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Nocardia pseudobrasiliensis sp. nov., a new species of Nocardia which groups bacterial strains previously identified as Nocardia brasiliensis and associated with invasive diseases.

We studied five strains of a new Nocardia taxon recently identified among Nocardia brasiliensis strains associated with invasive diseases (R. J. Wallace, Jr., B. A. Brown, Z. Blacklock, R. Ulrich, K. Jost, J. M. Brown, M. M. McNeil, G. Onyi, V. A. Steingrube, and J. Gibson, J. Clin. Microbiol. 33:1528-1533, 1995) to determine their taxonomic status. Several characteristics of these organisms, including the presence of chemotype IV cell walls, nocardomycolic acids, a predominant menaquinone similar to that of Nocardia asteroides ATCC 19247T (T = type strain), and G+C contents ranging from 67 to 68 mol%, are characteristics of the genus Nocardia. Phylogenies based on small-subunit ribosomal DNA sequences clearly confirmed that all five strains belong to the genus Nocardia and occur on a single branch that is clearly distinct from N. brasiliensis. This branch forms a clade with Nocardia vaccinii, Nocardia nova, Nocardia otitidiscaviarum, and Nocardia seriolae. The five new strains exhibited high levels of DNA relatedness with each other, as determined by DNA-DNA hybridization experiments (S1 nuclease procedure), but not with N. brasiliensis strains or with strains of the four phylogenetically related Nocardia species mentioned above. The five new strains differ from N. brasiliensis in the following characteristics: mycolic acid pattern, decomposition of adenine, nitrate reduction, and antimicrobial agent susceptibilities. Therefore, we propose that these strains belong to a new species, Nocardia pseudobrasiliensis. The type strain is strain ATCC 51512, which was isolated from a leg abscess on a patient suffering from ulcerative colitis.

DNA, Bacterial

Different degrees of morbidity, in the white mouse, induced by Nocardia brasiliensis, Nocardia asteroides and Nocardia caviae.

The respective degrees of morbidity induced by 6 strains each of Nocardia brasiliensis, N. asteroides and N. caviae were investigated by the method of inoculation in the plantar pad of the white mouse, using a suspension of these nocardiae at a concentration of 10 mg/0-1 ml., and inspecting the animals one month, two months and three months following inoculation. All of the strains of N. brasiliensis produced attack greater and more consistent than that of the other species, N. caviae and N. asteroides showing patterns of attack similar to each other, but substantially lower than those obtained with N. brasiliensis. The 6 strains of N. brasiliensis showed different rates of infection at different times of observation, but it was constantly high for all of them; on the other hand, the infectiousness of the different strains of N. caviae and N. asteroides was low, and there were no substantial differences between them. To sum up, these results with 6 strains of each species confirm those that were published in 1973 which dealt with only one strain of each species.

Animals

[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.

Animals

Determination of sensitivity and specificity of the Nocardia asteroides skin test for detection of bovine mammary infections caused by Nocardia asteroides and Nocardia caviae.

A total of 116 cows from 4 dairy herds in California were used to determine the sensitivity and specificity of extracellular antigen of Nocardia astero des for possible detection of bovine mastitis caused by N asteroides and N caviae. Three different positive criteria were used. These criteria gave different sensitivity and specificity to the skin test. The highest sensitivity of 80% and highest specificity of 96.04% were obtained by combining 2 criteria. The study found no cross-reaction between N asteroides antigen and Mycobacterium fortuitum or N asteroides and tuberculin skin tests. There was cross-reaction between N asteroides antigen and N caviae infection.

Animals

A phylogeny of the genus Nocardia deduced from the analysis of small-subunit ribosomal DNA sequences, including transfer of Nocardia amarae to the genus Gordona as Gordona amarae comb. nov.

According to phylogenetic analyses of nearly complete small-subunit ribosomal DNA sequences, the genus Nocardia should not comprise the two species Nocardia petroleophila and Nocardia amarae. N. amarae should be reassigned to the genus Gordona as Gordona amarae. All of the other Nocardia species form a monophyletic unit, closely related to species of the genus Rhodococcus. It is proposed to revive the name 'CMN' to comprise the genera Corynebacterium, Tsukamurella, Mycobacterium, Gordona, Rhodococcus and Nocardia that form a well identified and monophyletic unit. They are all characterized by a cell wall chemotype IV with mycolic acids.

DNA, Bacterial

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens.

Antibodies, Bacterial

DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex.

Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.

Bacterial Typing Techniques

[Distribution of pyrimidine blocks in the DNA of Brevibacterium linens, Arthrobacter globiformis, Nocardia corallina and Nocardia rubra].

The nucleotide composition and the frequency of pyrimidine blocks were studied in DNA of the following bacteria: Brevibacterium linens (Weignamm, 1910) Breed, 1953; Arthrobacter globiformis (Conn, 1928) Conn et Dimmick, 1947; Nocardia corallina (Bergey et al., 1923) Waksman et Henrici, 1948; Nocardia rubra (Krassilnikov, 1949) Waksman et Henrici, 1948. These organisms are classed by some microbiologists as mycobacteria (the Mycobacteriaceae family) while other authors regard them as representatives of three families belonging to two orders. About 60 percent of all pyrimidines in DNA of these bacteria are found in the sequences pur-pyr-pur and pur-pyr-pyr-pur, the number of dipyrimidines being higher than the amount of monopyrimidine nucleotides. The content of dipyrimidine nucleotides in DNA of Nocardia corallina and Nocardia rubra is higher (16.8 mole %) than the content of dipyrimidine blocks in DNA of Brevibacterium linens and Arthrobacter globiformis, in which the quantity of dipyrimidines is almost the same (13.9 and 14.4 mole %). A new characteristic, the selected mean value, is suggested to evaluate differences in the distribution of pyrimidines in DNA.

Arthrobacter

[Comparative electron microscopic study of cultures of Nocardia asteroides and of the Nocardia-like variant, Actinomyces chrysomallus].

The ultrastructural organization of the red-orange cultures of Nocardia (Proactinomyces) asteroides was compared with that of a Nocardia-like variant of Actinomyces (Streptomyces) chrysomallus isolated upon the inoculation of the submerged parent culture. The comparison has demonstrated for the first time that the major difference consists in the structure of their cross partitions. In Nocardia, the septa is split at all stages of its formation; in the Nocardia-like variant, the partitions have the same structure as the cultures of the genus Actinomyces (Streptomyces). Therefore, the accepted terminology is conventional.

Culture Media

Nocardia asteroides and Nocardia brasiliensis infections in mice.

A model for Nocardia asteroides and Nocardia brasiliensis infections in Swiss white mice has been established without the addition to the inocula of any form of adjuvant. Serial histopathological studies revealed that these two actinomycetes cause lesions that are quite different in their features. An acute suppurative abscess characterizes the lesions of N. asteroides. In the case of N. brasiliensis infections a granuloma is produced in which a striking feature is the presence of large numbers of foam-laden macrophages, although occasional exceptions to this pattern were noted. Electron microscopic studies demonstrated that these macrophages contain within their cytoplasm organisms in varying stages of degeneration. Repeated mortality studies in mice failed to demonstrate differences in mortality rates produced by N. asteroides and N. brasiliensis. Thus, despite relatively trivial biochemical and antigenic differences between these two species of Nocardia, the local pathogenic response is quite different. The presence in the "brasiliensis lesion" of foamy macrophages with intracellular organisms is reminiscent of the histopathological features of lepromatous leprosy and of disseminated Myocobacterium bovis infection when this occurs in the immune suppressed situation. It is possible that N. brasiliensis infection produces a depression of cellular immunity that modifies the local host response to the organism.

Animals

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG.

Different vaccination procedures to increase the mechanisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2 x 10(8) N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG-treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.

Animals

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis.

Growth curves were determined for three strains each of Nocardia asteroides and Nocardia brasiliensis. Two strains of N. brasiliensis and one strain of N. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours. The ultrastructure of the cell envelope of each Nocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains of N. brasiliensis and one strain of N. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degrees of virulence as well as the divergent pathologies exhibited by these organisms.

Cell Wall

New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease.

Nocardia brasiliensis, the second most frequently isolated aerobic actinomycete in the clinical laboratory, is usually associated with localized cutaneous infections. However, 22% of 238 N. brasiliensis isolates from the United States and 12% of 66 isolates from Queensland, Australia, which had been collected over a 17-year period, were associated with extracutaneous and/or disseminated diseases. Of the 62 invasive isolates, 37 (60%) were susceptible to ciprofloxacin and/or were susceptible to clarithromycin and resistant to minocycline, compared with only 6 (3%) of 242 localized cutaneous isolates. The 43 isolates with this susceptibility pattern appeared to define a new taxon. They were similar to Nocardia asteroides complex isolates clinically in proportions from persons with pulmonary (70%), central nervous system (23%), and/or disseminated diseases (37%) in the setting of corticosteroids (74%) or AIDS (14%). This putative new taxon differed from N. brasiliensis in the hydrolysis of adenine (92 versus 4%), beta-lactamase patterns on isoelectric focusing, and the presence of two early mycolic acid-ester peaks by high-performance liquid chromatography. Restriction analysis of a 439-bp fragment of the 65-kDa heat shock protein gene revealed that N. brasiliensis and the new taxon had different restriction patterns with 8 of the 11 enzymes tested. Screening of invasive isolates of N. brasiliensis for susceptibility to ciprofloxacin will identify most isolates of the new taxon, which likely represents a new Nocardia species.

Base Sequence

Molecular study of persistence of Nocardia asteroides and Nocardia otitidiscaviarum strains in patients with long-term nocardiosis.

Three Nocardia asteroides and five Nocardia otitidiscaviarum isolates recovered from three patients with long-term nocardiosis were compared by random amplification of polymorphic DNA fingerprinting, antibiotic susceptibility testing, and enzymatic characterization. Results obtained with primer DKU49 (P. Palittapongarnpim, S. Chomyc, A. Fanning, and D. Kunimoto, J. Infect. Dis. 167:975-978, 1993) provide evidence that patient A was infected by two N. asteroides strains during a single episode of nocardiosis and that patients B and C remained infected by the same strain, respectively. Resistance to minocycline that was present in the first isolate recovered from patient B reverted to intermediate resistance in the second isolate and reverted to susceptibility in the third isolate. Resistance to penicillin G and beta-lactams was acquired by the second isolate obtained from patient C.

DNA Fingerprinting

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

Presence and subcellular localization of two distinct mitogenic fractions in the cells of Nocardia rubra and Nocardia opaca: preparation of soluble mitogenic peptidoglycan fractions.

Fractionation of cells of Nocardia rubra and Nocardia opaca led to the separation of the cell wall and a "cytoplasmic" fraction. Both fractions were mitogenic for the splenocytes of AKR and nude mice and of rabbits. The peptidoglycan was the active part of the cell wall fraction. The products solubilized by the action of Streptomyces albus G peptidases on the peptidoglycan of N. rubra were mitogenic, but the products solubilized by lysoyme were not. Tentative structures are proposed for these fractions. The most active part of the cytoplasmic fractions could be sedimented by centrifugation and seemed to be related to the cytoplasmic membrane.

Animals