The Complexities of Nocardia Taxonomy and Identification.

Nocardia species are a complex group of organisms considered to belong to the aerobic actinomycetes. Of the validly described species, many have been implicated as the cause of serious human infections, especially in immunocompromised patients. The genus has a complicated taxonomic history; this is especially true for Nocardia asteroides, the type species of the genus and previously the most frequently reported nocardial taxon from human specimens. We provide background on the current taxonomy of Nocardia, with a focus on clinically relevant species, and discuss the currently available methods used to accurately identify isolates to the species, complex, or group level.

Laboratory evaluation of an outbreak of nocardiosis in immunocompromised hosts.

Seven patients in a renal unit were proved to have nocardiosis in an interval of nine months. Six of these patients had received renal transplants. Serologic investigation suggested that two additional cases of undiagnosed pulmonary disease were also nocardial, and that there were no subclinical cases in patients or staff. Clinical-serologic correlations indicate that serologic evaluation may be a useful adjunct in diagnosis of nocardiosis, if used early and repeatedly, and to follow response to therapy. Epidemiologic investigations yielded cultures of Nocardia asteroides from air and dust inside the unit and elsewhere in the hospital. Biochemical, metabolic, physical and immunologic characterization of the isolates indicated that those from patients and those from the unit environment were identical, whereas some from outside the unit could be differentiated from these. The "epidemic strain" had type III antigen, which surveys indicated is not the most common type in human nocardiosis (it occurs in association with a minority of human cases). The isolates were of subgroup B, which has been associated with virulence. The characterization methods employed could be useful in studies of nocardial epidemiology. The laboratory studies indicate epidemic spread within the unit of a single organism, and current epidemiologic guidelines, which do not recommend respiratory isolation of cases of pulmonary nocardiosis, may need reconsideration particularly when there are immunocompromised hosts in the environment.

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

Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified to 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.

Nocardial cerebral abscess associated with mycetoma, pneumonia, and membranoproliferative glomerulonephritis.

Nocardial brain abscesses remain a clinical challenge. We successfully treated a patient with nocardial brain abscess, mycetoma, pneumonia, and glomerulonephritis. Nocardial soft tissue involvement, mycetoma, is well known. However, the fact that actinomycetoma can metastasize may not be as well appreciated. The association between nocardiosis and glomerulonephritis should be better clarified.

Numerical classification of some named strains of Nocardia asteroides and related isolates from soil.

One hundred and forty-nine strains of nocardiae, freshly isolated from soil samples obtained from a number of countries with either tropical or temperate climates, and from rubber pipe seals, were compared with appropriate marker cultures in a numerical phenetic study using 156 unit characters. Marker strains were chosen to represent the Nocardia asteroides complex, other Nocardia species and related taxa in an effort both to classify the new soil isolates and, possibly, clarify the structure of the heterogeneous N. asteroides complex. The data were examined using the simple matching (SSM) and pattern (DP) coefficients, and clustering was achieved using both single and average linkage algorithms. Cluster composition was not markedly affected by either of the coefficients or clustering methods. The estimated test error of 7.1% was rather high and could account for a few apparently anomalous results. The 16 defined clusters, containing 185 of the 197 strains studied, were divided into seven major and nine minor clusters, four of which were further subdivided into two subclusters. Marker strains allowed four clusters to be designated as N. asteroides, seven as Nocardia species and one each as Nocardia carnea, Nocardia farcinica, Nocardia autotrophica, Mycobacterium farcinogenes and Rhodococcus species. Twelve strains formed single member clusters including the type strains of Nocardia aerocolonigenes, Nocardia amarae, Nocardia fukuyae, Nocardia orientalis and Nocardia otitidis-caviarum. The majority of the soil and rubber isolates were recovered in the major clusters labelled N. asteroides, N. carnea and Nocardia species and clusters of soil isolates without marker strains seem to represent new centres of variation. The study highlights the need for additional reproducible tests to help both define and determine the status of defined clusters within the N. asteroides complex which would considerably benefit both the ecological and epidemiological study of these organisms.

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.

Antigenic relationship among Nocardia asteroides immunotypes.

Culture filtrate antigens from seven Nocardia asteroides immunotypes, and N caviae and N. brasiliensis, were studied by agar gel diffusion using anti-Nocardia sera and immune sera absorbed with cross-reacting antigens. It was found that all seven immunotypes demonstrated specific antigenic determinants. A few immunotypes showed cross-reactivity with other immunotypes. However, this cross-reactivity could be completely removed by absorbing the antibody with the cross-reacting antigen without major change in the reactivity with homologous antigen and antisera. This study clearly demonstrates that the various immunotypes are antigenically distinct, and that for the demonstration of antibody in patients, antigens from various immunotypes are to be used.

Relationships between structure and biological activity of the mycolic acid-containing glycolipids from Nocardia asteroides "sensu stricto" and related species.

To reveal the taxonomical situation of Nocardia asteroides "sensu stricto", we compared the mycolic acid and mycolic acid-containing glycolipid composition and their granulomagenic activities in mice. The major glycolipids were glucose mono- and dimycolate, trehalose mono- and dimycolate and several unknown glycolipids, commonly, although the relative amount differed from strain to strains. On the other hand, molecular species composition of mycolic acids differed distinctively among the three closely related species: N. asteroides "sensu strico", N. farcinica and N. nova. GC/MS analysis showed the most abundant species of mycolic acids were C50(52) in N. asteroides, C54(52) in N. farcinica and C58(56) in N. nova, respectively with a different alpha-alkyl branch. Glucose mycolate and trehalose dimycolate possessing C50 mycolic acid showed a strong activity for granuloma formation in mice.

Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons.

Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.

Rapid identification of Nocardia farcinica clinical isolates by a PCR assay targeting a 314-base-pair species-specific DNA fragment.

Nocardia farcinica is the most clinically significant species within the Nocardia asteroides complex. Differentiation of N. farcinica from other members of N. asteroides complex is important because this species characteristically demonstrates resistance to several extended-spectrum antimicrobial agents. Traditional phenotypic characterization of this species is time- and labor-intensive and often leads to misidentification in the clinical microbiology laboratory. We previously observed a 409-bp product for all strains of N. farcinica by using randomly amplified polymorphic DNA analysis with the primer DKU49. In this investigation, the 409-bp fragment was sequenced and then used to design a specific primer pair, Nf1 (16-mer) and Nf2 (16-mer), complementary to the 409-bp fragment. PCR amplification of genomic DNA from 28 N. farcinica isolates with Nf1 and Nf2 generated a single intense 314-bp fragment. The specificity of the assay with these primers was verified, since there were no PCR amplification products observed from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41). Restriction enzyme digestion using CfoI and direct sequencing of the 314-bp fragment further confirmed the specificity of the assay for N. farcinica. This highly sensitive and specific PCR assay provides a rapid (within 1 day of obtaining DNA) method for identification of this medically important emerging pathogen. Rapid diagnosis of N. farcinica infection may allow for earlier initiation of effective therapy, thus improving patient outcome.

Bacteriophages of Nocardia asteroides.

101 Nocardia strains from different species and different sources were examined with respect to their lysogeny. 18 phages from 17 Nocardia asteroides strains could be isolated, purified, and propagated. All phages were released spontaneously. Induction experiments with mitomycin C and UV-rays brought no practicale advantages. Using 14 of the 18 phages, 38 of the 101 Nocardia strains tested could be grouped into 18 phage patterns. Nearly all of the typable strains belonged to the species Nocardia asteroides.

Distribution of serotypes of Nocardia asteroides from animal, human, and environmental sources.

The antigenic types of 129 isolates of Nocardia asteroides from diverse clinical, environmental, and geographic origins were determined. The majority of the isolates studied were of bovine (56) or human (44) origin; 11 were derived from six species of animals other than cattle, and 10 were isolated from environmental sources; the source of 8 strains could not be determined. Testing culture filtrate antigens against four standard reference sera in a gel diffusion precipitin test established the antigenic type of 95.3% of the isolates. After excluding strains that weighted the data because of common infection, the distribution of serotypes was examined according to the origin of the isolate. Type I was the most frequently encountered serotype (31.9%); types III (15.0%) and IV (20.4%) were also observed frequently, as was the antigenic mixture III + IV (14.2%). There was an apparent difference in frequency of type III and IV antigens among isolates of bovine and human origin; type III made up 20.0% of the bovine isolates and 13.6% of the human isolates, whereas type IV constituted 10.0% of bovine and 27.3% of human isolates.

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, alpha-glucosidase and beta-glucosidase. In addition, beta-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-beta-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in > 95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-glucoronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase and alpha-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.

A case of lung infection caused by an unusual strain of Nocardia farcinica.

A case of lung infection caused by an unusual strain of Nocardia farcinica is reported. This is the third case of the N. farcinica infection in this country. The strain failed to utilize rhamnose as sole carbon source, but could be identified by a numerical identification method. The mycolic acids contained 1-3 double bonds and the numbers of the carbon atoms of the mycolic acids were 50 to 60, average 56.

Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica.

A recent study of Nocardia asteroides revealed that 95% of clinical strains had one of five antibiotic resistance patterns. We found the pattern of resistance to cefotaxime and cefamandole in 19% of 200 clinical N. asteroides isolates. Isolates with this drug resistance pattern were from numerous geographic sources and were associated with significant clinical disease (56% of patients had disseminated infections). Phenotypic studies revealed that these isolates were relatively homogeneous and matched previous descriptions and reference strains of the controversial species N. farcinica. Growth at 45 degrees C, acid production from rhamnose, ability to utilize acetamide as a nitrogen and carbon source, and resistance to tobramycin and cefamandole were features of N. farcinica that could be tested in the clinical laboratory and allowed their distinction from N. asteroides. The serious nature of disease due to N. farcinica and its resistance to the newer cephalosporins suggest a clinical need for laboratory identification of this species. (Current tests used in clinical laboratories do not distinguish N. farcinica from N. asteroides.) This is the first recognition that N. farcinica has a specific drug resistance pattern and confirms the previously described concept that drug resistance patterns of N. asteroides may be associated with specific taxonomic groups.

Characterization of Nocardia asteroides isolates from different ecological habitats on the basis of repetitive extragenic palindromic-PCR fingerprinting.

Thirteen isolates of Nocardia asteroides from both soils and aquatic samples (lake and moat sediments, as well as scum from activated sludge), together with a type strain and two known clinical isolates of this species, were characterized by repetitive extragenic palindromic-PCR fingerprinting with the BOX-A1R primer. The resulting DNA fingerprint patterns proved to be strain specific, and cluster analysis distinguished the soil isolates, the aquatic isolates, and the known strains as being in separate groups.