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.

Confocal microscopy for Nocardia keratitis.

PURPOSE: To assess the role of confocal microscopy for in vivo detection of Nocardia in patients with microbial keratitis. DESIGN: Retrospective interventional case series. PARTICIPANTS: Three patients with microbiologically proven Nocardia keratitis. INTERVENTION: Confocal microscopy. METHODS: We performed confocal microscopy in microbiologically proven cases of Nocardia asteroides keratitis. A masked observer examined the images. For better understanding of the images, we also performed confocal microscopy on a blood agar culture plate with Nocardia growth. RESULTS: Confocal microscopy of infiltrated cornea revealed highly reflective, short, thin branching filaments with bright inflammatory cells in the background. The filaments were seen clearly at the edge of the infiltrate. In scans with faint images, these filaments became more visible on inversion of bright and dark components. Confocal microscopy of cultured organisms also revealed filamentous beaded structures with a morphology identical to that of those seen in vivo. CONCLUSION: Nocardia, a filamentous bacterium, produces a distinct image on confocal microscopy. This in vivo examination technique may be useful in cases of deep-seated infiltrates where routine microbiology workup does not yield positive results.

Case report: Nocardia asteroides mycetoma.

Primary cutaneous infections with Nocardia asteroides are rare and have been reported in immunocompromised patients. Herein, we report a case of primary cutaneous Nocardia asteroides mycetoma of the skin in an immunocompetent individual. The infection was treated successfully with trimethoprim-sulfamethoxazole. Because a prolonged incubation time is required for the cultures and since additional biochemical tests are necessary for identification of this species, the clinician should alert the microbiology laboratory when such an infection is suspected clinically.

Antimicrobial susceptibility profile of soil isolates of Nocardia asteroides from Kuwait.

OBJECTIVES: To find the antimicrobial susceptibility profile of 42 soil isolates of Nocardia asteroides against 14 antimicrobial agents representing beta-lactams, aminoglycosides, ciprofloxacin, minocycline, erythromycin and third generation cephalosporins. METHODS: The antimicrobial susceptibility was determined by the disk diffusion method using Mueller-Hinton agar medium. A homogeneous suspension giving an inoculum of 106-108 CFU/mL was used to streak the plates. The zone of inhibition was read after 36-48 h of incubation at 37 degrees C. RESULTS: All the soil isolates of N. asteroides were susceptible to amikacin, imipenem and tobramycin. Susceptibility to cephalosporins was quite variable; 86% of the isolates were susceptible to cefotaxime, 57% to ceftriaxone and 40% to cefamandole. Fifty-seven per cent of the isolates showed intermediate susceptibility to cefamandole, 33% to ceftriaxone and 5% to cefotaxime. Ninety-three per cent of the isolates were resistant to sulfamethoxazole alone or in combination with trimethoprim. CONCLUSIONS: The study reports a wide variation in the antimicrobial susceptibility profile of soil isolates of N. asteroides originating from a single geographical area. Of interest is the finding that over 90% of N. asteroides isolates were resistant to sulfamethoxazole without any previous exposure to this drug. This may have serious therapeutic implications as sulphonamides or the combination of trimethoprim-sulfamethoxazole is the therapy of choice for nocardiosis. Demonstration of resistance to beta-lactam antibiotics may be attributed to the presence of beta-lactamases which was detectable in > 90% of the soil strains of N. asteroides. The study underscores the importance of antimicrobial susceptibility testing for clinical isolates of Nocardia since individual strains show considerable differences in their susceptibility patterns necessitating therapeutic adjustments.

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.

Ultrastructural analysis of growth of Nocardia asteroides during invasion of the murine brain.

BALB/c mice were infected with 10(6) CFU of log-phase cells of Nocardia asteroides GUH-2 by tail vein injection (at this lethal inoculum dose, approximately 800 to 1,000 CFU becomes deposited in the brain). At 24 h after infection, the ultrastructural interactions of the nocardiae during growth within the murine brain were investigated. The nocardiae grew perivascularly in the pons, substantia nigra, hypothalamus, and thalamus portions of the brain, where they were either within or associated with most brain cell types. There appeared to be a propensity for growth within the soma of neurons and their axonal extensions. The nocardial cells were surrounded by 1 to 30 layers of membrane, and the innermost membrane was usually tightly adherent to the cell wall. This compartmentalization of nocardiae within brain cells could contribute to their failure to induce an inflammatory response or a cytopathic effect. Nevertheless, the bacteria were able to obtain adequate nutrients from the host to grow within the brain. The nocardiae were not completely inert, since some of the brain cells showed signs of degeneration. The myelin sheaths of axons were the most strongly affected, and there was evidence of demyelinization and axonal degeneration. Nocardiae growing within brain cells were phagocytized by compact, dense cells that were probably microglia. There was no ultrastructural evidence that the nocardiae were damaged by these phagocytes 24 h after infection; nevertheless, these cells may be important for the elimination of nocardiae from the brain during a nonlethal infection.

Interactions of Nocardia asteroides with murine glia cells in culture.

Astrocytes and microglia were obtained from brains of newborn mice and infected with Nocardia asteroides. Scanning electron micrographs showed nocardiae adhering to the astrocyte cell surface and entering the cytoplasm. After 6 h of incubation the intracellular nocardiae appeared as filaments, demonstrating that growth was occurring. In contrast, the microglia phagocytized nocardiae, but after 6 h the presence of coccoid cells indicated that nocardial growth was inhibited.

Natural occurrence of Nocardia in soil of Tucumán: physiological characteristics.

This is the first study initiated in Argentina to establish the presence of species of Nocardia from soil samples. These samples were gathered in different areas of Tucumán. Thirty three pathogenic strains of Nocardia were isolated by the paraffin bait method. Out of them, 28 were N. brasiliensis, 3 N. asteroides and 2 N. caviae. N. brasiliensis was widely distributed in the soil of the areas tested. It is proved that N. caviae, so rarely found in other regions of the world, occurs in Tucumán. A detailed study of the morphological and physiological characteristics for identification is discussed.

Variations in properties of Nocardia asteroides resulting from growth in the cell wall-deficient state.

Several revertants possessing a cell wall were obtained from L-forms of Nocardia asteroides 10905. These L-form revertants differed from the parent in respect to colonial and cellular morphology, pigmentation, metabolic, capacities, cell wall structure, and mycolic acid composition. These data suggest a possible mechanism for the taxonomic heterogeneity and phenotypic diversity observed among N. asteroides strains.

Effects of activated macrophages on Nacardia asteroides.

The mechanism(s) of host resistance against Nocardia asteroides has not been well defined. Since disease due to N. asteroides frequently occurs in patients with impaired cell-mediated immunity, we studied the interaction of N. asteroides with activated and control mouse peritoneal macrophages. Activated macrophages were from mice infected with Toxoplasma gondii or injected with Corynebacterium parvum. N. asteroides in the early stationary phase (>99% in the coccobacillary form) was used for challenge of macrophage monolayers. Growth of two strains of N. asteroides was markedly inhibited in activated macrophages, whereas N. asteroides grew well in control macrophages. Quantitation of macrophage-associated N. asteroides indicated that activated macrophages killed 40 to 50% of N. asteroides within 6 h (P < 0.002). In control macrophage preparations, it appeared as if Nocardia filaments extended from within macrophages to the outside, and many of these filaments appeared to have extended to and then grown through neighboring macrophages. In activated macrophage preparations, Nocardia remained in the coccobacillary form in most macrophages. Control macrophage monolayers were almost completely overgrown with and destroyed by Nocardia 20 h after challenge, whereas activated macrophage monolayers remained intact. Nocardia that grew in control macrophages were not acid-alcohol fast or only weakly so, whereas the few Nocardia that grew in activated macrophages were strongly acid-alcohol fast. Our results indicate that activated macrophages may be important in host defense against N. asteroides.

Cytoplasmic antigens from Nocardia elicting a specific delayed hypersensitivity.

Purified cytoplasmic extracts from Nocardia asteroides and N. brasiliensis elicit delayed hypersensitivity in Nocardia-sensitized guinea pigs. The differences in skin reactivity clearly show that it is possible to distinguish between different Nocardia species. When peritoneal exudate cells from the latter animals were obtained and treated with the purified cytoplasmic extracts, their migration was inhibited to a significant degree only by means of the homologous antigen. A mild delayed reactivity was observed when the cytoplasmic antigens were used as skin test materials on animals sensitized with BCG. On the other hand, no inhibition of migration was observed when peritoneal exudate cells from BCG-sensitized guinea pigs were exposed to the cytoplasmic antigens.