JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wilson, R. W.
Right arrow Articles by Wallace, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, R. W.
Right arrow Articles by Wallace, R. J., Jr.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 1998, p. 148-152, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Clinical Application of PCR-Restriction Enzyme Pattern Analysis for Rapid Identification of Aerobic Actinomycete Isolates

Rebecca W. Wilson,1 Vincent A. Steingrube,2,* Barbara A. Brown,2 and Richard J. Wallace Jr.1,2

Center for Pulmonary and Infectious Disease Control1 and Department of Microbiology,2 The University of Texas Health Center at Tyler, Tyler, Texas

Received 9 July 1997/Returned for modification 23 September 1997/Accepted 17 October 1997

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The accuracy and practicality of PCR-restriction enzyme pattern analysis (PRA) for routine identification of aerobic actinomycete clinical isolates were evaluated for 299 cultures submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler. PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by traditional methods, including growth characteristics, susceptibility patterns, biochemical testing, and high-performance liquid chromatography analysis. Microbiological examination of six cultures ruled out aerobic actinomycetes, and they were omitted from the study. Amplicons were analyzed with BstEII, HaeIII, MspI, HinfI, and BsaHI. When necessary, AciI, HhaI, and NarI were also used. From March 1995 through May 1997 (27 months), 274 of the remaining 293 (93.5%) isolates were accurately identified by PRA. Major diagnostic groups included 170 mycobacteria, 93 nocardiae, and 30 other aerobic actinomycetes. Mixed cultures were readily recognized by PRA, including a wound culture that contained two Nocardia taxa that were indistinguishable morphologically. Mycobacterium mucogenicum was identified in three cultures heavily contaminated with gram-positive cocci. The 19 isolates that produced PRA patterns that did not match those in the current PRA database were differentiated into 8 Mycobacterium species and 11 other aerobic actinomycetes by the presence or absence of BstEII recognition sites. Identification of 15 of these 19 isolates was also equivocal by traditional methods. PRA results were reportable within 2 to 5 working days and were as accurate as and faster and less expensive to obtain than those of traditional methods.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interest in the identification and taxonomy of aerobic actinomycetes, nocardiae and mycobacteria in particular, has been increasing as a result of the increasing number of immunocompromised individuals in the population who are at greater risk for actinomycoses, especially those with advanced human immunodeficiency virus disease (1, 4, 5). Traditional methods for differentiation of species and taxa of aerobic actinomycetes are laborious and time-consuming and frequently require specialized testing that is beyond the capabilities of clinical laboratories (2-5, 9, 11, 19, 21). The occurrence of clinical isolates of aerobic actinomycetes that are inherently resistant to specific antimicrobials increases the significance of timely and accurate species and taxon recognition (4, 9, 17, 22).

Successful application of molecular biological methodology to the development of protocols for rapid differentiation of mycobacterial species was demonstrated by Telenti et al. in 1993 (13). These authors used PCR-restriction enzyme pattern analysis (PRA) of an amplified 439-bp segment of the 65-kDa heat shock protein (hsp-65) gene and introduced the abbreviation PRA for this method that has now gained wide acceptance (16). Application of this methodology has since been expanded to include 50 commonly encountered pathogenic species and taxa of aerobic actinomycetes comprising the genera Mycobacterium (10, 13); Nocardia (9, 17, 22); and Actinomadura, Gordona, Rhodococcus, Streptomyces, and Tsukamurella (11). The rapidity and accuracy of PRA prompted the current study (9-11, 13, 17). Clinical isolates of aerobic actinomycetes submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler (UTHCT) for identification and susceptibility testing were subjected to PRA for identification in an effort to evaluate the efficacy and cost-effectiveness of this methodology for routine clinical use.

(This study was presented in part at the 97th General Meeting of the American Society for Microbiology, Miami Beach, Fla., 1997.)

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Organisms. The present study included 293 clinical isolates of aerobic actinomycetes submitted to the Mycobacteria/Nocardia Research Laboratory at the UTHCT for identification and susceptibility testing during the 27-month period from March 1995 through May 1997. All clinical isolates used in this study were subcultured onto Trypticase soy and Middlebrook 7H10 agar plates. One culture of each isolate was used for identification by colonial morphology and antimicrobial susceptibility patterns (1, 17-21). Selected biochemical testing was done in order to differentiate species or taxa with similar susceptibility patterns (6, 10, 14, 15, 17, 19, 21). High-performance liquid chromatography (HPLC) and additional biochemical testing were kindly performed by the Bureau of Laboratories, Texas State Health Department (Austin).

The ATCC type strains of Mycobacterium fortuitum (ATCC 6841) and Nocardia brasiliensis (ATCC 19296) were utilized as internal controls for PRA.

PCR amplification. DNA was prepared from cells harvested from the initially submitted agar slants and/or the second subculture, when necessary, according to methods previously described (9-11, 13). A 439-bp segment of the hsp-65 gene was amplified from ground cell supernatants by PCR with 1.0 U of Taq DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.) in optimized buffer E (1.5 mM MgCl2 [pH 9.0]; Invitrogen, San Diego, Calif.) containing 83 µM (each) deoxynucleoside triphosphates, 9% dimethyl sulfoxide, and 1 µM (each) primers TB11 (5'-ACCAACGATGGTGTGTCCAT) and TB12 (5'-CTTGTCGAACCGCATACCCT) (Midland Certified Reagent Co., Midland, Tex.), together with the appropriate positive and negative controls according to a modification of the method of Telenti et al. (13). The PCR mixtures were run for 45 cycles of 94, 55, and 72°C for 1 min each and then for a 10-min extension period at 72°C.

Restriction enzyme analysis. Data from previous studies (9-11, 13) resulted in the selection of five commercially available restriction endonucleases, BstEII, HaeIII, MspI, HinfI, and BsaHI (New England Biolabs, Beverly, Mass., and Promega, Madison, Wis.), for routine use in the production of PRA band patterns. When indicated (references 10 and 22 and unpublished data), one or more of a secondary set of endonucleases that included AciI, HhaI, and NarI was utilized. Restriction digests were incubated for the appropriate time periods, at the appropriate temperatures, and with the buffers recommended by the manufacturers, with the exception of the temperature and digest mixture for BsaHI. To achieve complete digestion with BsaHI, acetylated bovine serum albumin was substituted for bovine serum albumin and the digestion mixture was incubated at 60°C for 1 h.

Restriction fragments were electrophoresed on 3% Metaphor agarose (4-bp resolution; FMC Bioproducts, Rockland, Maine), containing ethidium bromide (0.625 µg/ml), in a Mini-Sub-Cell electrophoresis system (Bio-Rad Laboratories, Richmond, Calif.) at 95 V for 1.5 to 2.0 h.

Isolate identification. PRA band sizes (base pairs) from each isolate were estimated visually by comparison with a 100-bp ladder (Life Technologies, Grand Island, N.Y.), a pGEM base pair ladder (Promega), and the PRA patterns obtained for control strains on each gel. Each isolate was then initially identified by one member of the staff (R.W.W.) to the species or taxon level by comparison of visually estimated PRA band sizes with those of species- and taxon-specific patterns contained in the PRA database (9-11, 13, 17, 22). Visual PRA isolate identifications were made prior to, and without knowledge of, identification results by traditional methods.

The PRA database was developed primarily for clinically significant nonpigmented rapidly growing mycobacteria, nocardiae, and other clinically significant aerobic actinomycetes (9-11, 13). With the exception of Mycobacterium avium and Mycobacterium intracellulare, for which 83 and 129 isolates, respectively, have been studied by PRA, the entries in the database for slow-growing mycobacteria have been less extensively developed. This portion of the database (unpublished data) represented PRA patterns obtained from 2 to 10 isolates of each slow-growing mycobacterial species most commonly encountered in clinical samples, including Mycobacterium celatum, Mycobacterium kansasii, Mycobacterium scrofulaceum, and Mycobacterium triviale, in addition to the 10 species listed in Table 1. These patterns were very similar to those published by Telenti et al. (13). To date, the PRA database has not been expanded to include pigmented rapidly growing mycobacterial species owing to their predominantly environmental origin and infrequent clinical occurrence as agents of traumatic wound infections.

Species or genus (e.g., Streptomyces) identification was considered conclusive when the PRA pattern matched that of a single species or taxon of aerobic actinomycete in the PRA database and the resulting identification was in agreement with that based on traditional methods.

To test whether precise PRA band size measurements were required for accurate isolate identification, band sizes were measured on a computerized Bio Image system (Millipore, Bedford, Mass.) with the same molecular size standards as noted above, and isolate identifications were made independently by a different member of the staff (V.A.S.) without prior knowledge of the initial visual identifications (R.W.W.). The two independent PRA identifications were then compared to one another and with identifications based on traditional methods including growth characteristics, susceptibility patterns, biochemical tests, and HPLC analysis.

Time studies of the PRA identification protocol were performed in order to provide an estimate of the amount of time and labor involved in applying this methodology under routine clinical conditions.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Organisms. On microbiological evaluation, six of the cultures submitted to the UTHCT laboratory for identification and susceptibility testing were not aerobic actinomycetes. These cultures did not yield PCR amplification products (amplicons) and were excluded from further study. The distribution of species and taxa identified among the remaining 293 isolates is listed in Table 1. The seven predominant species of aerobic actinomycetes identified in this study comprised over half, 174 of 293 (59%), of the isolates submitted and included the following: Mycobacterium abscessus, 47 of 293 (16%) isolates; M. fortuitum, 32 of 293 (11%) isolates; Nocardia nova, 24 of 293 (8%) isolates; Mycobacterium mucogenicum (formerly Mycobacterium chelonae-like organism) (8), 20 of 293 (7%) isolates; M. chelonae, 18 of 293 (6%) isolates; Nocardia asteroides type VI (20), 17 of 293 (6%) isolates; and N. brasiliensis, 16 of 293 (5%) isolates.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Clinical isolates identified by PRA in this study

PRA identification. As shown in Table 1, 274 of the 293 (93.5%) isolates produced PRA patterns that matched species- or taxon-specific patterns in the PRA database and resulted in an identification that correlated with the identification by traditional methods. The remaining 19 isolates produced PRA patterns that did not match any of the patterns currently available in the database. Eight of these isolates produced amplicons that contained BstEII recognition sites and were therefore tentatively identified as Mycobacterium species (9-11). Only 4 of these 19 isolates were unequivocally identified to the species level by traditional methods, as shown in Table 2. One isolate (Mo 816) produced an amplicon lacking BstEII recognition sites and an HaeIII pattern resembling that published for Mycobacterium vaccae (13) but was not identified as such due to the lack of adequate data in the PRA database at the time that the isolate was received. A second unique pattern was observed for isolate N 1426, which was identified as Nocardia sp., most likely N. asteroides complex, and which produced an amplicon containing BstEII recognition sites. This sputum isolate gave unique PRA patterns with all enzymes tested and was the first and only Nocardia isolate, among 210 Nocardia isolates examined by PRA in this laboratory, that demonstrated BstEII recognition sites. Traditional methods unequivocally identified 278 of the 293 (94.9%) clinical isolates studied, while PRA correctly identified 274 isolates, resulting in a comparative accuracy of 98.6%.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Identification of clinical isolates that exhibited patterns that did not match those in the current PRA database

Three cultures that contained small numbers of gram-positive rods mixed with heavy overgrowth of gram-positive cocci were submitted. Direct PRA was carried out on cells taken from the mixed cultures submitted, and in all three cases, the amplicons obtained produced patterns that were typical of M. mucogenicum. This identification was later confirmed by traditional microbiological methods. Two additional cultures that appeared to be pure produced an excessive number of bands on PRA gels that gave total base pair values in excess of the expected 439 bp. One of these cultures was identified at a reference laboratory as containing both Corynebacterium aquaticum and Actinomyces viscosus (Table 2). The second mixed culture from a multiply infected wound site yielded two distinct PRA patterns that, when reanalyzed on individual colony picks, were typical for isolates of the N. asteroides complex antibiogram type I (20) and the Nocardia transvalensis new taxon 2 of the proposed N. transvalensis complex (9, 22).

Comparison of visual PRA band size estimates and isolate identifications from gel photographs (Fig. 1) with independent isolate identifications based on computer-generated measurements of PRA band sizes resulted in 100% agreement between the two identification methods. Comparison of measured PRA band size (base pair) values from Fig. 1 with published values for the matching species and taxon (9-11) corroborated the visual estimations of PRA band sizes and the identification of the clinical isolates. For example, clinical isolate 96-113 gave PRA band patterns of 235, 115, and 80 bp with BstEII (Fig. 1A, lane 4) and 145 and 125 bp with HaeIII (Fig. 1A, lane 8), which matched the published PRA patterns of M. fortuitum (10). These patterns also matched those of the control strain (ATCC 6841) of M. fortuitum shown in Fig. 1A, lanes 5 and 9, respectively. Likewise, clinical isolates 96-110, 96-111, and 96-112 were identified by matching their PRA band patterns from BstEII, MspI, HinfI, and BsaHI digests with those published (9, 11) for N. nova, N. asteroides complex antibiogram type I, and N. brasiliensis, respectively.


View larger version (89K):
[in this window]
[in a new window]
  		FIG. 1.   PRA patterns from BstEII, HaeIII, MspI, HinfI, and BsaHI digests of amplicons from clinical isolates and reference strains of aerobic actinomycetes. (A) Lanes 1 to 5, BstEII digests; lanes 8 and 9, HaeIII digests. Amplicon digests: lanes 1 to 5, clinical isolates 96-110, 96-111, 96-112, and 96-113 and reference strain ATCC 6841, respectively; lanes 6 and 7, size markers (100-bp and pGEM-bp ladders, respectively); lanes 8 and 9, clinical isolate 96-113 and reference strain ATCC 6841, respectively. (B) Lanes 1 to 4, MspI digests; lanes 5 to 8, HinfI digests; lanes 11 to 14, BsaHI digests. Amplicon digests: lanes 1 to 4, clinical isolates 96-110, 96-111, and 96-112 and reference strain ATCC 19296, respectively; lanes 5 to 8, clinical isolates 96-110, 96-111, and 96-112 and reference strain ATCC 19296, respectively; lanes 9 and 10, size markers (100-bp and pGEM-bp ladders, respectively); lanes 11 to 14, clinical isolates 96-110, 96-111, and 96-112 and reference strain ATCC 19296, respectively. Clinical isolates 96-110, 96-111, 96-112, and 96-113 were identified as N. nova, N. asteroides complex antibiogram type I (20), N. brasiliensis, and M. fortuitum, respectively, by matching each of their PRA band patterns with published values (9-11).

A time course study was performed with the PRA procedure in order to better define the amount of time and labor involved and to provide a stepwise description of the procedure (Table 3). Final identification results can be achieved within 24 h of receiving a culture under optimal conditions. As a routine practice, however, separate analysis of individual isolates was neither cost-effective nor practical. As a practical routine, identification results based on PRA were generally achievable on a 2- to 5-day schedule.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Timed protocol for aerobic actinomycete clinical isolate identification by PRAa

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PRA correctly identified 274 of 293 (93.5%) aerobic actinomycete clinical isolates, compared to traditional identification methods that unequivocally identified 278 of those isolates (94.9%). Comparison of the two identification methods resulted in a 98.6% relative accuracy for the molecular biological identification method. PRA proved highly specific, enabling identification of aerobic actinomycetes from cultures heavily contaminated with other bacteria as well as identifying mixed cultures that contained more than one taxon of aerobic actinomycete that was not readily recognizable on isolation plates. The specificity of PRA was further demonstrated with six cultures that failed to yield amplicons on PCR and were found not to contain aerobic actinomycetes on further microbiological evaluation.

Identification of species and taxa of aerobic actinomycete isolates commonly encountered in clinical specimens was accomplished with virtually 100% accuracy. Only very rarely occurring species presented difficulties for identification, most frequently as a result of the unavailability of reference patterns in the PRA database. As noted above, the current PRA database has been well developed for species and taxa of nonpigmented rapidly growing mycobacteria, nocardiae, and other clinically significant aerobic actinomycetes (9-11). PRA data have not been as well developed for species of slow-growing mycobacteria, and to date, data from PRA of pigmented rapidly growing mycobacteria remain to be developed. As this study has progressed over the past 2 years, data for less frequently occurring species have been constantly added to the database as sufficient numbers of isolates and reference strains have been accumulated and examined by PRA. This steady development of the scope and breadth of the PRA database should continually expand the number of aerobic actinomycete species identifiable by this method.

Seven categories listed in Table 1 appear as complex or genus identifications and warrant further consideration. The M. abscessus-M. chelonae complex; M. fortuitum third biovariant complex; M. fortuitum third biovar, sorbitol-positive-Mycobacterium peregrinum Pipr complex (10); and the Mycobacterium terrae-Mycobacterium nonchromogenicum complex each represent two groups containing very closely related mycobacterial taxa that are not readily differentiated by either traditional methods or PRA (10, 13). Three isolates listed as M. abscessus-M. chelonae complex could not be differentiated further by traditional methods and exhibited hybrid PRA patterns that contained features of both M. abscessus and M. chelonae. These isolates may represent a heretofore-unrecognized taxon closely related to these mycobacterial species. The isolate listed as an undifferentiated taxon of the N. asteroides complex gave patterns with MspI and BsaHI that resembled those of the N. asteroides complex antibiogram type I (20) but produced a pattern with HinfI that was not represented in the PRA database and, therefore, may represent a minor pattern for isolates within this taxon, similar to the occurrence of major and minor patterns previously observed for isolates of this taxon with BsaHI (9). The single isolate listed as Gordona sp. exhibited a PRA pattern with HinfI that was unique to this genus but gave patterns with all other endonucleases tested that matched those of the previously reported isolate Mo 315 (11). These two isolates could be identified only to the genus level at the Centers for Disease Control and Prevention (Atlanta, Ga.) and were neither Gordona bronchialis nor Gordona sputi. As previously discussed (11), identification of Streptomyces isolates beyond the genus level was considered to be clinically irrelevant and was not pursued in this study.

Identification of clinical isolates of aerobic actinomycetes by PRA, performed on a continuing daily basis common to the routine of clinical laboratories, provided final identification results within 2 to 5 working days, compared to traditional identification methods that required from 2 to 6 weeks for final results. Prompt and accurate identification of pathogenic aerobic actinomycete isolates is particularly important when invasive species such as Nocardia pseudobrasiliensis (7, 17) are encountered or when innately drug-resistant species such as those of the N. transvalensis complex, which are resistant to all aminoglycosides (9, 20, 22), and Nocardia farcinica, which is resistant to all extended-spectrum cephalosporins (12, 21), are involved. This is particularly significant when immunocompromised patients such as those with advanced human immunodeficiency virus disease are infected with these organisms (4, 5, 9, 17, 22).

Although absolute PRA band sizes have been found to vary from 5 to 10 bp between laboratories (10, 13), the overall patterns have proven highly reproducible and species- and taxon-specific. Visual comparison of PRA patterns with molecular size standards and patterns produced by internal control isolates, such as M. fortuitum and N. brasiliensis reference strains used in this study, resulted in successful clinical isolate identifications that correlated perfectly with identifications based on measured band size values. Consequently, there is no requirement for costly computerized measurement systems, a major expense consideration, in implementing this methodology for routine clinical use.

PRA was cost-effective, with the expenses of specialized equipment and reagents being more than compensated for by savings in time and labor, and could be economically incorporated into the clinical laboratory setting. This methodology has proven both practical and cost-effective as a rapid, efficient, and highly accurate identification system for use in identifying clinically significant species and taxa of aerobic actinomycetes.

    ACKNOWLEDGMENTS

This work was supported by the Department of Microbiology and the Center for Pulmonary and Infectious Disease Control at UTHCT.

We express our appreciation to Phyllis Pienta, Collection Manager of Bacteriology, American Type Culture Collection, Rockville, Md., who kindly provided the reference strains used in the development of the PRA database; to the Bureau of Laboratories of the Texas State Department of Health (Austin, Tex.); and to Kenneth C. Jost, Jr., for his expertise with HPLC in assisting with the identification of clinical isolates of aerobic actinomycetes evaluated in this study.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, The University of Texas Health Center at Tyler, P.O. Box 2003, Tyler, TX 75710-2003. Phone: (903) 877-7685. Fax: (903) 877-7652. E-mail: vsteingr{at}UTHCT.edu.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Beaman, B. L., M. A. Saubolle, and R. J. Wallace, Jr. 1995. Nocardia, Rhodococcus, Streptomyces, Oerskovia, and other aerobic actinomycetes of medical importance, p. 379-399. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. ASM Press, Washington, D.C.
2. Collins, C. H., M. D. Yates, and A. H. C. Uttley. 1988. Presumptive identification of nocardias in a clinical laboratory. J. Appl. Bacteriol. 65:55-59[Medline].
3. Georghiou, P. R., and Z. M. Blacklock. 1988. Infection with Nocardia species in Queensland. A review of 102 clinical isolates. Med. J. Aust. 156:692-697.
4. McNeil, M. M., J. M. Brown, P. R. Georghiou, A. M. Allworth, and Z. M. Blacklock. 1992. Infections due to Nocardia transvalensis: clinical spectrum and antimicrobial therapy. Clin. Infect. Dis. 15:453-463[Medline].
5. McNeil, M. M., J. M. Brown, C. H. Magruder, K. T. Shearlock, R. A. Saul, D. P. Allred, and L. Ajello. 1992. Disseminated Nocardia transvalensis infection: an unusual opportunistic pathogen in severely immunocompromised patients. J. Infect. Dis. 165:175-178[Medline].
6. Mishra, S. K., R. E. Gordon, and D. A. Barnett. 1980. Identification of nocardia and streptomycetes of medical importance. J. Clin. Microbiol. 11:728-736[Abstract/Free Full Text].
7. Ruimy, R., P. Riegel, A. Carlotti, P. Boiron, G. Bernardin, H. Monteil, R. J. Wallace, Jr., and R. Christen. 1996. Nocardia pseudobrasiliensis sp. nov., a new species of nocardia which groups bacterial strains previously identified as Nocardia brasiliensis and associated with invasive diseases. Int. J. Syst. Bacteriol. 46:259-264[Abstract/Free Full Text].
8. Springer, B., E. C. Böttger, P. Kirschner, and R. J. Wallace, Jr. 1995. Phylogeny of the Mycobacterium chelonae-like organism based on partial sequencing of the 16S rRNA gene and proposal of Mycobacterium mucogenicum sp. nov. Int. J. Syst. Bacteriol. 45:262-267[Abstract/Free Full Text].
9. Steingrube, V. A., B. A. Brown, J. L. Gibson, R. W. Wilson, J. Brown, Z. Blacklock, K. Jost, R. F. Ulrich, and R. J. Wallace, Jr. 1995. DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the N. asteroides complex. J. Clin. Microbiol. 33:3096-3101[Abstract].
10. Steingrube, V. A., J. L. Gibson, B. A. Brown, Y. Zhang, R. W. Wilson, M. Rajagopalan, and R. J. Wallace, Jr. 1995. PCR amplification and restriction endonuclease analysis of a 65-kilodalton heat shock protein gene sequence for taxonomic separation of rapidly growing mycobacteria. J. Clin. Microbiol. 33:149-153[Abstract].
11. Steingrube, V. A., R. W. Wilson, B. A. Brown, K. C. Jost, Jr., Z. Blacklock, J. L. Gibson, and R. J. Wallace, Jr. 1997. Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis. J. Clin. Microbiol. 35:817-822[Abstract].
12. Steingrube, V. A., R. J. Wallace, Jr., B. A. Brown, Y. Zhang, L. C. Steele, G. Young, and D. R. Nash. 1993. Partial characterization of Nocardia farcinica beta -lactamases. Antimicrob. Agents Chemother. 37:1850-1855[Abstract/Free Full Text].
13. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Böttger, and T. Bodmer. 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175-178[Abstract/Free Full Text].
14. Tsukamura, M. 1977. Extended numerical study of Nocardia. Int. J. Syst. Bacteriol. 27:311-323[Abstract/Free Full Text].
15. Tsukamura, M. 1978. Numerical classification of Rhodococcus (formerly Gordona) organisms recently isolated from sputa of patients: description of Rhodococcus sputi Tsukamura sp. nov. Int. J. Syst. Bacteriol. 28:169-181[Abstract/Free Full Text].
16. Wallace, R. J., Jr. 1997. Presented at the 18th Annual Congress of the European Society of Mycobacteriology and the 2nd International Symposium on Mycobacteria of Clinical Interest, Córdoba, Spain, 17 to 20 June 1997 .
17. Wallace, R. J., Jr., B. A. Brown, Z. Blacklock, R. Ulrich, K. Jost, J. M. Brown, M. M. McNeil, G. Onyi, V. A. Steingrube, and J. L. Gibson. 1995. New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease. J. Clin. Microbiol. 33:1528-1533[Abstract].
18. Wallace, R. J., Jr., and L. C. Steele. 1988. Susceptibility testing of nocardia species for the clinical laboratory. Diagn. Microbiol. Infect. Dis. 9:155-166[Medline].
19. Wallace, R. J., Jr., B. A. Brown, M. Tsukamura, J. M. Brown, and G. O. Onyi. 1991. Clinical and laboratory features of Nocardia nova. J. Clin. Microbiol. 29:2407-2411[Abstract/Free Full Text].
20. Wallace, R. J., Jr., L. C. Steele, G. Sumter, and J. M. Smith. 1988. Antimicrobial susceptibility patterns of Nocardia asteroides. Antimicrob. Agents Chemother. 32:1776-1779[Abstract/Free Full Text].
21. 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[Abstract/Free Full Text].
22. Wilson, R. W., V. A. Steingrube, B. A. Brown, Z. Blacklock, K. C. Jost, Jr., A. McNabb, W. D. Colby, J. R. Biehle, J. L. Gibson, and R. J. Wallace, Jr. 1997. Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides, including amikacin, and PCR-restriction fragment length polymorphism analysis. J. Clin. Microbiol. 35:2235-2242[Abstract].

Journal of Clinical Microbiology, January 1998, p. 148-152, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wilson, R. W.
Right arrow Articles by Wallace, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, R. W.
Right arrow Articles by Wallace, R. J., Jr.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS