Abstract
Nocardia isolates that share the property of in vitro amikacin resistance are grouped together by some authors in the Nocardia transvalensis complex. Our examination of 13 isolates that are amikacin resistant has revealed the existence of three distinct species. Sequence analysis of the 16S rRNA, 65-kDa heat shock protein, and secA1 genes, coupled with DNA-DNA hybridization, indicated that “N. asteroides drug pattern IV,” “N. transvalensis new taxon 1,” and N. transvalensis sensu stricto should each be considered a distinct species. The phenotypic and molecular characteristics of the proposed new species Nocardia wallacei (N. asteroides drug pattern IV) and N. blacklockiae (N. transvalensis new taxon 1) are presented and compared with those of N. transvalensis sensu stricto. The relative genetic diversity of isolates best placed with the species N. blacklockiae is also discussed. Case studies demonstrating the pathogenicity of N. wallacei and N. blacklockiae are presented. The type strain of N. wallacei is ATCC 49873 (DSM 45136), and that of N. blacklockiae is ATCC 700035 (DSM 45135).
Nocardia transvalensis was first described by Pijper and Pullinger in 1927 as the causative agent of mycetoma of the foot in a South African patient (17); this isolate was later characterized biochemically by Gordon et al. (11). N. transvalensis has since been found to be an uncommon but significant pathogen capable of causing disseminated infections, particularly in immunocompromised patients. In 1992, McNeil et al. reported on the recovery of N. transvalensis from 16 patients from Australia, Canada, Nigeria, Spain, and the United States, 10 of whom had clinical and/or histopathologic evidence of infection (16). Of these patients with proven pulmonary or disseminated infection, six had definable underlying immunosuppressive conditions. While susceptibility to specific antimicrobials varied among the isolates, it was apparent that these N. transvalensis isolates showed high levels of antibiotic resistance, especially to the aminoglycosides. In addition, the results of biochemical testing showed considerable variability among the isolates, suggesting the presence of several biotypes within the N. transvalensis group.
In 1988, Wallace et al. reported the presence of six patterns of antibiotic susceptibility among Nocardia isolates identified biochemically as N. asteroides (19). One of these patterns, drug pattern IV, showed a high level of resistance to aminoglycosides, including amikacin. Using amplification and subsequent restriction endonuclease analysis (REA) of a portion of the 65-kDa heat shock protein gene (HSP), Steingrube et al. were able to differentiate three restriction fragment length polymorphisms (RFLPs) in isolates determined to be amikacin resistant (18). Isolates identified phenotypically as N. asteroides drug pattern IV showed a single unique RFLP pattern, while isolates identified as N. transvalensis showed one of two different RFLP patterns.
In 1997, Wilson et al. (23) examined the biochemical characteristics, molecular differences (as determined by REA of a portion of the HSP gene), and cell wall composition of Nocardia isolates that were determined to be resistant to amikacin or that had been determined phenotypically to belong to N. transvalensis. On the basis of their results, isolates identified as N. asteroides drug pattern type IV were determined to be more closely related to N. transvalensis than to other members of the N. asteroides complex. Isolates of their N. transvalensis complex were assigned to one of four distinct groups: N. transvalensis sensu stricto, N. asteroides drug pattern IV, and N. transvalensis new taxons 1 and 2. Wilson et al. found that these four groups were best defined using REA of the HSP gene with six restriction endonucleases (23).
Here we describe our findings regarding the molecular, biochemical, and susceptibility characteristics of the reference strains of N. asteroides drug pattern type IV, and N. transvalensis new taxon 1, and clinical isolates assignable to two of these three taxonomic groups. In addition, we compare these findings to the molecular and biochemical characteristics of N. transvalensis sensu stricto. As no reference strain for N. transvalensis new taxon 2 is available, we did not pursue the study of this taxon. We also propose species designation for N. asteroides drug pattern IV (N. wallacei) and N. transvalensis new taxon 1 (N. blacklockiae). Additionally, we provide data confirming the clinical significance of these two proposed species.
CASE REPORTS
Case 1 (isolate 1, N. wallacei).
The patient was a 57-year-old male who presented in 1979 with a 3-week history of chills, fever, cough, and left pleuritic pain. He had a history of carcinoma of the tongue that had been treated with radiotherapy and Pneumocystis jirovecii pneumonia that had been diagnosed by an open lung biopsy 2 years earlier. On admission, the patient was found to have a new extensive left lung infiltrate and a left pleural effusion. No results could be found for an acid-fast stain, but Gram stain results indicated sparse Nocardia-like organisms and many polymorphonuclear cells. Cultures of pleural fluid and sputum grew a Nocardia species, as determined by phenotypic methods. The organism was later identified as N. asteroides drug pattern IV by REA of the HSP gene. Susceptibility testing was performed by disk diffusion at a reference laboratory and indicated resistance to amikacin. Subsequent testing at the Centers for Disease Control and Prevention (CDC) confirmed the resistance of this isolate to multiple drugs, including amikacin. The patient became afebrile within 24 h of beginning trimethoprim-sulfamethoxazole treatment. Immunologic evaluation revealed defective macrophage function, but lymphocyte studies revealed no abnormalities. The patient became febrile again, with progression of his lung infiltrates, after 4 months of therapy. He developed an altered mental status and aseptic meningitis. Therapy with multiple additional agents including amikacin failed, and he died of his disease.
Case 2 (isolate 6, N. blacklockiae).
A 37-year-old Australian aborigine with a history of chronic alcoholism presented with a 3-week history of fever, chest pain, and cough with purulent sputum production. A chest radiograph showed consolidation and volume loss of the left upper lobe. Microscopy of Gram-stained sputum showed gram-positive branching filamentous organisms, and testing with a modified acid-fast stain was positive. Cultures of sputum grew a Nocardia species that was identified as N. transvalensis by phenotypic characteristics. The organism was later identified as a member of the N. transvalensis complex by REA of the HSP gene. MICs were determined in a reference laboratory and indicated resistance to amikacin. The patient was initially treated with intravenous gentamicin and sulfadiazine; after 1 week the antibiotics were changed to oral sulfadimidine and trimethoprim-sulfamethoxazole. A chest radiograph after 14 days of therapy demonstrated persistent left upper lobe consolidation and new cavitation. The drug regimen was again modified to include sulfadimidine, amoxicillin, and amikacin. Clinical and radiographic improvements were evident after 3 weeks of antibiotic treatment; however, considerable residual fibrosis remained in the left upper lobe. This case was previously reported in a discussion of the antibiotic susceptibility of N. transvalensis and of the spectrum of clinical illness attributed to the organism (16).
MATERIALS AND METHODS
Organisms.
The ATCC reference strains of N. wallacei (ATCC 49872 and ATCC 49873 [proposed as the type strain of N. wallacei], and ATCC 700035 [proposed as the type strain of N. blacklockiae]) and the type strain of N. transvalensis sensu stricto (ATCC 6865) were used in this study. Patient isolates were obtained from the Children's Hospital and Regional Medical Center, Seattle, WA (one isolate), from isolates referred for identification to ARUP Laboratories (three isolates), or from isolates referred for identification and/or susceptibility testing to the University of Texas Health Center at Tyler (five isolates).
N. wallacei isolate 1 (case study 1) and isolates 2 through 5 were all recovered from sputum samples; N. blacklockiae isolate 6 (case study 2) and isolates 7, 8, and 9 were recovered from sputum, a brain abscess, a corneal ulcer, and a bronchial wash specimen, respectively. Except for N. blacklockiae isolate 6 (case study 2), the clinical significance of the respiratory isolates has not been determined; however, the N. blacklockiae isolates from the brain abscess and the corneal ulcer are presumably clinically relevant.
Phenotypic identification.
Colonies were examined for aerial hyphae by using a dissecting microscope; microscopic characteristics were determined by Gram stain and by modified Kinyoun acid-fast stain. Biochemical tests were performed at the Actinomycete Reference Laboratory of the CDC by the methods of Berd (1). Utilization of acetamide as the sole carbon and nitrogen sources was determined as described by Wallace et al. (20); utilization of citrate as the sole carbon source was determined as described by Yassin et al. (24). Arylsulfatase production tests were performed as described by Kent and Kubica (12). Esculin hydrolysis testing was performed on solid medium (Remel, Lenexa, KA), as described by Williams et al. (22).
Susceptibility testing.
Susceptibility testing was performed in two laboratories; isolates considered to belong to the species N. blacklockiae were tested in duplicate in each laboratory; isolates considered to belong to the species N. wallacei were tested only once in each laboratory. In laboratory 1 (CDC), the organisms were grown at 35°C in 25 ml of Middlebrook 7H9 broth (Difco, Sparks, MD) with albumin-dextrose-catalase enrichment (Gibco, Carlsbad, CA) by using glass beads and continuous shaking (generally for 3 days) until good growth was achieved. A standardized suspension was prepared from the supernatant by using Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) guidelines (4). In laboratory 2 (National Institutes of Health [NIH]), Nocardia colonies from a 3- to 5-day growth on sheep blood agar (Remel) were crushed in 0.5 ml of sterile water with a pellet pestle (Kimble Chase, Vineland, NJ). The larger organism clumps were allowed to settle; a standardized suspension was prepared from the supernatant by using CLSI guidelines (4). In each laboratory, 10 μl of a standardized inoculum was added to each well of a commercially prepared, frozen microdilution plate (PML Microbiologicals, Wilsonville, OR) containing antimicrobial agents diluted in 100 μl of cation-supplemented Mueller-Hinton broth per well. The plates were incubated at 35°C for 72 h in ambient air; endpoints were determined according to the CLSI guidelines (4). The interpretive breakpoints used for all drugs were those recommended by the CLSI.
Gene sequencing.
DNA was extracted from all reference strains, type strains, and patient isolates as described previously (7). 16S rRNA gene sequences (1460 bp), HSP gene sequences (441 bp), and secA1 gene sequences (520 bp) were determined as described previously (6, 7, 9). The sequences of all genes were assembled and aligned by using Lasergene SeqMan II and MegAlign software (DNAStar, Inc., Madison, WI). The deduced amino acid sequence of the SecA1 protein was determined with MegAlign software. To determine sequence similarity, all sequences were cut to the size of the shortest sequence. Percent similarity was determined by counting the number of base differences and relating the number of these differences to the sequence length. For determination of percent similarity, any ambiguous bases were counted as mismatches.
DNA-DNA hybridization.
Purified DNA of the designated type strains of N. wallacei, N. blacklockiae, and N. transvalensis and from the patient isolates was prepared from lysed protoplasts, as described previously (8, 14). In two separate experiments, reference strains were labeled with [32P]dCTP by using a nick translation system (Invitrogen, Carlsbad, CA). In experiment 1, labeled DNA from the type strain of N. wallacei (ATCC 49873) was hybridized with the unlabeled DNA from isolates 1 through 5 and with the unlabeled DNA from N. transvalensis. In experiment 2, labeled DNA from the type strain of N. blacklockiae (ATCC 700035) was hybridized with the unlabeled DNA from isolates 6 through 9 and with the unlabeled DNA from N. transvalensis. Hybridization was performed as described previously (3). All reactions were performed in duplicate at 70°C. The relative binding ratio (RBR) was calculated by the method of Brenner et al. (2) as follows: (percentage of heterologous DNA bound to hydroxyapatite/percentage of homologous DNA bound to hydroxyapatite) × 100. The percent divergence (D; calculated to the nearest 0.5%) was determined by assuming that each degree of heteroduplex instability compared to the melting temperature of the homologous duplex was caused by 1% unpaired bases (2). An RBR greater than 70% with less than 6% D is considered indicative of conspecificity (21).
G+C content.
The G+C content of DNA for the type strains of N. wallacei and N. blacklockiae was determined spectrophotometrically by thermal denaturation, as described previously (15).
Nucleotide sequence and culture collection accession numbers.
The 16S rRNA, HSP, and secA1 gene sequences of the N. wallacei and N blacklockiae type strains and of patient isolates 1, 6, and 7 have been submitted to GenBank and assigned accession numbers EU099357 through EU099371, respectively.
The N. wallacei and N. blacklockiae strains have been deposited in culture collections under the indicated strain numbers: N. wallacei, ATCC 49873T and DSM 45136T; N. blacklockiae, ATCC 700035T and DSM 45135T.
RESULTS
Phenotypic identification.
For the five patient isolates considered to belong to the species N. wallacei, the biochemical results were identical to those obtained for the type strain except for the production of acid from the oxidation of sucrose and of salicin (for which the patient isolates gave variable results) (Table 1).
TABLE 1.
Biochemical characteristics of the type strains of N. transvalensis sensu stricto, N. wallacei, and N. blacklockiae; the reference strain of N. wallacei; and the patient isolates of N. wallacei and N. blacklockiae
| Test | Result fora:
|
|||||
|---|---|---|---|---|---|---|
| N. transvalensis ATCC 6865T | N. wallacei ATCC 49873T | N. wallacei reference strain ATCC 49872 | N. wallacei patient isolates 1 to 5 (n = 5) | N. blacklockiae ATCC 700035T | N. blacklockiae patient isolates 6 to 9 (n = 4) | |
| Acid from oxidation of: | ||||||
| Adonitol | + | − | − | − | + | + |
| Arabinose | − | − | − | − | − | − |
| Cellobiose | − | − | − | − | − | − |
| Dulcitol | − | − | − | − | − | − |
| i-Erythritol | + | − | − | − | + | + |
| d-Fructose | + | − | − | − | + | + |
| d-Galactose | + | + | + | + | − | + |
| d-Glucose | + | + | + | + | + | + |
| Glycerol | + | + | + | + | + | + |
| i-myo-Inositol | + | − | − | − | − | − |
| Lactose | − | − | − | − | − | − |
| Maltose | + | + | + | + | + | + |
| d-Mannitol | + | − | − | − | + | + |
| Mannose | − | − | − | − | + | V |
| Melibiose | − | − | − | − | − | − |
| Raffinose | − | − | − | − | − | − |
| l-Rhamnose | − | − | − | − | − | − |
| Salicin | + | + | + | V | + | V |
| d-Sorbitol | + | − | − | − | + | + |
| Sucrose | − | W+ | − | − | − | V |
| Trehalose | + | + | + | + | + | + |
| d-Xylose | − | − | − | − | − | − |
| Growth on heart infusion agar: | ||||||
| At 25°C | 3+ | 2+ | 2+ | 2 + to 3 + | 3+ | 3+ |
| At 35°C | 3+ | 3+ | 3+ | 2 + to 3 + | 3+ | 3+ |
| At 45°C | − | 1+ | 2+ | 1+ to 2 + | − | − |
| Growth in lysozyme | + | + | + | + | + | + |
| Arylsulfatase production at: | ||||||
| 3 days | − | − | − | − | − | − |
| 14 days | − | − | − | − | − | − |
| Hydrolysis of: | ||||||
| Adenine | − | − | − | − | − | − |
| Casein | − | − | − | − | − | − |
| Esculin | + | + | + | + | + | + |
| Hypoxanthine | + | + | + | + | + | + |
| Tyrosine | − | − | − | − | − | − |
| Urea | + | + | + | + | + | + |
| Xanthine | − | − | − | − | − | − |
| Utilization of acetamide as the sole carbon and nitrogen source | − | − | − | − | − | − |
| Utilization of citrate (1%) as a sole carbon source | + | + | + | + | + | + |
Abbreviations: +, positive; −, negative; W+, weakly positive; V, variable; 1+, scant growth; 2+, moderate growth; 3+, heavy growth.
For the four patient isolates considered to belong to the species N. blacklockiae, variable results were obtained for the production of acid from the oxidation of mannose, salicin, and sucrose; and all patient isolates produced acid from the oxidation of d-galactose. Otherwise, the biochemical results for the patient isolates were identical to those for the N. blacklockiae type strain.
Susceptibility testing.
As was expected for the members of the N. transvalensis complex, all type and reference strains and all patient isolates showed in vitro resistance to amikacin (data not shown) (23). For the type strain of N. wallacei, the susceptibility testing results (obtained once for each isolate in each laboratory) varied between laboratories for amoxicillin-clavulanic acid, imipenem, sulfamethoxazole, and trimethoprim- sulfamethoxazole (Table 2).
TABLE 2.
Susceptibility results for the type strain, the reference strain, and patient isolates of N. wallaceia
| Antimicrobial agent | Result for N. wallacei:
|
|||
|---|---|---|---|---|
| ATCC 49873T | Reference strain ATCC 49872 | Patient isolate 1 | Patient isolates 2-5 | |
| Amikacin | R | R | R | R |
| AMC | S/Ib | S | S | S |
| Ceftriaxone | S | S | S/Ib | S/Ib |
| Ciprofloxacin | S | S | S | S/Rb |
| Clarithromycin | R | R | R | R |
| Imipenem | I/Rb | R | R | R |
| Linezolidc | S | S | S | S |
| Minocycline | I | I | I | S/Ib |
| SMX | S/Rb | S | S/Rb | S/Rb |
| SXT | S/Rb | S/Rb | S/Rb | S/Rb |
Based on a single susceptibility test of each strain performed in two laboratories. Breakpoints are based on those reported in CLSI document M24-A (4). Abbreviations: AMC, amoxicillin-clavulanic acid; SMX, sulfamethoxazole; SXT, trimethoprim-sulfamethoxazole; R, resistant; S, susceptible; I, intermediate.
Results varied between laboratories.
Based on the proposed breakpoint (4).
The susceptibility testing results for the type strain and patient isolates of N. blacklockiae showed not only interlaboratory variation but also intralaboratory variation, with the exception of amikacin resistance and linezolid susceptibility (data not shown).
Gene and amino acid sequences.
Comparison of a 1,388-bp region of the 16S rRNA gene sequences of the type strains of N. blacklockiae, N transvalensis, and N. wallacei showed between 98.0 and 98.9% similarity among the strains (Table 3). Comparison of a 394-bp region of the HSP genes of these three isolates showed between 94.4 and 97.7% sequence similarity. Comparison of a 464-bp region of the secA1 genes of the three isolates showed between 95.9 and 97.0% sequence similarity; the deduced SecA1 protein amino acid sequences showed between 97.4 and 98.7% sequence similarity (Table 3).
TABLE 3.
Comparison of 16S rRNA, HSP, and secA1 gene sequences and SecA1 amino acid sequences of type strains of N. wallacei (ATCC 49873), N. blacklockiae (ATCC 700035), and N. transvalensis sensu stricto (ATCC 6865)
| Species | % Similarity
|
|||||||
|---|---|---|---|---|---|---|---|---|
|
N. blacklockiae
|
N. transvalensis sensu stricto
|
|||||||
| 16S rRNA gene (1,388 bp) | HSP gene (394 bp) | secA1 gene (464 bp) | SecA1 amino acid (155 residues) | 16S rRNA gene (1,388 bp) | HSP gene (394 bp) | secA1 gene (464 bp) | SecA1 amino acid (155 residues) | |
| N. wallacei | 98.0 | 97.7 | 97.0 | 98.7 | 98.6 | 94.4 | 95.9 | 97.4 |
| N. blacklockiae | 98.9 | 95.9 | 96.6 | 97.4 | ||||
The 16S rRNA gene sequences of five patient isolates (isolates 1 through 5; 1,353 bp) showed ≥99.9% sequence similarity to the 16S rRNA gene sequence of the N. wallacei type strain (ATCC 49873) (Table 4). The 16S rRNA gene sequence of N. wallacei reference strain ATCC 49872 showed 99.8% similarity to that of the N. wallacei type strain (Table 4); the sequence discrepancies noted between these two strains (3 bp) were due in part to the presence of two ambiguous bases in the 16S rRNA gene sequence of ATCC 49872. Patient isolates 1 through 5 showed ≥99.8% gene sequence similarity to the N. wallacei type strain for both the HSP gene (394 bp) and the secA1 gene (464 bp) (Table 4). The deduced amino acid sequence of the SecA1 protein for all patient isolates and the N. wallacei reference strain (ATCC 49872) showed 100% similarity to the deduced amino acid sequence of the N. wallacei type strain (data not shown).
TABLE 4.
Comparison of the 16S rRNA, HSP, and secA1 gene sequences and DNA-DNA hybridization results for five patient isolates and one ATCC reference strain of N. wallacei with those for N. wallacei ATCC 49873T
| Isolate | % Similarity to N. wallacei ATCC 49873T
|
DNA-DNA hybridization with N. wallacei ATCC 49873T (RBR/% D) | ||
|---|---|---|---|---|
| 16S rRNA gene sequence (1,353 bp) | HSP gene sequence (394 bp) | secA1 gene sequence (464 bp) | ||
| ATCC 49872 | 99.8 | 100 | 100 | 86/1.0 |
| 1 | >99.9 | 100 | 100 | 89/1.0 |
| 2 | >99.9 | 100 | 100 | 100/2.0 |
| 3 | 100 | 100 | 100 | 100/1.5 |
| 4 | >99.9 | 99.8 | 100 | 84/1.5 |
| 5 | >99.9 | 100 | 100 | 100/1.5 |
The 16S rRNA gene sequence of isolate 6 (case 2) showed 99.9% similarity to that of the type strain of N. blacklockiae (1407 bp) (Table 5); the only differences between the two sequences were two base insertions in the sequence of the type strain. Analysis of the HSP gene (394 bp) and the secA1 gene (464 bp) sequences showed that the two gene sequences of isolate 6 were 99.8 and 99.1% similar to the two gene sequences of the N. blacklockiae type strain, respectively (Table 5). The amino acid sequence of the SecA1 protein was identical to that of the type strain (data not shown).
TABLE 5.
Comparison of the 16S rRNA, HSP, and secA1 gene sequences and DNA-DNA hybridization results for four patient isolates with those for the type strain of N. blacklockiae ATCC 700035T
| Isolate | % Sequence similarity to N. blacklockiae ATCC 700035T
|
DNA-DNA hybridization with N. blacklockiae ATCC 700035T (RBR/% D) | ||
|---|---|---|---|---|
| 16S rRNA gene sequence (1,407 bp) | HSP gene sequence (394 bp) | secA1 gene sequence (464 bp) | ||
| 6 | 99.9 | 99.8 | 99.1 | 93/0.0 |
| 7 | 99.3 | 99.8 | 98.7 | 85/0.5 |
| 8 | 99.3 | 99.5 | 98.5 | 93/0.0 |
| 9 | 99.3 | 99.8 | 98.7 | 98/1.0 |
Isolates 7, 8, and 9 each showed 99.3% 16S rRNA gene sequence similarity to the 16S rRNA gene sequence of the N. blacklockiae type strain, with 10 base differences each, and they were more similar to each other than to the N. blacklockiae type strain (99.4 to >99.9% similarity). In addition, isolates 8 and 9 contained one and two ambiguous bases, respectively, that could not be resolved by repeat testing. Analysis of the HSP and secA1 gene sequences for isolates 7, 8, and 9 showed ≥99.5 and ≥98.5% sequence similarities to the sequences of these two genes of the N. blacklockiae type strain, respectively. For isolates 7, 8, and 9, the deduced amino acid sequences of the SecA1 protein were 100% similar to the deduced amino acid sequence of the SecA1 protein of the N. blacklockiae type strain (data not shown).
DNA-DNA hybridization.
DNA-DNA hybridization studies performed with the type strains of N. wallacei, N. blacklockiae, and N. transvalensis showed the strains to be sufficiently different to warrant their inclusion in three separate species (Table 6).
TABLE 6.
DNA relatedness among the type strains of N. blacklockiae, N. transvalensis, and N. wallacei
| Source of labeled DNA | Source of unlabeled DNA | RBR (% D) |
|---|---|---|
| N. wallacei | N. wallacei | 100 |
| N. wallacei | N. transvalensis | 44 (10.5) |
| N. wallacei | N. blacklockiae | 53 (8.5) |
| N. blacklockiae | N. blacklockiae | 100 |
| N. blacklockiae | N. wallacei | 45 (7.5) |
| N. blacklockiae | N. transvalensis | 34 (8.5) |
DNA-DNA hybridization of patient isolates 1 through 5 with the N. wallacei type strain showed RBRs of ≥84 and D values of ≤2.0% (Table 4). DNA-DNA hybridization of isolates 6 through 9 with the N. blacklockiae type strain showed RBRs of ≥85 and D values of ≤1% (Table 5).
G+C content.
The G+C content of the type strains of both N. blacklockiae (ATCC 700035) and N. wallacei (ATCC 49873) was 65.0 mol%. This value is consistent with the G+C content observed in the genus Nocardia (64 to 72 mol%) (13).
DISCUSSION
The results of the present study confirm the differences among the type and reference strains of three of the four groups of the N. transvalensis complex. The type strains of N. wallacei, N. blacklockiae, and N. transvalensis sensu stricto show biochemical differences similar to those noted by Wilson et al. (23) (Table 1). Sequence analysis of the 16S rRNA, HSP, and secA1 genes showed considerable molecular differences among these type and reference strains (Table 3). In addition, the DNA-DNA hybridization results for the type strains of these species confirm the molecular distinctness of these three groups (Table 6).
In the United States, N. wallacei is the most commonly isolated member of the N. transvalensis complex (23). In the 1988 study of Wallace et al. (19), this organism represented 5% of the Nocardia isolates submitted for susceptibility testing to the University of Texas Health Center in Tyler. In the present study, isolates determined to belong to the species N. wallacei showed considerable homogeneity in their biochemical characteristics (Table 1) and, except for the sulfonamides, in their susceptibility testing results (Table 2). The 16S rRNA, HSP, and secA1 gene sequences of the clinical isolates of N. wallacei were extremely similar to those of the type strain of the species, in addition to being molecularly similar to each other. DNA-DNA hybridization confirmed their conspecificity.
N. blacklockiae is similar to other members of the N. transvalensis complex in its resistance to amikacin; the results of molecular analysis presented here show that this species is distinct from the other members of the complex.
Three of the four isolates (patient isolates 7, 8, and 9) have been designated here as belonging to the species N. blacklockiae, despite the presence of some notable 16S rRNA gene sequence differences from the type strain. Although the HSP and secA1 gene sequences and the DNA-DNA hybridization results all place these isolates within the species N. blacklockiae, the similarity of the 16S rRNA gene sequences of these isolates to the 16S rRNA gene sequence of the N. blacklockiae type strain is only 99.3%. This is a lower percentage of similarity than that seen for other isolates unequivocally belonging to the same Nocardia species, which are usually ≥99.9% similar. The 99.3% similarity of the 16S rRNA genes of isolates 7, 8, and 9 to the 16S rRNA gene sequence of the type strain of N. blacklockiae is also less than the 16S rRNA gene sequence similarity of some distinct species to each other. For example, the species N. kruczakiae and N. veterana and the species N. paucivorans and N. brevicatena show 99.8 and 99.5% similarity to each other, respectively (5, 25). The results of a BLAST (Basic Local Alignment Search Tool; NCBI, Bethesda, MD) analysis of the 16S rRNA gene sequences of isolates 7, 8, and 9 indicate that the next most similar type strains to these isolates are those of N. transvalensis and N. wallacei, with 98.6 and ≤98.4% similarities, respectively, which are significantly less than the similarities of these isolates to the N. blacklockiae type strain. As the criteria for determining conspecificity by 16S rRNA gene sequence similarity are undefined and given the DNA-DNA hybridization results, we think that all three of these isolates should be considered N. blacklockiae isolates.
Sequence analysis of the 16S rRNA genes of a reference strain and two patient isolates examined in this study revealed the presence of ambiguous bases that could not be resolved with repeat testing. The 16S rRNA gene of N. wallacei reference strain ATCC 49872 and patient isolate 9 each contained two such ambiguous bases; patient isolate 8 contained one ambiguous base. This finding suggests that these isolates may contain multiple different copies of the 16S rRNA gene (8). The presence of ambiguous bases in the 16S rRNA gene sequence of patient isolates 8 and 9 does not appear to have been the reason for the lower level of 16S rRNA gene sequence similarity to the N. blacklockiae type strain; ignoring the ambiguous bases in the sequences of the patient isolates when mismatches were counted does not significantly change the percent similarity of that organism's sequence to the sequence of the type strain. One organism noted as a reference strain of N. transvalensis new taxon 1 (ATCC 700034) by Wilson et al. (23) contains nine ambiguous bases (data not shown) and was not included in this evaluation of members of the N. transvalensis complex.
There does appear to be geographic variability in the frequency of isolation of the different species included in this complex. Wilson et al. (23) noted that organisms identified as N. transvalensis comprised 4.2% of the Nocardia isolates recovered in Queensland, Australia, but only 1.1% of the isolates referred for susceptibility testing to two Texas reference laboratories. A previous study noted that 4.9% of the 102 Nocardia isolates from Queensland belonged to N. transvalensis (10). In the study of Wilson et al. (23), no isolates of N. wallacei and six of eight isolates (75%) of N. blacklockiae were isolated from Australian patients. All isolates of N. wallacei in the study of Wilson et al. (23) were isolated from patients in the United States.
The discrimination among the species considered to be members of the N. transvalensis complex is best achieved by gene sequencing methods. Phenotypic characterization with biochemicals is difficult and time-consuming, the number of tests available is small, and the data for basing species identification on phenotypic results are not robust. Initial studies that used amplification of the HSP gene and subsequent restriction endonuclease assays were able to show clear distinctions among members of this complex (23). Unfortunately, this method requires the use of numerous restriction endonucleases; it is also unclear if newly described Nocardia species would give RFLP patterns similar to those of members of the N. transvalensis complex.
The susceptibility testing results presented here illustrate some of the problems inherent in the testing of Nocardia species. Susceptibility testing of the same N. wallacei isolates in different laboratories gave various results for some drugs; if duplicate testing had been performed in each laboratory, even more variation might have been observed. In the case of the patient isolates of N. blacklockiae, considerable inter- and intralaboratory susceptibility test result variation was observed. It is unclear whether technical factors or differences in endpoint interpretation resulted in the variations in the susceptibility testing results. Clearly, the interpretation of endpoints with the sulfa drugs is particularly problematic; the interpretation of an 80% reduction in growth as the MIC endpoint, as is required for those drugs, is a subjective judgment and may result in disparate results.
In both case reports described above, susceptibility testing was performed in reference laboratories. It is presumed that the susceptibility testing results were not immediately available to the patients' physicians at the time of initial diagnosis and treatment. Hence, both patients received treatment with amikacin, despite the in vitro resistance of the organisms to this drug. In addition, species-related susceptibility patterns were not widely recognized at the time that treatment was initiated.
Generally, we would urge caution in assigning Nocardia isolates to a given species if the full 16S rRNA gene sequence differs from that of the type strain by ≤99.3%. However, given that the three patient isolates of N. blacklockiae (isolates 7, 8, and 9) described here have nearly identical HSP gene sequences, very similar secA1 gene sequences, and identical SecA1 amino acid sequences to those of the type strain of N. blacklockiae and because the 16S rRNA gene sequences are more similar to the 16S rRNA gene sequence of N. blacklockiae than to the 16S rRNA gene sequences of any other species, we think they are currently best regarded as belonging to this species. In addition, DNA-DNA hybridization between each patient isolate and the type strain of N. blacklockiae gave RBRs of ≥85%, significantly higher than the 70% that is usually used to determine species conspecificity.
Because of its in vitro resistance to commonly used antibiotics, especially amikacin, recognition of organisms belonging to this group that are isolated from clinical specimens is particularly important and also may suggest some problems with susceptibility test performance if the susceptibility testing results deviate significantly from the results expected for these species. However, as our results with the very similar isolates investigated here show, correct species assignment of some isolates may remain problematic even after extensive study of an isolate.
Description of Nocardia wallacei sp. nov.
wallacei (wal.lace′e.i, M.L. gen. masc. wallacei, of Wallace, in honor of Richard J. Wallace, Jr., in recognition of his contributions to the understanding of the taxonomy and drug susceptibility of Nocardia species). The organism is an aerobic, modified acid-fast-positive, branching gram-positive rod. Colonies produce aerial hyphae. Acid is produced from the oxidation of d-galactose, d-glucose, glycerol, maltose, salicin, sucrose, and trehalose but not from adonitol, arabinose, cellobiose, dulcitol, i-erythritol, d-fructose, i-myo-inositol, lactose, d-mannitol, mannose, melibiose, raffinose, l-rhamnose, d-sorbitol, or d-xylose. It grows at 25, 35, and 45°C, with the best growth at 35°C. It grows in the presence of lysozyme. It does not produce arylsulfatase at 3 or 14 days. It is able to hydrolyze esculin, hypoxanthine, and urea but not adenine, casein, tyrosine, or xanthine. It is not able to utilize acetamide as a sole source of carbon and nitrogen. It is able to utilize citrate (1%) as a sole source of carbon. The type strain is susceptible to ceftriaxone, ciprofloxacin, and linezolid and is resistant to amikacin and clarithromycin. The organism is a pathogen of immunocompromised patients.
The type strain of the species is ATCC 49873 (DSM 45136); and the sequences of a 1,414-bp region of the 16S rRNA gene, a 394-bp region of the HSP gene, and a 464-bp region of the secA1 gene have been deposited in GenBank under accession numbers EU099357, EU099358, and EU099359, respectively.
Description of Nocardia blacklockiae sp. nov.
blacklockiae (black.lock′.e.ae, M.L. gen. fem. blacklockiae, of Blacklock, in memory of Zeta M. Blacklock, in honor of and in recognition for her contributions to the study of Nocardia taxonomy). The organism is an aerobic, modified acid-fast-positive, branching gram-positive rod. Colonies produce aerial hyphae. Acid is produced from the oxidation of adonitol, i-erythritol, d-fructose, d-glucose, glycerol, maltose, d-mannitol, mannose, salicin, d-sorbitol, and trehalose but not from arabinose, cellobiose, dulcitol, d-galactose, i-myo-inositol, lactose, melibiose, raffinose, l-rhamnose, sucrose, or d-xylose. It grows at 25 and 35°C but not at 45°C. It grows in the presence of lysozyme. It does not produce arylsulfatase at 3 or 14 days. It is able to hydrolyze esculin, hypoxanthine, and urea but not adenine, casein, tyrosine, or xanthine. It is not able to utilize acetamide as a sole source of carbon and nitrogen. It is able to utilize citrate (1%) as a sole source of carbon. The type strain is susceptible to amoxicillin-clavulanic acid, ceftriaxone, and linezolid and is resistant to amikacin and clarithromycin. The organism is a pathogen of immunocompromised patients.
The type strain of the species is ATCC 700035 (DSM 45135); and the sequences of a 1,414-bp region of the 16S rRNA gene, a 394-bp region of the HSP gene, and a 464-bp region of the secA1 gene have been deposited in GenBank under accession numbers EU099360, EU099361, and EU099362, respectively.
Acknowledgments
We thank Joann L. Cloud, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT; Linda Mann and Rebecca Wilson of the University of Texas Health Center at Tyler; and Xuan Qin, Children's Hospital and Regional Medical Center, Seattle, WA, for providing clinical isolates for this study. We thank Patrick R. Murray, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, NIH, for critically reviewing the manuscript.
This research was supported in part by the Intramural Research Program of the NIH Warren G. Magnuson Clinical Center.
The views expressed herein are those of the authors and should not be construed as those of the U.S. Department of Health and Human Services.
Footnotes
Published ahead of print on 6 February 2008.
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