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JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 103–107 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 1

Novel Method for Rapid Identification of Nocardia Species by Detection of Preformed Enzymes JON R. BIEHLE,1* STEPHEN J. CAVALIERI,1 TRACY FELLAND,2

AND

BARBARA L. ZIMMER2

Department of Pathology, Creighton University Medical Center, Omaha, Nebraska 68131,1 and Dade MicroScan Inc., West Sacramento, California 956922 Received 27 June 1995/Returned for modification 12 September 1995/Accepted 12 October 1995

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The b-naphthylamide-labeled substrate L-pyrrolidonyl-b-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-a-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.

Identification of the aerobic actinomycetes by standard methods is a lengthy process and involves tests for the hydrolysis of casein, tyrosine, xanthine, and hypoxanthine, the production of urease, resistance to lysozyme, and a partial acidfast property when the organism is stained with carbolfuchsin (3). Incubation of the hydrolysis substrates for a minimum of 2 weeks after inoculation may be necessary for the most common isolate, Nocardia asteroides complex, since this species fails to hydrolyze any of the substrates. Additional tests are required to identify N. farcinica and N. nova, two recently recognized species contained within the N. asteroides complex (7, 9). These include susceptibility to several antimicrobial agents as well as other biochemical and physiologic characteristics. Although all media used to perform these tests are commercially available and the methods have been adequately described, identification of N. farcinica and N. nova may be beyond the scope of many clinical microbiology laboratories. The purpose of the study described here was to devise a technically simple and reproducible method for the identification of Nocardia species that can be completed within 3 to 4 days. We focused on a commercially available bacterial identification system that contained chromogenic test substrates which, when hydrolyzed, produce an obvious color change. This system tests for preformed enzymes, which eliminates the need for extended incubation times to obtain results. Rapid identification is of critical importance in the selection of appropriate antimicrobial therapy.

Samaritan Regional Medical Center, Phoenix, Ariz.; Centers for Disease Control and Prevention, Atlanta, Ga.; and Microbial Diseases Laboratory, California Department of Health Services, Berkeley. The distribution of species was N. asteroides (n 5 24), N. otitidiscavarium (n 5 10), N. brasiliensis (n 5 15), N. farcinica (n 5 25), and N. nova (n 5 11). The type strains used were N. asteroides ATCC 19247, N. nova ATCC 33726, N. farcinica ATCC 3318, N. brasiliensis ATCC 19296, and N. otitidiscavarium ATCC 14629. All strains were maintained during the course of the study on brain heart infusion slants and were subcultured onto sheep blood agar plates (BAPs) prior to biochemical or susceptibility tests. Traditional biochemical identification. All isolates were identified as Nocardia species by the use of standard methods (3). All isolates were observed to produce branched vegetative hyphae and aerial hyphae, two important criteria for the establishment of an organism as a member of the genus Nocardia. Resistance to lysozyme was determined with glycerol broth containing 50 mg of lysozyme (Remel, Lenexa, Kans.) per ml. Fifty microliters of a McFarland no. 0.5 suspension prepared in sterile distilled water was inoculated into glycerol broth with and without lysozyme. Tubes were incubated at 358C for 7 days in an ambient air incubator. Strains were considered resistant to lysozyme when the turbidity of the lysozyme-containing broth was equal to that of the glycerol-containing control broth. Hydrolysis tests were performed for casein, xanthine, tyrosine, and urease (Remel). Hydrolysis plates were inoculated with 1 drop of a McFarland no. 1 suspension in sterile distilled water. The suspension was prepared from active growth on BAPs. Hydrolysis plates were incubated at 308C and were examined daily for clearing of the medium. The plates were held for 14 days before they were considered negative and discarded. Strains that hydrolyzed casein and tyrosine but not xanthine were identified as N. brasiliensis. Strains that hydrolyzed xanthine only were identified as N. otitidiscavarium. Strains that failed to hydrolyze any substrate were tentatively identified as ‘‘N. asteroides complex.’’ Further tests were required in order to separate N. farcinica and N. nova from N. asteroides sensu stricto, since these three species all have negative hydrolysis reactions and are resistant to lysozyme. These consisted of equivalent growth on BAPs at 35 and 458C, utilization of acetamide as a carbon and nitrogen source with prepared acetamide agar slants and phenol red as the indicator (BBL Microbiology Systems, Inc., Cockeysville, Md.), acid production from cystine trypticase agar (CTA) medium with L-rhamnose with phenol red as the indicator (BBL), 7- and 14-day arylsulfatase tests (Remel), and studies of susceptibility to the antimicrobial agents tobramycin (10 mg), cefamandole (30 mg), and erythromycin (15 mg) by a disk diffusion method (7–9). Equivalent growth at 458C was determined by placing 1 drop of a McFarland no. 1 suspension of the organism onto duplicate BAPs, streaking for isolation, and incubating at 45 and 358C for 3 days. Equivalent growth at 458C was considered to be positive when the amount of growth at 458C was identical to that at 358C. The L-rhamnose test was considered positive when the upper portion of the

MATERIALS AND METHODS Study strains. A total of 85 clinical isolates representing five species of Nocardia were used in the study. The sources of the isolates were the culture collections of the Creighton University Medical Center, Omaha, Nebr.; Good

* Corresponding author. Mailing address: Department of Pathology, Creighton University Medical Center, 601 N. 30th St., Omaha, NE 68131. Phone: (402) 449-4952. Fax: (402) 280-5247. 103

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J. CLIN. MICROBIOL.

TABLE 1. Standard biochemical results for the five American Type Culture Collection type strains and 85 clinical strains of Nocardia % Positivea Strain Casein Tyrosine Xanthine

% Susceptible tob:

Equivalent Acid from Utilization Arylsulfatase Tobramycinc Cefamandolec Erythromycind growth at 458C L-rhamnose of acetamide

N. brasiliensis ATCC 19296 Clinical strains (n 5 15)

1 100

1 100

2 0

2 0

ND ND

ND ND

ND ND

ND ND

ND ND

ND ND

N. otitidiscavarium ATCC 14629 Clinical strains (n 5 10)

2 0

2 0

1 100

1 100

ND ND

ND ND

ND ND

ND ND

ND ND

ND ND

N. farcinica ATCC 3318 Clinical strains (n 5 25)

2 0

2 0

2 0

1 100

1 96

1 48

2 0

R 0

R 0

R 0

N. nova ATCC 33726 Clinical strains (n 5 11)

2 0

2 0

2 0

2 0

2 0

2 0

1 18

R 0

S 100

S 100

N. asteroides ATCC 19247 Clinical strains (n 5 24)

2 0

2 0

2 0

2 67

2 71

2 17

2 0

S 63

S 71

R 0

1, positive reaction; 2, negative reaction; ND, test was not performed. S, susceptible; R, resistant. c Breakpoint for susceptibility, .20 mm. d Breakpoint for susceptibility, .30 mm. a b

agar changed from red to a bright yellow. The acetamide test was considered positive if the entire slant changed from pale yellow to pink. For these two tests the tubes were examined daily for 2 weeks. For arylsulfatase, 6 drops of 2 N sodium carbonate was added to each tube of broth after 7 and 14 days of incubation, respectively, and the tubes were observed for a color change. Production of a pink to a red color throughout the broth indicated a positive reaction. The 7-day broth sample was discarded if no color change was observed after adding sodium carbonate. Disk diffusion susceptibility testing was performed with Mueller-Hinton agar (BBL) and commercial disks for tobramycin (10 mg), cefamandole (30 mg), and erythromycin (15 mg) as described previously (8). Plates were incubated at 358C for 72 h, after which the diameters of the clear zones of inhibition were measured and recorded. Strains were identified as N. farcinica if they exhibited equivalent growth at 45 and 358C in 3 days, were resistant to tobramycin and cefamandole (zone diameters, ,20 mm) and to erythromycin (zone diameters, ,30 mm), and had a positive result for either acetamide or L-rhamnose. Strains were identified as N. nova if they failed to exhibit equivalent growth at 458C after 3 days, were resistant to tobramycin, susceptible to cefamandole and erythromycin, and negative for both acetamide and L-rhamnose. Strains that did not meet the requirements given above for identification as N. farcinica or N. nova, had negative hydrolysis reactions, and were resistant to lysozyme were identified as N. asteroides sensu stricto. Chromogenic substrate identification. Two MicroScan (Dade MicroScan Inc., West Sacramento, Calif.) products were selected for use in the present study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification (ANA) and the HNID (Haemophilus-Neisseria) panels. Both panels are marketed as dehydrated microtiter plates, with each panel simultaneously rehydrated and inoculated by the addition of a McFarland no. 3 suspension of bacteria prepared in sterile distilled water. The ANA panel contains 24 modified conventional and chromogenic substrates and the HNID panel contains 18 modified conventional and chromogenic substrates. The nitrophenyl-labeled substrates contain either ortho- or para-nitrophenyl and are initially colorless when they are rehydrated with the bacterial suspension. If the organism produces the appropriate enzyme, the substrate is cleaved and free ortho- or para-nitrophenyl is released, producing a yellow color in the well. The amino acid b-naphthylamide substrates function in a similar manner, with various substrates linked to b-naphthylamide. Hydrolysis by the proper arylamidase liberates free b-naphthylamide, which is detected by the addition of paradimethylaminocinnamaldehyde, producing a complex that is red to magenta in color. The indoxyl phosphate (IDX) test contains 3-indoxyl phosphate which, when it is cleaved by a phosphatase, releases free indoxyl. The indoxyl combines with oxygen to form indigo blue, which is an insoluble blue or blue-green precipitate. Other conventional tests present on the panels include substrates for the detection of urease, nitrate reduction, indole production, b-lactamase production, carbohydrate utilization, and decarboxylation of ornithine.

Study strains were subcultured onto brain heart infusion slants and were incubated at 358C for 36 to 48 h in an ambient air incubator prior to inoculation of the MicroScan panels. Confluent growth was harvested from the slants by the use of a cotton-tipped swab, and an approximate McFarland no. 3 suspension was prepared in sterile distilled water. Three drops (50 to 75 ml) of the bacterial suspension was placed into each substrate well by the use of a sterile Pasteur pipette. One drop of the remaining bacterial suspension was placed onto duplicate blood agar plates and streaked for isolation, and the plates were incubated at 35 and 458C for 3 days as a means of ensuring the purity of the suspension and the viability of the organism, as well as determining equivalent growth. The inoculated plates were covered and incubated at 358C in an ambient air incubator for 18 to 24 h. After the appropriate incubation period, reagents were added to the wells containing the b-naphthylamide and nitrate reduction substrates. The plates were then visually examined by the use of a reverse light source, and the results for each well were recorded. Positive and negative reactions for the chromogenic substrates were determined by comparing each test well with the negative control well.

RESULTS Biochemical identification. The results of standard tests for the identification of the type strains and clinical isolates are presented in Table 1. All clinical isolates were found to be resistant to lysozyme. Identification of an isolate as either N. brasiliensis or N. otitidiscavarium by the use of hydrolysis tests generally required 5 to 7 days. Hydrolysis plates for isolates that failed to hydrolyze any of the three substrates were held for 14 days before they were considered negative and discarded. All clinical isolates of N. brasiliensis were negative for equivalent growth at 458C, whereas all clinical isolates of N. otitidiscavarium were positive. Of the 60 clinical isolates negative for any hydrolysis substrate, i.e., N. asteroides complex, 32% (19 of 60) were negative for equivalent growth at 458C. Of the 19 strains negative for equivalent growth at 458C, 11 strains were subsequently identified as N. nova by the additional test procedures. All clinical isolates of N. farcinica were positive for equivalent growth at 458C. The type strain of N. asteroides was negative for equivalent growth at 458C. Acid production from CTA L-rhamnose and utilization of

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TABLE 2. Results of discriminating substrates for the five American Type Culture Collection type strains and 85 clinical strains of Nocardia % Positivea Strain IDX

MNP

NGLU

GAL

NGL

ZAR

PYR

N. brasiliensis ATCC 19296 Clinical strains (n 5 15)

1 100

1 100

1 100

1 100

1 100

2 0

2 0

1 100

N. otitidiscavarium ATCC 14629 Clinical strains (n 5 10)

1 100

2 0

1 100

1 100

2 0

1 100

2 0

1 100

N. farcinica ATCC 3318 Clinical strains (n 5 25)

1 100

2 0

1 100

2 0

1 100

1 100

1 100

1 80

N. nova ATCC 33726 Clinical strains (n 5 11)

2 0

2 0

1 100

2 0

2 0

2 0

1 100

2 0

N. asteroides ATCC 19247 Clinical strains (n 5 24)

2 0

2 0

2 0

2 0

2 0

2 0

2 0

1 58

a

Urease

1, positive reaction; 2, negative reaction.

acetamide were not performed for isolates positive for hydrolysis substrates, i.e., N. brasiliensis and N. otitidiscavarium. All clinical isolates of N. nova were negative for acid production from CTA L-rhamnose, whereas 96% (24 of 25) of N. farcinica isolates and 71% (17 of 24) of N. asteroides sensu stricto isolates were positive. All clinical isolates of N. nova were negative for utilization of acetamide, whereas 48% (12 of 25) of the strains of N. farcinica and 17% (4 of 24) of the strains of N. asteroides sensu stricto were positive. Susceptibility to tobramycin, cefamandole, and erythromycin was critical for establishing an identification of N. nova, N. farcinica, and N. asteroides sensu stricto. All clinical isolates of N. nova were susceptible to erythromycin and cefamandole but resistant to tobramycin. All clinical isolates of N. farcinica were resistant to tobramycin, cefamandole, and erythromycin. For clinical isolates of N. asteroides sensu stricto, 63% (15 of 24) and 71% (17 of 24) were susceptible to tobramycin and cefamandole, respectively, and 42% (10 of 24) were susceptible to both antimicrobial agents. Two strains (8%) were resistant to both antimicrobial agents but were negative for rhamnose and acetamide. All clinical isolates of N. asteroides sensu stricto were resistant to erythromycin. Only two clinical isolates (18%) of N. nova were positive for the 2-week arylsulfatase test. Both strains were negative after 7 days of incubation. All clinical isolates of N. asteroides sensu stricto or N. farcinica were negative for arylsulfatase after 2 weeks. Chromogenic substrate identification. Data from the nitrophenyl- and b-naphthylamide-labeled substrates were analyzed to determine which reactions were highly specific ($95% positive or negative) and would enable us to separate the five species of Nocardia. The results for discriminating substrates obtained from the MicroScan panels for the type strains and clinical isolates are provided in Table 2. The b-naphthylamidelabeled substrates N-a-benzoyl-DL-arginine-b-naphthylamide (ZAR), N-a-L-glutamyl-b-naphthylamide (NGL), and L-pyrrolidonyl-b-naphthylamide (PYR) were found to be useful for identification purposes. All other b-naphthylamide-labeled substrates were positive for the five species, with the exception that reactions for glycylglycine-b-naphthylamide (GGLY)

were variable for N. asteroides sensu stricto (data not shown). N. otitidiscavarium and N. farcinica were positive for ZAR, whereas only N. brasiliensis and N. farcinica were positive for NGL. N. farcinica and N. nova were the only species positive for PYR. The nitrophenyl-labeled substrates p-nitrophenyl-a-D-mannopyranoside (MNP), p-nitrophenyl-N-acetyl-b-D-glucosaminide (NGLU), and o-nitrophenyl-b-D-galactoside (GAL) were also found to be useful for identification purposes. N. brasiliensis was the only species that hydrolyzed MNP; hence, this substrate appears to be specific for this species. All strains of N. asteroides sensu stricto were negative for NGLU, whereas the other four species were positive. N. brasiliensis and N. otitidiscavarium were the only species positive for GAL. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for IDX, whereas strains of N. nova and N. asteroides sensu stricto were always negative. In the very early stages of the study it was noted that the IDX reactions were variable for strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica if the inoculum was obtained from BAPs. Subsequently, all further testing with the MicroScan panels was performed with growth from brain heart infusion agar medium, which provided consistent strong positive IDX reactions for these species and consistent negative reactions for N. nova and N. asteroides sensu stricto. All of the other substrates, including the carbohydrates, had low specificities or were negative and thus did not contribute to the determination of the species (data not shown). DISCUSSION The primary objective of the present study was to compare the identification results obtained by the use of standard methods with those obtained by the use of chromogenic substrates. The standard techniques used for the identification of the Nocardia spp., particularly hydrolysis substrates and lysozyme resistance, have been in use for many years. Application of these standard techniques to the identification of the aerobic actinomycetes has been useful, but they are not regarded as the most accurate method available. Clinical microbiology labora-

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tories that use the standard methods should be cautioned that the related genera Mycobacterium, Rhodococcus, Gordona, Tsukamurella, and Corynebacterium may give results similar to those obtained for some Nocardia spp. These standard identification techniques have restricted laboratories to identifying strains of Nocardia as N. brasiliensis, N. otitidiscavarium, or the heterogeneous N. asteroides complex by a process that could require up to 2 to 4 weeks for completion. The recent work of Wallace et al. (7, 9) has demonstrated the need for the accurate determination of Nocardia spp., particularly N. farcinica and N. nova, since each species has been shown to have a unique antimicrobial susceptibility pattern. Several studies have demonstrated differences in enzymatic activity among species of Nocardia by using the API ZYM system (1, 2, 5). In the study by Richter et al. (5), the subtyping of two groups of N. asteroides sensu stricto (determined previously by use of bromcresol purple casein glucose agar) by using enzymatic profiles obtained from the API Yeast Ident kit was largely unsuccessful. Results for nine substrates were varied and indiscriminate, suggesting diverse heterogeneity within this group. No single substrate or combination of substrates that could be used to distinguish the two groups was identified. In the present study, strains of N. asteroides sensu stricto exhibited variable results for two nitrophenyl and b-naphthylamide-labeled substrates; these results also suggest heterogeneity within this group. However, a sufficient number of discriminate substrates were identified that separated N. asteroides sensu stricto from the other species. In contrast, isolates of N. brasiliensis have not been considered to be heterogeneous taxonomically, and for this species we observed no variation in the results obtained with the discriminating substrates. However, Wallace et al. (6) have recently described a new Nocardia taxon among isolates of N. brasiliensis associated with invasive disease. Differences between the new taxon and N. brasiliensis sensu stricto were reported to be susceptibility to ciprofloxacin and clarithromycin, resistance to minocycline, and differences in the highpressure liquid chromatography patterns of mycolic acids. It would be of considerable interest to determine the enzymatic profiles for a representative number of strains placed into this new taxon and compare the results with those obtained for N. brasiliensis sensu stricto. Highly specific (100% positive or negative) differences in preformed enzyme activity were observed between the five species tested. Strains initially identified by standard methods as N. asteroides complex, the most common clinical isolate of the genus (4), were separated into three distinct subgroups on the basis of the results obtained with the discriminating substrates. These were subsequently confirmed to be strains of N. farcinica, N. nova, and N. asteroides sensu stricto by the additional testing methods discussed above. The total time required to identify any one of the five most commonly isolated species of Nocardia by use of the enzymatic method was, on average, 5 days. This compares quite favorably to the total time required to identify strains of N. brasiliensis and N. otitidiscavarium by the use of hydrolysis media (5 to 7 days for positive results) and lysozyme resistance (7 days for positive results). The identification of an isolate as N. asteroides sensu stricto, N. farcinica, or N. nova by the use of standard methods may require 2 to 4 weeks, since hydrolysis medium often is held for up to 4 weeks before it is considered negative and discarded. Therefore, a substantial time savings may be realized by the use of the enzymatic identification method for these three species. However, the role of the lysozyme resistance test still remains critical in the separation of Nocardia spp. from other genera of aerobic actinomycetes, and such a

J. CLIN. MICROBIOL.

FIG. 1. MicroScan Rapid Anaerobe Identification panel inoculated with N. brasiliensis. Key substrates for the identification of this species are MNP (positive 5 yellow), IDX (positive 5 black), and PYR (negative 5 yellow). The negative control wells are NPC (nitrophenyl control) and BNAC (b-naphthylamide control).

test would be required in addition to the use of the enzymatic substrates. The technical performance of the enzymatic method was found to be uncomplicated. Growth of all species on brain heart infusion agar was determined to be satisfactory for inoculum preparation purposes, and all strains demonstrated sufficient growth after 48 h of incubation at 358C. The development of a color reaction for all chromogenic substrates and the IDX substrate was easily detected compared with that for negative control wells (Fig. 1). It was noted, however, that all strains of N. nova produced weaker (less intense yellow), but nonetheless positive, results for the nitrophenyl substrates, regardless of the inoculum density (Fig. 2). We also observed that it was not necessary for the turbidity of the inoculum suspension to equal that of a McFarland no. 3 standard, as described in the product insert. During the course of the study, a small percentage (ca. 15%) of the bacterial suspensions with turbidity judged to be less than that of a McFarland no. 3 standard (ca. McFarland no. 0.5 to 1 standards) were found to be adequate for the hydrolysis of the chromogenic substrates. The technical performance of the enzymatic method of identification would be enhanced by incorporating all discriminat-

FIG. 2. MicroScan Rapid Anaerobe Identification panel inoculated with N. nova. Key substrates for the identification of this species are MNP (negative 5 clear), IDX (negative 5 clear), and PYR (positive 5 red).

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FIG. 3. Flowchart scheme for the identification of five species of Nocardia by using the chromogenic substrates MNP and PYR, IDX, and equivalent growth at 458C.

ing substrates into one microtiter plate, since the MNP, NGLU, and PYR substrates are contained in the ANA plate and the NGL, ZAR, and GAL substrates are contained in the HNID plate. The IDX substrate is contained in both plates. After a thorough review of the results obtained with all discriminating substrates, a simple flowchart for the separation of the five species was devised by using the results obtained with the three substrates present on the ANA plate (MNP, PYR, and IDX) and equivalent growth at 458C. The flowchart is illustrated in Fig. 3. The MNP, PYR, and IDX substrates were selected from the eight discriminatory substrates (Table 2) used for the development of this scheme, because the results of these tests for each of the five species were always clearly positive or negative. In summary, preformed enzyme detection appears to be a simple and reproducible method for the identification of Nocardia spp. Agreement between the traditional biochemical and enzymatic identification methods was 100%. The MicroScan ANA and HNID panels used in the study contained substrates sufficient for the separation of the most commonly isolated species of Nocardia. Other commercial products may contain additional substrates which may contribute to the formulation of an unique identification system based on a database of preformed enzyme activity. Further studies by this method appear to be warranted and should also focus on the detection of preformed enzymes in other genera of the aerobic actinomycetes, particularly Streptomyces, Actinomadura, Rhodococcus, and Oerskovia.

ACKNOWLEDGMENTS We thank Tony Maltese and Remel for their generous support of the study. REFERENCES 1. Boiron, P., and F. Provost. 1990. Enzymatic characterization of Nocardia spp. and related bacteria by API ZYM profile. Mycopathologia 110:51–56. 2. Kilian, M. 1978. Rapid identification of Actinomycetaceae and related bacteria. J. Clin. Microbiol. 57:127–133. 3. Land, G., M. R. McGinnis, J. Staneck, and A. Gatson. Aerobic pathogenic actinomycetes, p. 340–360. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 4. McNeil, M. M., J. M. Brown, W. R. Jarvis, and L. Ajello. 1990. Comparison of species distribution and antimicrobial susceptibility of aerobic actinomycetes from clinical specimens. Rev. Infect. Dis. 12:778–783. 5. Richter, S., J. Kane, R. C. Summerbell, S. Krajden, and B. Diena. 1995. Application of the API YeastIdent system in determining the enzymatic profiles of Nocardia asteroides isolates. Mycopathologia 129:1–4. 6. Wallace, R. J., Jr., B. A. Brown, Z. Blacklock, R. Ulrich, K. Jost, J. M. Brown, M. McNeil, G. Onyi, V. Steingrube, and J. Gibson. 1995. New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease. J. Clin. Microbiol. 33:1528–1533. 7. Wallace, R. J., Jr., B. A. Brown, M. Tsukamura, J. Brown, and G. O. Onyi. 1991. Clinical and laboratory features of Nocardia nova. J. Clin. Microbiol. 29:2407–2411. 8. Wallace, R. J., E. J. Septimus, D. M. Musher, and R. R. Martin. 1977. Disk diffusion susceptibility testing of Nocardia species. J. Infect. Dis. 135:568–576. 9. Wallace, R. J., Jr., M. Tsukamura, B. A. Brown, J. Brown, V. A. Steingrube, Y. Zhang, and D. R. Nash. 1990. Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica. J. Clin. Microbiol. 28:2726–2732.