IL-17 and γδ T-lymphocytes play a critical role in innate immunity against Nocardia asteroides GUH-2.

The early host response during pulmonary nocardiosis is highly dependent on neutrophils and the successful clearance of bacteria in tissue. The data presented in this study showed that IL-17 mediated the neutrophil response following intranasal inoculation with Nocardia asteroides strain GUH-2. Flow cytometry revealed that neutrophil levels in C57BL/6 mice were increased by day 1 post inoculation and remained elevated until day 3, during which time the majority of bacterial clearance occurred. Intracellular cytokine staining for IL-17 showed a 3.5- to 5-fold increase in IL-17 producing T-lymphocytes that were predominately comprised by CD4(-)CD8(-) γδ T-lymphocytes. The importance of IL-17 and γδ T-cells was determined by the in vivo administration of antibody, capable of blocking IL-17 binding or TCR δ, respectively. Neutralization of either IL-17 or γδ T-cells in Nocardia treated mice resulted in attenuated neutrophil infiltration. Paralleling this impaired neutrophil recruitment, nearly a 10-fold increase in bacterial burden was observed in both anti-IL-17 and anti-TCR δ treated animals. Together, these data indicate a protective role for IL-17 and suggest that IL-17 producing γδ T-lymphocytes contribute to neutrophil infiltration during pulmonary nocardiosis.

Filterable forms of Nocardia: a preferential site of infection in the mouse brain.

Filterable forms of Nocardia (i.e., filterable nocardiae) are characterized as spherical structures containing many granules. They fluoresce brilliantly under ultraviolet light when stained with acridine orange and are acid-fast in paraffin-containing broth cultures. An accumulation of acid-fast lipochrome bodies, similar to filterable nocardiae, is observed in glial cells of the midbrain nigral lesions found in Parkinson's disease (PD). Preliminary results from experimental infection with filterable nocardiae revealed a possible site of infection in a region of the nervous system associated with movement disorders. Here we examined the preferential site of filterable nocardiae infection in the brain and investigated the effects of various chemicals and biological substances in relation to this preferential site on nocardiae growth in vitro. Examination of tissue samples immunostained for filterable nocardiae revealed that neurons in the olfactory bulbus and the midbrain periaqueductal gray area were immunopositive for filterable nocardiae. Several erythrocytes within blood vessels of the brain were also immunopositive. In addition, a dose-dependent relationship was observed between the growth of filterable nocardiae and erythrocyte lysates. These results suggest first that the preferential site of infection for filterable nocardiae might be erythrocytes, but could also be neurons in the bulbus olfactorius as well as in the midbrain periaqueductal gray area. Second, filterable nocardiae might grow to be branching cylindrical tubules such as that of mycelial bacteria in the presence of erythrocyte lysates.

Comparison of PCR and culture for detection of Nocardia asteroides in brain specimens from experimentally infected BALB/c mice.

Systemic infection of BALB/c mice with Nocardia asteroides strain GUH-2 results in widespread replication of the organism in the brain, followed by its immune-mediated clearance. The present study compared the sensitivity of polymerase chain reaction (PCR) to bacterial culture for detection of cerebral nocardial infection in this experimental system. Mice (n=4/time point) were administered N. asteroides by intravenous injection, and brain specimens were evaluated for Nocardia by PCR and culture at post-infection days 2, 7, 14 and 21. Nocardia was detected by PCR in all infected animals on post-infection days 2, 7, and 14, and in one of four mice on post-infection day 21; in contrast, the organism was detected by culture only on post-infection days 2 and 7. These findings suggest that PCR may be more sensitive than culture for the detection of low numbers of Nocardia in the brain.

Clinicomicrobiological review of Nocardia keratitis.

PURPOSE: To present the clinical profile, laboratory results, and outcome of treatment in 16 patients with Nocardia keratitis. METHODS: A retrospective review of 16 culture-proven cases of Nocardia keratitis was done. Clinical and microbiologic data were analyzed. RESULTS: Nocardia constituted 1.7% of laboratory-confirmed bacterial keratitis and was seen predominantly in male subjects (13 of 16) with a mean age of 39.1 years. Although the predisposing factor was unknown in the majority, a definite history of trauma was present in four (25%) cases. Patchy stromal infiltrates were seen in 12 patients. Wreath pattern of infiltrates (six of 16) and hypopyon (nine of 16) were notable features. Nocardia was detectable in corneal scrapings of 10 patients with Gram stain and all patients with 1% acid-fast preparation (six of six). Nocardia asteroides was the causative agent in all except one (N. caviae). All isolates were sensitive to gentamicin; however, topical 30% sulfacetamide was the preferred drug for treatment. Favorable outcome (healed scar) was achieved in 11. CONCLUSION: Although Nocardia keratitis is a rare condition, a high index of clinical suspicion should be kept in agricultural workers or in patients with trauma who have patchy stromal infiltrates. Sulfonamides are the initial drug of choice, and gentamicin could be an effective alternative. If recognized early, Nocardia keratitis responds to medical treatment with good visual recovery.

Biological potential of extraterrestrial materials. 2. Microbial and plant responses to nutrients in the Murchison carbonaceous meteorite.

Meteoritic materials are investigated as potential early planetary nutrients. Aqueous extracts of the Murchison C2 carbonaceous meteorite are utilized as a sole carbon source by microorganisms, as demonstrated by the genetically modified Pseudomonas fluorescence equipped with the lux gene. Nutrient effects are observed also with the soil microorganisms Nocardia asteroides and Arthrobacter pascens that reach populations up to 5 x 10(7) CFU/ml in meteorite extracts, similar to populations in terrestrial soil extracts. Plant tissue cultures of Asparagus officinalis and Solanum tuberosum (potato) exhibit enhanced pigmentation and some enhanced growth when meteorite extracts are added to partial nutrient media, but inhibited growth when added to full nutrient solution. The meteorite extracts lead to large increases in S, Ca, Mg, and Fe plant tissue contents as shown by X-ray fluorescence, while P, K, and Cl contents show mixed effects. In both microbiological and plant tissue experiments, the nutrient and inhibitory effects appear to be best balanced for growth at about 1:20 (extracted solid : H2O) ratios. The results suggest that solutions in cavities in meteorites can provide efficient concentrated biogenic and early nutrient environments, including high phosphate levels, which may be the limiting nutrient. The results also suggest that carbonaceous asteroid resources can sustain soil microbial activity and provide essential macronutrients for future space-based ecosystems.

Acid-fastness of Nocardia asteroides GUH-2 cultured in brain heart infusion broth supplemented with paraffin.

Nocardia asteroides GUH-2 was not acid-fast when grown in brain heart infusion (BHI) broth. When grown in BHI broth supplemented with paraffin, many filamentous cells showed acid-fastness after treatment with 1% acid-alcohol as the decolorizing agent. When treated with 3% acid-alcohol, filamentous cells were not acid-fast. In addition to the acid-fast filamentous cells of nocardiae, unknown acid-fast spherical bodies were observed in the paraffin-supplemented BHI broth cultures.

Opacification of Middlebrook agar as an aid in distinguishing Nocardia farcinica within the Nocardia asteroides complex.

Among 58 aerobic actinomycetes isolated from different sources and geographical locations, none of 23 Nocardia asteroides isolates, at 18 N. farcinica isolates, 1 of 5 N. otitidiscaviarum isolates, and 1 of 4 Rhodococcus species isolates opacified Middlebrook 7H10 medium. Within the N. asteroides complex, this characteristic, together with growth at 45 degrees C and resistance to each of erythromycin, cefotaxime, and tobramycin, provides a simple means of distinguishing N. farcinica from N. asteroides.

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines.

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.

Ammonium assimilation in Nocardia asteroides.

Specific enzymes of ammonium assimilation were measured in cell-free extracts of Nocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation in N. asteroides.

Influence of cultural conditions on growth and lipolytic activity in Nocardia asteroides.

The growth and the production of extracellular and intracellular lipases were measured from Nocardia asteroides grown under different cultural conditions. Maximal growth and intracellular and extracellular activities were observed at 3 d after inoculation. Among the tested media, synthetic medium induced maximal growth and extracellular activity, whereas tryptic soy broth induced the maximal intracellular lipase activity. The best carbon and nitrogen sources for growth and lipolytic activity were glucose, fructose, glutamate and nitrate, respectively. The optimal C:N ratio for growth was in the range of 1:4 to 2:3 and for lipase activity the range was 2:3 to 3:2. Anything above or below this range was detrimental to the organism and its enzyme activity. Under the conditions of this study, N. asteroides grew best and had the highest lipase activity when compared to N. brasiliensis and N. caviae.

Site-specific growth of Nocardia asteroides in the murine brain.

The growth of Nocardia asteroides GUH-2 and two mutants (NG-49 and I-38-syn) in regions of the brains of BALB/c mice was determined by microdissection and viable counting. GUH-2 grew throughout the murine brain but at different growth rates that depended on the specific location. The rate of increase in total CFU per brain during GUH-2 infection was unaffected by the inoculum size; however, in five of eight brain regions, an alteration in the inoculum size resulted in altered nocardial growth rates. Mutant NG-49 showed a significantly slower rate of increase in total CFU per brain than did the parental strain, GUH-2, and significantly decreased growth rates in seven brain regions. Mutant I-38-syn showed a rate of increase in total CFU per brain similar to that of the parental strain; however, this mutant grew significantly faster in the cerebellum and pons-medulla. Growth appeared to be a necessary precursor to the cellular damage that resulted in the variety of neurological disorders observed in mice infected with N. asteroides GUH-2, because mutant NG-49 exhibited a decreased ability to grow in specific regions of the brain and did not induce signs of neurological damage. In contrast, mutant I-38-syn induced neurological signs in a larger percentage of the infected animals than did parental strain GUH-2 and grew better in certain regions of the brain than did the parental strain. Furthermore, there appeared to be a relationship between the growth of N. asteroides in the substantia nigra and the induction of an L-dopa-responsive head shake that was observed in some of the mice following a sublethal intravenous injection of N. asteroides GUH-2.

Selective buffered charcoal-yeast extract medium for isolation of nocardiae from mixed cultures.

The recovery of Nocardia species from mixed cultures is facilitated by use of a selective medium. We show that buffered charcoal-yeast extract medium with polymyxin, anisomycin, and vancomycin can be used for the selective isolation of nocardiae from contaminated specimens.

Influence of nutritional factors on growth and hydrolytic enzyme production in Nocardia asteroides.

The growth and production of hydrolytic enzymes such as alpha-amylase, esterase and peroxidase as influenced by the type of media, carbon and nitrogen sources and C:N ratio were monitored in Nocardia asteroides at 37 degrees C. Sabouraud dextrose and the synthetic media yielded maximum growth compared with tryptic soy broth. Among the carbon sources (dextrose, fructose, sucrose, maltose, starch and citrate), monosaccharides supported maximum growth and induced higher alpha-amylase activity but repressed the peroxidase activity. On the other hand, the disaccharides and starch produced less growth but induced maximum esterase and peroxidase activities. Glutamate among the nitrogen sources (nitrate, nitrite, ammonium, hydroxylamine, glutamate and casein) supported maximum growth. Glutamate, nitrate and casein induced alpha-amylase and esterase activities but suppressed peroxidase activity. Nitrite, ammonium and hydroxylamine stimulated peroxidase activity to the maximum but repressed alpha-amylase and esterase activities. Low, medium and high C:N ratios induced maximum peroxidase, esterase and alpha-amylase activities, respectively.

Clinical demonstration of isolation of Nocardia asteroides on buffered charcoal-yeast extract media.

Nocardia asteroides was isolated only from sputum samples, obtained from three patients with pulmonary nocardiosis, that had been cultured onto buffered charcoal-yeast extract (BCYE) and selective BCYE media as part of laboratory workups for Legionella species. A decontamination procedure with low-pH pretreatment (KCl-HCl solution) had been performed on the sputa prior to culture onto the BCYE media because direct cultures on the media were overgrown with commensal microflora. Chalky white colonies, 0.5 to 1.0 mm in diameter, that were subsequently identified as N. asteroides grew well on the BCYE media. Thus, the techniques and the selective media used for Legionella species were useful for isolating Nocardia species from sputum.

Monoclonal antibodies demonstrate that superoxide dismutase contributes to protection of Nocardia asteroides within the intact host.

The importance of superoxide dismutase (SOD) in protecting cells of Nocardia asteroides from the oxidative killing mechanisms within the intact murine host was determined. Murine monoclonal antibodies specific for nocardial SOD and for another nocardial antigen were prepared. Both antibodies adhered to cell surface antigens, as shown by fluorescence-labeled-antibody staining. The anti-nocardial SOD antibody inhibited the effect of nocardial SOD on superoxide generated in vitro. Cells of N. asteroides GUH-2 in log phase of growth were incubated with monoclonal anti-nocardial SOD, another monoclonal antinocardial antibody (not reactive with SOD), or phosphate-buffered saline and then injected intravenously into mice. Total recovery of CFU and inhibition of growth were determined at 3, 24, and 48 h after infection. The brains, kidneys, spleens, lungs, and livers were weighed, homogenized, and plated in order to quantitate the number of organisms in each organ at each time period. There was an initial killing followed by enhanced clearance of N. asteroides from the lungs and livers of mice which had received anti-SOD antibody-treated nocardiae. There was also enhanced early killing in the spleen. At 48 h, there were fewer organisms recovered from the brains, kidneys, and livers of mice which had received anti-SOD antibody-treated nocardia. This was not true for mice which had received antinocardial antibody not specific for SOD. The data demonstrate that surface-associated SOD protects N. asteroides for oxidative killing in vivo during all stages of infection.

Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens.

Isolation of Nocardia spp. from clinical specimens can be enhanced by the use of paraffin baiting, which relies on the selective ability of the organism to metabolize paraffin. We evaluated 44 Nocardia isolates, 18 group IV mycobacterial isolates, and 4 Streptomyces isolates for growth on blood agar (BA) and on carbon-free agar containing single or double substrates as follows: paraffin agar (PA), gelatin agar (GA), urea agar (UA), PA-gelatin (PG), and PA-urea (PU). The growth rates of Nocardia spp. on BA, PA, PU, and PG were similar; but 3-day-old colonies were larger on BA for 20 (45%) isolates. After longer incubations (7 to 14 days), some Nocardia colonies were larger on PA, PG and PU than they were on BA. Despite variable morphologies on BA, colonies on PA, PG, and PU were consistently smooth, creamy, and raised. Compared with growth on BA, the growth of mycobacteria was much slower on PA, PG, and PU, with poor growth on UA and GA. The growth of Streptomyces spp. was greatly enhanced on GA, PG, UA, and PU and was poorest on PA. Twelve sputum specimens seeded with Nocardia asteroides (10(4) CFU/ml) were inoculated onto BA and all chemically defined media. Nocardiae were recovered from 6 to 12 specimens grown on BA, GA, and UA; 11 of 12 specimens grown on PG; and 12 of 12 specimens grown on PA and PU. Only PA was able to suppress the growth of other microorganisms that were present in sputum specimens. These results suggest that chemically defined media containing PA may be useful for the selective isolation of Nocardia spp. from contaminated clinical specimens.

Acid phosphatase stimulation of the growth of Nocardia asteroides and its possible relationship to the modification of lysosomal enzymes in macrophages.

Lysosomal acid phosphatase levels are reduced in murine macrophages by virulent strains of Nocardia asteroides. At the same time, other lysosomal enzymes either remain unchanged or increase in activity, indicating that acid phosphatase is not lost because of degranulation or membrane leakage. This study shows that acid phosphatase was utilized as a sole carbon source by Nocardia asteroides and that acid phosphatase combined with glutamate as a carbon source enhanced nocardial growth. As a consequence, the inverse relationship that was observed between acid phosphatase activity and the bactericidal capacity of macrophages infected with N. asteroides appears to be due to the ability of N. asteroides to preferentially metabolize this lysosomal enzyme during growth within phagocytes.

Acid phosphatase activity in Nocardia brasiliensis and Nocardia asteroides.

Nocardia asteroides and N. brasiliensis strains were found to possess acid phosphatase activity. This enzyme was found to be cell-associated and its activity paralleled the cell mass increase seen during the Nocardia growth cycle. Of the strains tested, N. brasiliensis exhibited the highest enzymatic activity. Implications of these findings are related to current evidence which indicates that other microbial acid phosphatases may constitute potential pathogenic factors for humans.

Acid phosphatase activity in Nocardia brasiliensis and Nocardia asteroides

Se encontró que cepas de Nocardia asteroides y N. brasiliensis poseenn actividad de fosfatasa ácida. Esta se encuentra asociada a las células y aumenta según éstas se reproducen durante su ciclo de crecimiento. De las cepas probadas, N. brasiliensis posee la mayor actividad enzimática. Las implicaciones de estos hallazgos se relacionan a evidencia reciente que sugiere que otras fosfatasas microbianas pueden constituir factores de patogenicidad para los humanos

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2.

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).