Abstract
Two different bacterial strains with different drug susceptibilities were isolated from the sputum and an inflammatory discharge from a swelling in the left thigh of a patient with rheumatoid arthritis. Both bacterial strains were provisionally assigned to the genus Nocardia on the basis of their morphological and chemotaxonomic characteristics and were further studied in order to establish their taxonomic status. One strain (IFM 10034) was identified as Nocardia farcinica on the basis of its physiological characteristics. The other strain, which was designated Nocardia sp. strain IFM 10035T, revealed a unique pattern of phenotypic properties that distinguished it from other representatives of established Nocardia species. Comparative 16S rRNA gene sequence studies of Nocardia sp. strain IFM 10035T also showed that the bacterium was closely related to the species Nocardia beijingensis. Determination of DNA-DNA relatedness, however, indicated that Nocardia sp. strain IFM 10035T could be delineated from N. beijingensis. The genotypic and phenotypic data combined indicated that the bacterium merits description as a new Nocardia species. The name proposed for the new species is Nocardia arthritidis sp. nov., the type strain being IFM 10035T (NBRC 100137T, JCM 12120T, DSM44731T). The present study suggests that Nocardia infections can be caused by multiple species of the bacterium.
Nocardiosis is a potentially life-threatening infection caused by several species of the genus Nocardia (33). Systemic disease is mostly due to infections caused by the Nocardia asteroides group, Nocardia asteroides sensu stricto, Nocardia farcinica, and Nocardia nova (2, 9, 22, 31, 34). N. asteroides is the major cause of nocardiosis; however, from many taxonomic studies N. asteroides is considered a heterogeneous species complex (25, 29, 32).
In the present study, two different strains from the N. asteroides group were isolated from the sputum and inflammatory exudates of a patient with rheumatoid arthritis (RA). One of the strains was readily identified as N. farcinica (strain IFM 10034) on the basis of its morphological and biochemical characteristics, while the other strain was not described as any reported Nocardia species of the N. asteroides group. The aim of this study was to clarify the taxonomic position of this novel Nocardia isolate, IFM 10035T (NBRC 100137T, JCM 10120T, DSM 44731T), by morphological, physiological, chemotaxonomic, and molecular biological methods; and the name Nocardia arthritidis is proposed (Biological Resource Center, Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Japan).
CASE REPORT
Strain IFM 10035T was isolated from the sputum of a 65-year-old Japanese male patient with RA and lung nocardiosis, whereas strain IFM 10034 was concurrently isolated from an inflammatory abscess in his left thigh. The patient had a history of diabetes mellitus and pneumoconiosis. He had been diagnosed with RA in 1995 and membranous glomerulopathy in 1996 and was treated with prednisolone (8 mg a day) at the time of diagnosis of lung nocardiosis (November 2000). The swelling in his thigh was increasing in size and caused the patient considerable pain. Laboratory findings showed the following: hemoglobin concentration, 11.3 g/dl; leukocyte count, 15,200/μl (82% neutrophils, 9% lymphocytes); platelet count, 461 × 109/liter; C-reactive protein concentration, 7.6 mg/dl; total protein concentration, 5.4 g/dl; albumin concentration, 2.2 g/dl; creatinine concentration, 0.54 mg/dl; blood glucose concentration, 205 mg/dl; hemoglobin A1C level, 6.8%; serum immunoglobulin G titer, 748 mg/dl; and urine protein level, 0.5 to 1.0 g/day. Flow cytometry analysis revealed 22.4% CD4-positive T lymphocytes (306/μl).
MATERIALS AND METHODS
Morphology.
Strain IFM 10035T and Nocardia beijingensis IFM 10174T were cultured on Mueller-Hinton II (MH II; Difco, Detroit, Mich.) slants with 1% glucose and 1% glycerol for 1 week at 30°C. Strain IFM 10035T and N. beijingensis IFM 10174T were then cultured on MH II agar plates with 1% glucose and 1% glycerol for 1 week at 30°C for colonization experiments. Morphological observations, performed under a scanning electron microscope (model S-5200; Hitachi), were performed with cultures grown on MH II agar at 30°C for 7 to 10 days.
Physiological characteristics and drug susceptibility testing.
The decomposition of adenine, casein, hypoxanthine, tyrosine, urea, and xanthine was examined by using the methods of Gordon et al. (6). Acid production from various carbohydrates and growth at 37 and 45°C were determined by the modified methods of Poonwan et al. (26). Arylsulfatase activity was determined by the method of Miyado (23).
MICs were obtained by the broth dilution method on the basis of an experimental report by Mikami and Yazawa (19) and the tentative NCCLS guidelines (24) with an MIC 2000 autoinoculator (Nagase Inc. Co., Tokyo, Japan).
Chemotaxonomic characteristics.
Whole-cell hydrolysates were analyzed for diaminopimelic acid isomers and sugars by thin-layer chromatography (21, 23). 3-Hydroxy fatty acids (mycolic acids) and menaquinones were prepared as reported previously (15, 23). The strain was grown for fatty acid analysis for 7 days at 28°C in petri dishes on Trypticase soy broth agar (medium 535; Deutsche Sammlung von Mikroorganismen und Zellkulturen). Three to four inoculation loops of cell material were scraped from the plates and used for the analyses for the other fatty acids. Fatty acid methyl esters were obtained from cells after saponification, methylation, and extraction as described previously (20). The fatty acid methyl ester mixtures were separated by using a 5% phenyl-methyl silicone capillary column (0.2 mm by 25 m) and a gas chromatograph (model 5898A; Hewlett-Packard, Avondale, Del.) controlled with MIS software (MIDI Inc., Newark, Del.). Peaks were automatically integrated, and fatty acid names and percentages were determined with the Microbial Id standard software package (28). The following components and conditions were applied: carrier gas, ultra-high-purity hydrogen; column head pressure, 60 kPa; injection volume, 2 μl; column split ratio, 100:1; septum purge, 5 ml/min; column temperature, 170 to 270°C at 5°C/min; injection port temperature, 250°C; and detector temperature, 300°C.
16S rDNA sequencing and phylogenetic analysis.
A nearly complete 16S rRNA gene (rDNA) sequence of the isolated strain was obtained. The 16S rDNA was amplified and sequenced by PCR with prokaryotic 16S rDNA universal primer pairs 8F and 691R, 520F and 1100R, and 926F and 1542R. PCR was performed in a DNA thermal cycler (TaKaRa Biochemicals, Osaka, Japan) by using 35 cycles, each of which consisted of denaturation at 94°C for 60 s, primer annealing at 60°C for 60 s, and primer extension at 72°C for 120 s. The strains were cultured on brain heart infusion (BHI; Difco) broth with 0.1% glucose and 0.1% glycerol for 5 days at 30°C for DNA extraction and sequencing. Preparation of genomic DNA samples for sequencing was performed by the guanidine thiocyanate method (10, 11). The DNA sequences were determined with an automated sequence analyzer (ABI PRISM 3100; PE Applied Biosystems, Foster City, Calif.) and a dye terminator cycle sequencing kit (PE Applied Biosystems). The sequence data for related species were retrieved from GenBank. Nucleotide substitution (KNUC) rates were calculated (12), and phylogenetic trees were constructed by the neighbor-joining method (27). The topology of the trees was evaluated by bootstrap analysis of the sequence data with CLUSTAL W software (33).
DNA base composition and DNA-DNA hybridization.
The bacterial strain was cultured on BHI broth with 2% glucose and 2% glycine for 3 days at 30°C for DNA-DNA hybridization analyses. The DNA base composition was estimated by high-pressure liquid chromatography (23). The levels of DNA-DNA relatedness were determined by the method of Ezaki et al. (3) by using photobiotin and a microplate format.
Nucleotide sequence accession number.
The DDBJ accession number for the 16S rDNA sequence of N. arthritidis IFM 10035T is AB108781.
RESULTS AND DISCUSSION
Taxonomic studies.
The chemotaxonomic and morphological characteristics of strain IFM 10035T were consistent with its assignment to the genus Nocardia (4, 5, 14, 17). This strain also contained galactose and arabinose as characteristic whole-cell sugars, in addition to meso-diaminopimelic acid as the dominant cell wall diamino acid. The strain also contained mycolic acid, which is characteristic of the genera Nocardia and Rhodococcus. The major menaquinone was MK-8(H4ω-cycle). The analyses of the fatty acids by gas-liquid chromatography revealed the expected pattern diagnostic for members of the genus Nocardia and related taxa, i.e., straight-chain saturated and unsaturated fatty acids together with a diagnostic amount of tuberculostearic acid (10-methyl branched octadecanoic acid). Strain IFM 10035T could be separated from the closely related species N. farcinica and N. beijingensis and from N. nova and N. asteroides by a combination of qualitative and quantitative differences in its fatty acid pattern (Table 1). Strain IFM 10035T was also examined for a set of phenotypic characteristics (Table 2), and it was clear from this information that the present strain could be distinguished from other validly described Nocardia species by a combination of phenotypic characteristics, such as decomposition of xanthine, utilization of carbohydrates, and maximum growth temperature. Comparison of the nearly complete 16S rDNA sequence from strain IFM 10035T and the corresponding sequences of other Nocardia species (1, 7, 8) showed that the strain forms a monophyletic clade with N. beijingensis in the neighbor-joining tree (Fig. 1). The sequence similarity value between strain IFM 10035T and N. beijingensis IFM 10174T was 98.9%, and a high bootstrap value (98%) was obtained by neighbor-joining analysis. However, strain IFM 10035T and N. beijingensis IFM 10174T share a DNA-DNA relatedness value of only 14 to 17%. This value was well below the 70% cutoff point for a species classification, as recommended by Wayne et al. (36). The unique 16S rDNA, low level of DNA-DNA hybridization, and phenotypic results supported the conclusion that strain IFM 10035T is a new species of the genus Nocardia. We therefore propose the name N. arthritidis sp. nov.
TABLE 1.
Fatty acid compositions of N. arthritidis IFM 10035T, N. beijingensis IFM 10174T, N. farcinica DSM 43665T, N. nova DSM 44481T, and N. asteroides DSM 43757T
| Fatty acida | Composition (%)
|
||||
|---|---|---|---|---|---|
| IFM 10035T | IFM 10174T | DSM 43665T | DSM 44481T | DSM 43757T | |
| 14:0 | 1.71 | 1.06 | 0.88 | 0.90 | 0.36 |
| Unknown (ECL 14.959) | 1.50 | 1.22 | |||
| 15:1 | 0.39 | ||||
| 15:0 | 0.25 | trb | 1.58 | ||
| cis-7-16:1 | 0.30 | 0.27 | |||
| cis-8-16:1 | 1.17 | 0.87 | |||
| cis-9-16:1 | 9.35 | 8.69 | 15.35 | 9.74 | 18.46 |
| 16:0 | 32.47 | 30.01 | 22.88 | 36.87 | 28.79 |
| 10-Methyl-16:0 | 0.48 | ||||
| cis-9-17:1 | 1.36 | tr | 3.67 | ||
| 17:0 | 3.41 | 3.40 | 1.15 | 5.08 | |
| 10-Methyl-17:0 | 1.44 | ||||
| cis-9-18:1 | 20.04 | 20.65 | 22.01 | 24.69 | 21.34 |
| cis-11-18:1 | 0.79 | ||||
| 18:0 | 19.43 | 13.59 | 9.25 | 10.02 | 4.44 |
| 10-Methyl-18:0 | 12.59 | 15.39 | 14.96 | 16.63 | 12.58 |
| cis-9 19:1 | 0.69 | 1.39 | |||
| 19:0 | 0.49 | ||||
| cis-11 20:1 | 0.59 | 5.08 | tr | ||
| 20:0 | 0.94 | tr | 0.46 | ||
cis-9-18:1, cis-9 octadecenoic acid (oleic acid); 10-methyl-18:0, 10-methyl-octadecanoic acid (tuberculostearic acid).
tr, < 0.25%.
TABLE 2.
Phenotypic properties that distinguish isolate strains from type strains of Nocardia speciesa
| Property | Result for species
|
|||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IFM 10035T | Nocardia abscessus DSM 44432T | Nocardia africana DSM 44491T | Nocardia asteroides ATCC 19247T | Nocardia brasiliensis ATCC 19296T | Nocardia brevicatena DSM 43024T | Nocardia beijingensis JCM 10666T | Nocardia carnea DSM 43397T | Nocardia cerradoensis DSM 44546T | Nocardia crassostreae ATCC 70418T | Nocardia cummidelens DSM 44490T | Nocardia cyriacigeorgica DSM 44484T | Nocardia farcinica ATCC 3318T | Nocardia flavorosea JCM 3332T | Nocardia fluminea DSM 44488T | Nocardia ignorata DSM 44496T | Nocardia nova JCM 6044T | Nocardia otitidiscaviarum NCTC 1934T | Nocardia paucivorans DSM 44386T | Nocardia pseudobrasiliensis ATCC 51512T | Nocardia salmonicida JCM 4826T | Nocardia seriolae JCM 3360T | Nocardia soli DSM 44488T | Nocardia transvalensis DSM 43405T | Nocardia uniformis JCM 3224T | Nocardia vaccinii DSM 43285T | Nocardia veterana DSM 44445T | Nocardia vinacea JCM 10302T | |
| Nitrate reduction | + | − | + | + | + | − | + | + | + | + | + | − | + | + | + | − | + | + | + | + | + | + | − | + | ||||
| Decomposition of: | ||||||||||||||||||||||||||||
| Adenine | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | − | − | ||
| Casein | − | − | + | − | + | − | − | − | − | − | − | − | − | − | − | + | − | − | − | + | − | − | − | − | − | − | − | − |
| Hypoxanthine | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | − | − | + | + | − | − | + |
| Tyrosine | − | − | − | − | + | − | − | − | − | − | − | − | − | − | + | − | − | − | − | + | + | − | − | − | + | − | − | − |
| Urea | + | − | − | − | − | − | − | − | − | − | − | + | − | − | + | + | − | − | − | + | + | − | + | |||||
| Xanthine | − | − | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | + | − | − | − |
| Esculin | w | − | − | + | + | + | + | + | + | + | + | + | − | + | + | + | + | − | + | + | + | + | + | + | + | |||
| Arbutine | + | − | + | + | + | − | + | + | + | + | + | − | + | + | + | + | + | − | ||||||||||
| Elastine | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | + | + | − | − | |||
| Testosterone | + | + | + | + | + | + | + | + | + | + | + | − | − | − | + | − | + | + | + | − | + | |||||||
| Utilization of: | ||||||||||||||||||||||||||||
| Acetate | + | + | − | + | + | + | + | + | + | + | + | + | − | + | + | + | + | + | + | + | + | + | + | − | − | |||
| Adonitol | − | |||||||||||||||||||||||||||
| Arabinose | − | − | − | − | − | − | − | − | − | − | − | − | − | + | ||||||||||||||
| Erythritol | − | − | − | − | − | − | − | − | − | − | − | − | + | − | ||||||||||||||
| Galactose | − | − | − | + | − | − | − | − | + | − | − | + | + | + | ||||||||||||||
| Glucose | + | + | + | + | + | − | + | + | + | + | − | + | + | + | ||||||||||||||
| Inositol | − | − | − | + | − | − | − | − | − | + | − | − | − | − | ||||||||||||||
| Maltose | − | + | − | + | − | + | + | − | − | − | − | + | − | |||||||||||||||
| Mannose | − | − | − | + | ||||||||||||||||||||||||
| Rhamnose | − | + | − | − | − | + | + | − | + | − | − | + | − | + | − | − | − | − | − | − | − | + | + | − | + | + | − | |
| Sorbitol | − | − | − | − | − | − | + | + | + | − | − | − | − | + | − | − | + | + | − | − | + | − | − | + | ||||
| Citrate | + | + | − | + | + | − | + | − | − | − | − | − | − | − | + | − | − | − | − | + | + | + | − | − | − | − | − | |
| Adipic acid | − | |||||||||||||||||||||||||||
| Acetamide | − | |||||||||||||||||||||||||||
| Gluconate | − | − | + | |||||||||||||||||||||||||
| Arylsulfatase activity | − | + | ||||||||||||||||||||||||||
| Growth at: | ||||||||||||||||||||||||||||
| 37°C | + | + | + | + | + | + | − | + | + | + | + | + | + | − | − | − | + | + | + | + | ||||||||
| 45°C | + | − | + | − | + | − | − | − | − | − | − | + | + | − | + | − | + | − | − | − | − | − | − | − | + | − | ||
FIG. 1.
Phylogenetic tree derived from 16S rDNA sequences. The tree was created by the neighbor-joining method with KNUC values (12). The numbers on the tree indicate bootstrap values for the branch points. Only values above 50% significance are indicated.
Therapy and drug susceptibility.
Trimethoprim-sulfamethoxazole was chosen as treatment for this patient after a tentative diagnosis of pulmonary nocardiosis as a result of finding a filamentous gram-positive bacillus in his sputum. However, the patient had adverse reactions, such as general fatigue, loss of appetite, and renal dysfunction. Therefore, imipenem therapy was started, and the effect was only marginal because the C-reactive protein value decreased from 7.6 to 2.4 mg/dl 10 days after administration but returned to 7.6 mg/dl in the next 16 days even with continuous treatment with imipenem. Since our susceptibility study indicated that both the N. arthritidis and the N. farcinica strains isolated from the same patient were susceptible to minocycline (MICs, 1 μg ml−1), minocycline therapy was started. The patient responded well to minocycline, and the nocardiosis resolved 6 months after the administration of minocycline. No recurrence of the infection has been noted for about 2 years.
The drug susceptibility patterns of N. farcinica IFM 10034 and N. arthritidis IFM 10035T were also compared (Table 3). N. farcinica IFM 10034 was highly susceptible to ampicillin and gentamicin, but it was only slightly susceptible to erythromycin. N. arthritidis IFM 10035T was resistant to ampicillin, gentamicin, clindamycin, and erythromycin. Differences in drug susceptibilities were observed between the two strains; and these data indicate the importance of careful drug selection for the treatment of the concurrent nocardial infections, because each Nocardia species shows species-specific drug susceptibility patterns.
TABLE 3.
Drug susceptibilities of two clinical isolates of N. arthritidis IFM 10035T and N. farcinica IFM 10034
| Drugs used | MIC (μg/ml)
|
|
|---|---|---|
| IFM 10035T | IFM 10034 | |
| Imipenem | 4 | 4 |
| Ampicillin | >16 | 0.5 |
| Piperacillin | >32 | 16 |
| Oxacillin | >32 | >32 |
| Cefaclor | 16 | >32 |
| Cefazolin | 8 | >32 |
| Cefotaxime | 16 | 16 |
| Gentamicin | >16 | 0.5 |
| Arbekacina | 0.5 | 0.5 |
| Minocycline | 1 | 1 |
| Ofloxacin | 4 | 16 |
| Erythromycin | >16 | 8 |
| Clindamycin | >16 | 8 |
| Vancomycin | >16 | >16 |
| Fosfomycin | >32 | >32 |
An aminoglycoside antibiotic (16).
Phylogenetic and pathogenic relationship of N. arthritidis to other pathogenic Nocardia.
N. beijingensis, a recently established species, was isolated from soil in China (35). A recent report (10) described the first case of human infection due to N. beijingensis, and it was proposed that members of the species N. beijingensis be categorized as an opportunistic infection group, regardless of their original isolation from soil. Phylogenetically, N. arthritidis is most closely related to the recently characterized species N. beijingensis. Most clinical manifestations due to N. beijingnsis isolates were found in the lungs of patients from Japan and Thailand (10), suggesting the high degree of association of N. beijingensis with pulmonary or systemic disease. N. arthritidis IFM 10035T was also an isolate from a patient with pulmonary nocardiosis, indicating that both Nocardia species can be classified into the members of a pathogenicity group that invade the lungs in a manner similar to that for N. farcinica and Nocardia pseudobrasiliensis. Although N. arthritidis IFM 10035T and N. beijingensis are phenotypically and pathologically closely related, both bacteria are differentiated by characteristics such as the decomposition of xanthine, nonutilization of rhamnose and sorbitol, and growth at 45°C.
Nocardia species can usually be distinguished in most clinical laboratories by use of a combination of biochemical, chemical, and morphological features. However, most laboratories have difficulty with identification of the species of the N. asteroides group using these methods, necessitating the introduction of genotypic methods into several clinical laboratories. Such genotypic tests may show that concurrent infections (30) are more common than generally believed in immunocompromised patients.
Description of N. arthritidis sp. nov.
Nocardia arthritidis (ar.thri′ti.dis. Gr. n. arthron, joint; L. suff. -itis or -itidis, inflammation; N.L. gen. n. arthritidis, of arthritis).
The bacterium is an aerobic, gram-positive, acid-alcohol-fast, nonmotile actinomycetes which forms a branched orange substrate mycelium that fragments into irregular rod-shaped elements (0.3 to 0.6 by 1.4 to 1.7 μm in diameter). It carries pale orange aerial hyphae on BHI agar medium. Melanin pigments were not produced on the organic agar media tested.
The dimensions of the colonies are 0.5 to 1.2 mm after growth for 7 days at 30°C on MH II medium with 0.2% glucose. The organism utilizes glucose but not adonitol, arabinose, erythritol, galactose, inositol, maltose, rhamnose, or sorbitol. It hydrolyzes arbutine and urea and weakly hydrolyzes esculin; but it does not hydrolyze adenine, casein, elastine, hypoxanthine, tyrosine, or xanthine. The organism utilizes sodium citrate and testosterone, but not adipic acid or gluconate. Arylsulfatase is not produced.
The strain grows at 45°C. The G+C content of total genomic DNA is 68 mol%. The strain was isolated from a patient with RA. IFM 10035T is the type strain of N. arthritidis.
Acknowledgments
We thank T. Tamura and K. Suzuki (National Institute of Technology and Evaluation) for advice on scanning electron microscopic observation.
This work was partly supported by the program “Frontier Studies and International Networking of Genetic Resources in Pathogenic Fungi and Actinomycetes (FN-GRPF)” through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (2001-2003) (to Y.M.).
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