The addition of low concentrations of streptomycin (5-10mug/ml of medium) to Serratia marcescens caused significant alterations in the lipid composition of this organism, but neither growth nor pigmentation was affected. The acetone-soluble cyclic depsipeptides, which comprise on average 15% of the total lipid, were decreased almost to zero and the total lipid phosphorus was more than doubled in the presence of streptomycin. Most of the phospholipid increase was due to an increase in phosphatidylethanolamine. Cyclic depsipeptides were not leached from the cell in the presence of streptomycin, indicating a definite inhibition of the biosynthetic pathway. The effect of streptomycin on the reported peptidolipids of Rhodopseudomonas spheroides, Halobacterium halobium, Nocardia asteroides and Pseudomonas tabaci was investigated. In the case of the only strictly comparable cellular cyclic depsipeptide (that of N. asteroides) the biosynthesis was strongly inhibited by streptomycin, but cell weight was maintained or even slightly increased. A possible mode and site of action of low concentrations of streptomycin on bacterial lipids is discussed.     

Immunodiffusion studies of some Nocardia strains

Forty-three strains of Nocardia, one of Actinomadura and two of Nocardiopsis were studied using the comparative immunodiffusion technique. Three reference precipitation systems were employed: one represented Nocardia asteroides N10, one N. asteroides ATCC 19247, and one N. otitidis-caviarum ATCC 14629. One tight cluster was formed by the N. otitidis-caviarum strains and another tight cluster was formed by some of the N. asteroides strains studied. However, other strains of N. asteroides were distinct from the latter cluster. Furthermore, N. asteroides ATCC 19247, which is the type strain, differed from most ot the N. asteroides strains tested. Strains of the species N. asteroides, N. brasiliensis, N. farcinica and N. otitidis-caviarum were found to be closely related, while N. amarae strains differed slightly from this group. The strains referred to Actinomadura and Nocardiopsis were clearly distinct from the three Nocardia reference strains; nevertheless, three antigens common to these genera were revealed.     

Characterization of 2,3-dihydroxybenzoic acid from Nocardia asteroides GUH-2.

Culture filtrates of virulent Nocardia asteroides GUH-2 after growth in acetate minimal medium displayed an absorbance maximum at 320 nm. After isolation by polyamide extraction and anion chromatography, a UV-active compound with this absorbance was shown to be 2,3-dihydroxybenzoic acid (DHB) by nuclear magnetic resonance, gas chromatographic, and mass spectrometric techniques. DHB production under several culture conditions was quantified by a standard high-pressure liquid chromatography assay. Under iron deficiency conditions, N. asteroides GUH-2 excreted up to 11 mg of DHB per liter into the culture medium. No DHB was detected when N. asteroides GUH-2 was grown in an iron-rich medium. With the less virulent strain N. asteroides 10905, DHB was not found under any condition tested.     

Pathogenic and virulence characterization of colonial mutants of Nocardia asteroides GUH-2

The pathogenicities in mice (comparing LD50 determinations) of two mutant strains and one wild-type strain of Nocardia asteroides GUH-2, each possessing a colonial morphology distinct from the other, were compared at respective stages of growth. Despite the three strains' colinear growth curves and similar physiological properties, unique patterns of pathogenicity emerged for each strain upon analysis. Ultrastructural and fatty acid profiles of cultures at the various growth stages were monitored. The mutant strain SCII-A1 was consistently less virulent than the other strains of N. asteroides GUH-2 (SCII-P and SCII-C). Further, its fatty acid profiles as well as the shape and consistency of its colonies differed greatly from those of the wild-type strain. The fatty acid composition and the colonial morphology of strain SCII-C more closely resembled those of the parent, although its virulence was both greater than (before 28 h of growth) and less than the parent's depending upon the specific stage of growth. The comparative degrees of cellular fragmentation and complexity, as determined by scanning and transmission electron microscopy, were found to coincide with changes in relative degrees of pathogenicity.     

Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates

Abstract Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified on the species level. Of 117 N. asteroides complex organisms, 34 (29%) were N. farcinica , 28 (24%) were N. nova , and 55 (47%) were N. asteroides sensu stricto . An analysis of the specimen sites from which the organisms were derived showed that isolates derived from blood, brain, or bone marrow were more likely to be N. farcinica than the other two species. A study of the virulence of ten strains of each species was undertaken, using a mouse model with intravenous inoculation. The 50% lethal doses (LD₅₀) for N. farcinica were significantly lower than those of the other two species. LD₅₀ values for N. nova and N. asteroides were not significantly different. The above data confirming the greater virulence of N. farcinica support the identification of species within the N. asteroides complex.     

Immunodiagnosis of nocardiosis.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.     

Optimal Combination and Concentration of Antibiotics in Media for Isolation of Pathogenic Fungi and Nocardia asteroides

Strains of Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Cryptococcus neoformans, Nocardia asteroides, and Coccidioides immitis were tested for in vitro susceptibility to polymyxin, gentamicin, kanamycin, chloramphenicol, and neomycin at concentrations of 1, 2, 4, 8, and 16 mug/ml. Polymyxin was the most inhibitory and gentamicin was the least inhibitory of the five antibiotics. Two Histoplasma mycelial strains were partially inhibited by 2 and 8 mug of gentamicin per ml and showed at least a 2+ growth at the higher antibiotic concentration. Kanamycin and neomycin produced significant inhibition of N. asteroides but otherwise were noninhibitory. A combination of chloramphenicol and kanamycin, each at 16 mug/ml, and gentamicin, at 4 mug/ml, was noninhibitory to the strains tested except for N. asteroides. Chloramphenicol at 16 mug/ml was not inhibitory for N. asteroides. The results suggest that the optimal antibiotic combination to use in the isolation of fungi and higher bacteria is chloramphenicol, 16 mug/ml, and gentamicin, 4 mug/ml. Addition of sheep blood (5%) had no effect on antibiotic susceptibility of the organisms studied.     

Nocardia asteroides (Eppinger) Blanchard in the soils of the Ukrainian SSR]

The distribution of Nocardia asteroides (Eppinger) Blanchard, the main causative agent of nocardiosis in humans and animals, in the soils of the Ukrainian SSR was studied. This species was found to inhabit cultivated soils in various soil-climatic zones of the Ukrainian SSR. The isolated strains proved to be heterogeneous in their properties. N. asteroides fell into 2 groups: acetamidase-negative (A) and acetamidase-positive (B). Newly isolated N. asteroides belonging to the acetamidase-positive group were shown to be always pathogenic, whereas the organisms of the acetamidase-negative group could be pathogenic and non-pathogenic in white mice.     

Changa in the structure and activity of a soil microbial community caused by inorganic nitrogen fertilization

The changes in the structure and activity of a soil microbial community caused by addition of moderate and high rates of the mineral nitrogen fertilizer (KNO₃) were studied in a laboratory incubation experiment. The structure of the microbial community was evaluated from the phospholipid fatty acid (PLFA) profile; specific growth rate of the microorganisms was determined by the method of the kinetics of substrate-induced respiration; the total pool of microbial carbon was estimated by the fumigation-extraction method. The amounts of nitrogen fertilizer applied in three treatments of the experiment were 0 (control), 100, and 2000 ??g N/g soil. Even in the absence of additional sources of organic carbon, a considerable portion of the added ¹⁵N (up to 74%) was immobilized. No significant increase in the amount of microbial carbon was observed during incubation. The specific growth rate of the microbial community in soil supplemented with glucose decreased twofold after addition of 2000 ??g N/g soil. In this treatment, the ratio of cyclic fatty acids to their monoenoic precursors also increased, indicating the adaptation of microbial cells to extremely high amounts of nitrogen fertilizer. Moreover, considerable changes in the structure of the soil microbial community, such as an increase in the ratio of fungalto bacterial markers and a decrease in the ratio between PLFA of gram-positive and gram-negative bacteria, were observed in the treatment with addition of 2000 ??g N/g soil. Our data clearly indicate that mineral nitrogen fertilization of soil under carbon limitation has a pronounced impact on the structure and activity of soil microbial communities.     

Changes in population structure and reproduction during a 3-yr population cycle of voles

Cyclic changes in population growth rate are caused by changes in survival and/or reproductive rate. To find out whether cyclic changes in reproduction are an important part of the mechanism causing cyclic fluctuations in small mammal populations, we studied changes in the population structure and reproduction of field voles ( Microtus agrestis ), sibling voles ( M. rossiaemeridionalis ), bank voles ( Clethrionomys glareolus ), and common shrews ( Sorex araneus ) in western Finland during 1984–1992, in an area with 3-yr vole cycles. We also modelled the population growth of voles using parameter values from this study. The animals studied were collected by snap trapping in April, May, June, August, September, and, during 1986–1990, also in October. We found several phase-related differences in the population structure (age structure, sex ratio, proportion of mature individuals) and reproduction (litter size, length of the breeding season) of voles. In non-cyclic common shrews, the only significant phase-related difference was a lower proportion of overwintered individuals in the increase phase. According to the analyses and the vole model, phase-related changes in litter size had only a minor impact on population growth rate. The same was true for winter breeding in the increase phase. The length and intensity of the summer breeding season had an effect on yearly population growth but this impact was relatively weak compared to the effect of cyclic changes in survival. The population increase rates of Microtus were delayed dependent on density (8–12-month time lag). Our results indicate that cyclic changes in reproduction are not an important part of the mechanism driving cyclic fluctuations in vole populations. Low survival of young individuals appeared to play an important role in the shift from the peak to the decline phase in late summer and early autumn.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.     

Fatty Acid Activation by a Lipophilic Bacterium

Cell-free extracts of Nocardia asteroides activated saturated fatty acids from octanoate to octadecanoate, plus docosanoate; maximal activation occurred with dodecanoate. No activation of short-chain fatty acids was observed. The activating enzyme, characterized as an acyl-coenzyme A (Co A) synthetase (acid: Co A ligase [adenosine monophosphate]; EC 6.2.1.3), was localized in the cytoplasm of the cells and had absolute requirements for Co A, adenosine 5'-triphosphate, and Mg(2+). Kinetic data suggested that N. asteroides possessed at least two synthetases: one specific for short-chain fatty acids, and the other specific for medium- and long-chain fatty acids.     

Experimental pathogenicity of isolates of Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae from different sources

The experimental pathogenicity of 14 isolates of Nocardia brasiliensis, 15 of N. asteroides, and 5 of N. caviae was investigated for the white Swiss mice inoculated intraperitoneally and in the foot pad, and for the guinea-pig and the hamster (Mesocricetus auratus) both inoculated intratesticularly. The guinea-pig was remarkably sensitive to N. asteroides, with an apparent relationship between pathogenicity and thermotolerance, confirming previous observations. Mice were in general less susceptible to this species. In both guinea-pigs and hamsters it was possible to observe typical granules with or without clubs. N. caviae was highly pathogenic for the guinea-pig and the hamster but no mycetomas were produced in the mice inoculated in the foot pad. Isolates of N. brasiliensis from natural sources were scarcely virulent for the different animals. Those of human origin produced significant lesions in the mice inoculated intraperitoneally with granules. Foot pad inoculation of mice with N. brasiliensis caused mycetomas in several animals.     

Inactivation of kanamycin A by phosphorylation in pathogenic Nocardia.

Among the five species of pathogenic Nocardia, i.e., N. asteroides, N. brasiliensis, N. farcinica, N. nova and N. otitidiscaviarum, all strains of N. brasiliensis and N. farcinica showed resistance to an aminoglycoside antibiotic, kanamycin A, showing the MIC (minimum inhibitory concentration) values of more than 100 micrograms/ml. This species-specific difference in sensitivity was found to be explained by the production of an inactivation enzyme, aminoglycoside 3'-phosphotransferase APH(3'). Structural studies by mass and NMR spectroscopy on the inactivated substance produced by a cell-free extract of the Nocardia confirmed the conversion of kanamycin A to an inactive substance, kanamycin A 3'-phosphate. The MIC values of N. otitidiscaviarum and N. nova for kanamycin A, on the other hand, ranged from 0.78 micrograms/ml to 100 micrograms/ml, and both species were non-producers of APH(3'). Sensitivity to the antibiotic and APH(3') productivity of N. asteroides varied depending on the strain.     

A study of the enzymatic profile of soil isolates of Nocardia asteroides

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, α-glucosidase and β-glucosidase. In addition, β-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-β-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in >95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-glucoronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nocardia as a pathogen of the brain: mechanisms of interactions in the murine brain--a review.

Nocardia asteroides strain GUH-2 invades the brain without inducing an early inflammatory response in normal mice. This strain can grow within the neurons as well as adjacent areas causing neurological damage without abscess formation. At low inoculum doses, this organism is gradually cleared from the brain after the initial burst of growth. Therefore, at two weeks, the brains of these mice appear to become sterile, but the animals begin to develop a variety of neurological signs including an L-dopa responsive headshake. It was found that mice that recovered from sublethal infection with N. asteroides strain GUH-2 had progressive and permanent neuron damage in regions of the brain, the extent of which appeared to correlate with specific neurological signs. The mechanisms of this response are not known, but current data indicate that the level of nonspecific phagocyte activation (microglia?) within the brain represents an important first line of defense. Next, T-lymphocytes are important in the secondary response in the brain to nocardial invasion. Finally, B-lymphocytes and a humoral immune response may be involved in these complex interactions; however, it is not clear how these responses relate to the permanent and progressive brain damage induced by Nocardia. It is possible that these latter responses may exacerbate neurodegeneration.     

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.