Effect of presensitization of Nocardia asteroides with specific antibody on the viability of the organism

Nocardia asteroides from various growth phases was treated in vitro with normal rabbit sera, immune rabbit sera containing nocardial polyclonal antibodies and a monoclonal antibody. At intervals, samples were grown in broth or on blood agar plates to determine their viability. Log and stationary phase cells were injected intra-peritoneally into female BALB/c mice and their survival rates in the liver and spleen were determined. Presensitization with antibodies reduced the viability of the log phase cells by 48% and that of the late stationary phase by 4%. The antibody-treated log phase organisms were less viable on the blood agar medium and in the spleen and liver than the control organisms. This indicates that pretreatment with antibody has a lethal effect on N. asteroides and affects its survival in vivo.     

Antibody response to sulfolipids in experimental tuberculosis

Live Mycobacterium tuberculosis H₃₇Rv, when injected into guinea pigs, induced antibodies to sulfolipids whereas antibodies were not detected in animals injected with heat killed cells. The antibody titre was found to be related to the degree of infection. A significant decrease in the titre was noted after streptomycin treatment, suggesting that antibodies were produced in response to an increasing bacterial load that occurred as the infection progressed.     

Primary subcutaneous Nocardia asteroides infection in a renal allograft recipient

Nocardia asteroides is an important opportunistic pathogen in immunocompromised hosts. The primary infection is usually in the lungs and is followed by dissemination to other parts of the body. Primary subcutaneous infection with Nocardia asteroides has been reported rarely (three reports) and no such case has been reported in a renal transplant recipient. We describe here a case of renal transplant recipient who developed primary subcutaneous infection with Nocardia asteroides within one and half years of the transplantation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of the Live and Heat-Killed Cells of Staphylococcus aureus on the Resistance of Mice to the Infection with Nocardia asteroides

When living cells of Staphylococcus aureus were introduced into mice by various routes within a few hours before or after infection with Nocardia asteroides, a marked increase in mortality and enhanced severity of lesions were observed. This effect was presumably caused by a heat-labile substance(s) liberated from the cells of S. aureus. No increase in resistance occurred when S. aureus was given 2 to 7 days before the nocardial infection, contrary to the effect of E. coli as reported previously. An increased resistance was noted when heat-killed cells of E. coli were administered two to four days before concurrent infection with N. asteroides and S. aureus.     

Pathogenicity of Nocardia caviae,N. asteroides and N. brasiliensis

The pathogenicity ofNocardia caviae, N. asteroides andN. brasiliensis has been tested for white mice, guinea pigs and rabbits and chorio-allantoic membrane of the developing chick embryo. Altogether, 14 strains belonging to the 3Nocardia species originating from soil, human and animal sources in India or abroad were tested. All of them proved pathogenic though the degree of virulence varied from strain to strain. Incorporation of hog gastric mucin in the inoculum enhanced the virulence of all the 3Nocardia species for white mice.N. caviae strains were uniformly more virulent than those ofN. asteroides andN. brasiliensis.In the white mice inoculated intraperitoneally, a greater dissemination of the disease was apparent withN. caviae than withN. asteroides. Of the 6 strains ofN. caviae tested, 5 disseminated to the lung, 3 to the heart and 2 to the brain. InN. asteroides dissemination of the disease to the brain was observed with 2 of its 3 strains.N. brasiliensis showed no dissemination.N. caviae was found to be equally virulent for white mice, guinea pigs and rabbits. On the other hand,N. asteroides andN. brasiliensis were more virulent for white mice than for guinea pigs and rabbits. The lesions caused byN. caviae in mice, guinea pigs and rabbits persisted up to 4 weeks. In strong contrast to this the lesions due toN. asteroides andN. brasiliensis found in the guinea pigs and rabbits showed a strong tendency towards spontaneous clearance.Histologically, the lesions caused byN. caviae, N. asteroides andN. brasiliensis in mice, guinea pigs and rabbits were in the form of abscesses which showed an acute or chronic reaction. In the case ofN. caviae these abscesses showed both granules and freely dispersed cocco-bacillary bodies or filaments. As forN. asteroides it occurred in the form of cocco-bacillary bodies or filaments whereasN. brasiliensis consistently produced granules in the lesions.The lesions caused by the 3Nocardia species on the chorio-allantoic membrane of the developing chick embryo were in the form of abscesses which contained cocco-bacillary bodies and branching filaments but no granules.This forms a part of the thesis submitted by P.V.K. for Ph. D. degree, of the University of Delhi.     

Brain abscess due to Nocardia

Cerebral Nocardiosis is a rare disease, usually occurring in immunocompromised hosts. We report here two cases of brain abscess due to Nocardia species-one due to usual N. asteroides and other by uncommon N. caviae. N. asteroides affected the brain in a post renal transplant patient, whereas N. caviae caused infection of brain in an apparently healthy individual. To the best of our knowledge, all the previous cases of brain abscess due to Nocardia caviae have been reported in compromised hosts. Agar dilution antimicrobial testing showed relatively higher resistant pattern in N. asteroides. In spite of antimicrobial therapy, both the patients succumbed, one within 4 days and other after an initial improvement for four weeks due to drainage of abscess.The technical assistance of Mr. R.K. Sapra and Mr. Pawan Kumar is greatfully acknowledged.     

Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

Application of the API YeastIdent system in determining the enzymatic profiles ofNocardia asteroides isolates

Glucose represses production of ammonium in many clinical isolates ofNocardia asteroides growing on bromcresolpurple casein glucose agar. Strains exhibiting this property are designated as group A, while group B represents isolates showing a high degree of proteolytic activity and a resulting rapid increase in pH. Twenty isolates ofN. asteroides were characterized as group A or B. Enzymatic profiles obtained using the API YeastIdent system showed significant enzymatic variation between 12 group B and 8 group A isolates. Proteolytic enzymes which most varied in activity between groups were glycine aminopeptidase, histidine aminopeptidase and leucyl glycine aminopeptidase. As some of theN. asteroides isolates were isolated from asymptomatic patients, it is of interest to consider the possibility of one group being of low virulence while the other is more strongly associated with infection.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.     

Infecc Ón Humana PorNocardia Asteroides,En México

Summary Although the existence ofNocardia asteroides as a human pathogen in Mexico has been known for several years, up to now there are only few reports in our country concerning the isolation of this microorganism both as a saprophyte as well as human or animal pathogen. This fact is more interesting when compared with the high incidence ofN. brasiliensis, causal agent of actinomycotic mycetoma, the most frequently observed deep mycosis in Mexico.In this paper, the isolation of eigth strains ofN. asteroides from clinical specimens is reported. Five strains were isolated from sputa, one from a fistulous lesion of the scrotum, one from a brain abscess and one more from a foot mycetoma.One of the strains isolated from sputum was in a case of pulmonary nocardiasis; another of these strains was isolated from a patient with coexistent pulmonary tuberculosis and nocardiasis. With the last three strains isolated from sputa, it was not possible to establish any correlation between the isolations and the clinical and radiological findings of the patients; two of the patients had pulmonary tuberculosis and the isolation ofN. asteroides was achieved only in one occasion in each case, with scant growth, and observed in routine cultures made for tubercle bacilli. The last strain was isolated from the sputum of a patient with chronic bronchitis. Some comments are made to the possibility that these three last isolations merely represent a saprophytic phase of the fungus.Some clinical and other features are discussed concerning the other three cases of scrotal fistula, brain abscess and mycetoma produced byN. asteroides.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

Monoamine Changes in the Brain of BALB/c Mice Following Sub-Lethal Infection with Nocardia asteroides (GUH-2)

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included head-shake, stooped posture, bradykinesia, and hesitation to forward movement (6). The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.     

Exploration of Respiratory Chain of <Emphasis Type="Italic">Nocardia asteroides:</Emphasis> Purification of Succinate Quinone Oxidoreductase

Nocardia asteroides is a pathogenic bacterium that causes severe pulmonary infections and plays a vital role in HIV development. Its electron transport chain containing cytochromes as electron carriers is still undiscovered. Information regarding cytochromes is important during drug synthesis based on cytochrome inhibitions. In this study we explored the electron transport of N. asteroides. Spectroscopic analysis of cytoplasm and membranes isolated from N. asteroides indicates the presence of soluble cytochrome-c, complex-II and the modified a ₁ c ₁ complex as the terminal oxidase. The molecular weight of the respiratory complex-II isolated and purified from the given bacterium was 103 kDa and was composed of three subunits, of 14, 26 and 63 kDa. Complex-II showed symmetrical α-absorption peaks at 561 nm in the reduced state. Spectral analysis revealed the presence of only one heme b molecule (14-kDa subunit) in complex-II, which was confirmed by heme staining. Heme b content was found to be 9.5 nmol/mg in complex-II. The electron transport chain of N. asteroides showed the presence of soluble cytochrome-c, cytochrome-a ₁ c ₁ and cytochrome-b.     

CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice

Gamma interferon (IFN-γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day ?1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-γ production. Moreover, the administration of anti-IFN-γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day ? 1, while these antibodies reduced significantly the ability of mice to produce IFN-γ. Indeed, simultaneous depletion of CD4⁺ and CD8⁺ cells had no greater suppressive effect on host defense and endogenous IFN-γ production than depletion of CD8⁺ cells alone. Together, these results suggest that CD8⁺ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-γ production. The possible role of early produced IFN-γ in the development of protective immune response to T. gondii is also discussed.     

The use of cortisone-treated mice in the screening of soil for pathogenic fungi

Summary Fifty-seven samples of desert soils were screened for pathogenic fungi by injection into cortisone-treated and non-treated mice. Four samples yielded pathogenic fungi in the cortisone-treated mice only.Nocardia asteroides was isolated from the cortisone-treated mice and from the controls. All of the cortisone-treated and the control mice injected with soil from the root zone of a dead century plant became critically ill very shortly after injection and cultures from all of the animals showed a variety of fungi. Seven species were isolated from the cortisone-treated animals and 5 species from the controls.The yeast,Torulopsis glabrata, was found in 69 of the 225 control mice and in 115 of the 213 cortisone-treated animals that came to autopsy. This organism was not considered to have been derived from the soil.The results appear to justify the conclusion that this method of soil screening deserves further consideration and study.     

Observations on paraffin baiting as a laboratory diagnostic procedure in nocardiosis

The efficacy of paraffin bait technique in the isolation ofNocardia asteroides from clinical specimens has been investigated. In a comparative study 1091 clinical specimens, mostly sputa and bronchial aspirates collected from 639 patients of bronchopulmonary diseases and 11 of meningitis, were examined by paraffin baiting and the conventional technique. Thirty-six clinical specimens originating from 12 of the patients yieldedN. asteroides by the paraffin bait technique but only 4 by the conventional technique. Approximately 95 % of 125 sputum samples inoculated withN. asteroides yielded the pathogen by paraffin baiting as against 49 % by the conventional technique. Paraffin baiting was more productive than the conventional technique in the isolation ofN. asteroides from mixed suspensions with a number of fungi and bacteria. It is concluded that paraffin baiting can be profitably adopted as a suitable technique for the isolation ofN. asteroides from clinical specimens, such as, sputum, gastric lavage, etc., which are often contaminated. The technique has no particular advantage with non-contaminated specimens.This work forms a part of the Ph. D. thesis of S.K.M. submitted in 1971 to the University of Delhi, and was presented at the Vth meeting of ISHAM held in Paris from 5th–10th July, 1971.     

3′-Phosphorylation of Aminoglycoside Antibiotics by Some Strains of Nocardia

Some strains of Nocardia were found to contain weak activities to phosphorylate aminoglycoside antibiotics in cell-free extracts. Properties of butirosin A resistant mutants derived from N. asteroides IFO 3423 were examined. An increase in their resistance to aminoglycoside antibiotics and their aminoglycoside 3′-phosphotransferase [APh(3′)] contents were shown to be well closely comparable. The findings indicate that APh(3′) of N. asteroides can be a biochemical mechanism in resistance to aminoglycoside antibiotics.

The mutant, BUR-38 with the largest increase in APh(3′) was examined for preparation of 3′-phosphate derivatives of aminoglycoside antibiotics. The derivatives were known to be useful intermediates in the chemical transformation of aminoglycoside antibiotics to more potent 3′-deoxy forms against resistant clinically-isolated bacteria. A nonionic detergent, sodium dodecyl sulfate was found to be very effective on 3′-phosphorylation of xylostasin and butirosin A by intact cells.     

Numerical Analysis of the Taxonomy of Nocardiae and Rhodococci

The genera Nocardia and Rhodococcus were clearly differentiated in the present study. Eleven characteristics were shown to be useful for differentiation between these two genera. Nocardia asteroides sunsu stricto previously defined by Tsukamura was divided into two taxa. One contained the type strain and was considered to retain the name Nocardia asteroides in a new sense. Another was named in the present study as Nocardia nova sp. nov. Tsukamura. The type strain of this species is ATCC 33726. The following seven characters were useful for differentiating N. nova from newly defined N. asteroides: 1) arylsulfatase activity after 14 days; 2) catalase activity (semiquantitative); 3) β-esterase activity; 4) pyrazinamidase activity; 5) utilization of citrate as a sole source of carbon; 6) utilization of 2,3-butylene glycol as a sole carbon source; and 7) resistance to 5-fluorouracil (20 μg/ml). The name Nocardia farcinica for Tsukamura's Kyoto-I group should be rejected. This taxon has been named Nocardia paratuberculosis sp. nov. Tsukamura. The type strain is ATCC 23826. Three new species of the genus Rhodococcus were proposed: Rhodococcus aichiensis sp. nov. Tsukamura (type strain, ATCC 33611); Rhodococcus chubuensis sp. nov. Tsukamura (type strain, ATCC 33609); Rhodococcus obuensis sp. nov. Tsukamura (type strain, ATCC 33610).     

Focus: Antimicrobial Resistance: Brain Abscess Caused by Nocardia asteroides in a Diabetic Patient: A Rare Case Report

Nocardial brain abscess is usually associated with immunodeficiency, but can sometimes emerge in healthy individuals. This infection can be acquired through inhalation or direct inoculation of the bacteria, followed by dissemination to various organs, including the brain, through blood circulation. Mortality rate due to nocardial cerebral abscess is three times higher than that associated with cerebral abscess caused by other types of bacteria. Moreover, patients with Nocardia asteroides-associated brain abscess show poorer prognosis compared to patients with brain abscess caused by other Nocardia species, which is probably due to the high tendency of N. asteroides to become resistant to numerous antibiotics. It is, therefore, of paramount importance to diagnose and treat N. asteroides cerebral abscess in patients as soon as possible. The current paper is a rare report of a brain abscess caused by N. asteroides in a diabetic patient who failed to respond to multiple antibiotics (trimethoprim/sulfamethoxazole and amikacin), but improved by receiving imipenem and linezolid, and was finally successfully treated by surgical operation and long-term antibiotic therapy (imipenem and linezolid).