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

Copyright © 1998, American Society for Microbiology

Clinical Application of PCR-Restriction Enzyme Pattern Analysis for

Rapid Identification of Aerobic Actinomycete Isolates

REBECCA W. WILSON,

_{VINCENT A. STEINGRUBE,}

_{* BARBARA A. BROWN,}

AND

RICHARD J. WALLACE, JR.

Center for Pulmonary and Infectious Disease Control

_{and Department of Microbiology,}

The University of Texas Health Center at Tyler, Tyler, Texas

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

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.

Interest in the identification and taxonomy of aerobic

acti-nomycetes, nocardiae and mycobacteria in particular, has been

increasing as a result of the increasing number of

immunocom-promised individuals in the population who are at greater risk

for actinomycoses, especially those with advanced human

im-munodeficiency virus disease (1, 4, 5). Traditional methods for

differentiation of species and taxa of aerobic actinomycetes are

laborious and time-consuming and frequently require

special-ized testing that is beyond the capabilities of clinical

laborato-ries (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

abbrevi-ation PRA for this method that has now gained wide

accep-tance (16). Application of this methodology has since been

expanded to include 50 commonly encountered pathogenic

species and taxa of aerobic actinomycetes comprising the

gen-era Mycobacterium (10, 13); Nocardia (9, 17, 22); and

Actino-madura, Gordona, Rhodococcus, Streptomyces, and

Tsukamu-rella (11). The rapidity and accuracy of PRA prompted the

current study (9–11, 13, 17). Clinical isolates of aerobic

actino-mycetes 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

Meet-ing of the American Society for Microbiology, Miami Beach,

Fla., 1997.)

MATERIALS AND METHODS

Organisms.The present study included 293 clinical isolates of aerobic actino-mycetes 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 antimi-crobial 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 addi-tional 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

Nocar-dia 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 83mM (each)

deoxynucleoside triphosphates, 9% dimethyl sulfoxide, and 1mM (each) primers

TB11 (59-ACCAACGATGGTGTGTCCAT) and TB12 (59-CTTGTCGAACCG

CATACCCT) (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 pat-terns. When indicated (references 10 and 22 and unpublished data), one or more

* Corresponding author. Mailing address: Department of

Microbi-ology, 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: [email protected].

148

on May 15, 2020 by guest

http://jcm.asm.org/

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 man-ufacturers, 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

(0.625mg/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 nonpig-mented 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 or-igin 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 agree-ment 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 stan-dards 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

Organisms.

On microbiological evaluation, six of the

cul-tures submitted to the UTHCT laboratory for identification

and susceptibility testing were not aerobic actinomycetes.

These cultures did not yield PCR amplification products

(am-plicons) and were excluded from further study. The

distribu-tion 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%)

iso-lates; M. fortuitum, 32 of 293 (11%) isoiso-lates; 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

aster-oides type VI (20), 17 of 293 (6%) isolates; and N. brasiliensis,

16 of 293 (5%) isolates.

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

tra-ditional methods. The remaining 19 isolates produced PRA

patterns that did not match any of the patterns currently

avail-able in the database. Eight of these isolates produced

ampli-cons that contained BstEII recognition sites and were

there-fore 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

recog-nition sites and an HaeIII pattern resembling that published

TABLE 1. Clinical isolates identified by PRA in this study

Sp. and taxon No. of clinical_isolates

Genus Mycobacterium Rapidly growing spp.

M. abscessus ... 47

M. abscessus-M. chelonae complex... 4

M. chelonae ... 18

M. fortuitum ... 32

M. fortuitum, third biovariant complex... 5

M. fortuitum, third biovar, sorbitol-positive– M. peregrinum Pipr_{complex... 1}

M. mucogenicum (MCLO)a_{... 20}

M. smegmatis... 10

Slow-growing spp. M. avium... 3

M. gordonae... 1

M. haemophilum... 2

M. intracellulare ... 4

M. marinum... 7

M. simiae ... 2

M. szulgai... 1

M. terrae-M. nonchromogenicum complex... 2

M. tuberculosis ... 3

Mycobacterium sp. (BstEII sites present; taxon not distinguishable)... 8

Mycobacterium subtotal ... 170

Genus Nocardia Nocardia asteroides complex Type Ib_{... 9}

Type II... 1

Type VI ... 17

Taxon not further differentiated ... 1

N. brasiliensis ... 16

N. farcinica... 11

N. nova ... 24

N. otitidiscaviarum... 3

N. pseudobrasiliensisc_{... 2}

N. transvalensis complex... 9

Nocardia subtotal ... 93

Genera of other aerobic actinomycetes Actinomadura madurae... 1

Gordona sputi ... 2

Gordona sp... 1

Rhodococcus equi ... 1

Streptomyces sp. ... 10

Tsukamurella paurometabolum ... 4

Other aerobic actinomycete taxa (BstEII sites absent; taxon not distinguishable) ... 11

Actinomycete subtotal ... 30

Total clinical isolates ... 293

a_{Described by Springer et al. (8). MCLO, M. chelonae-like organism.}

b_{Antibiogram types of N. asteroides complex were described by Wallace et al.}

(20).

c_{Described by Ruimy et al. (7) and Wallace et al. (17).}

on May 15, 2020 by guest

http://jcm.asm.org/

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

No-cardia sp., most likely N. asteroides complex, and which

pro-duced 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%.

Three cultures that contained small numbers of

gram-posi-tive rods mixed with heavy overgrowth of gram-posigram-posi-tive 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

tradi-tional microbiological methods. Two additradi-tional 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

ref-erence laboratory as containing both Corynebacterium

aqua-ticum 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

measure-ments 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,

respec-tively. 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.

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

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 ref-erence 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).

TABLE 2. Identification of clinical isolates that exhibited patterns

that did not match those in the current PRA database

Isolate no. Identificationa

Isolates distinguished as

Myco-bacterium sp. based on

the presence of BstEII

recognition sitesb,c

Mo 702 ...Mycobacterium sp. (pigmented rapid grower) Mo 748 ...Mycobacterium sp. (pigmented rapid grower) N 1426 ...Nocardia sp.

Mo 783 ...M. simiae

Mo 821 ...Mycobacterium sp. (pigmented rapid grower) Mo 830 ...Mycobacterium sp. (slow grower)

Mo 855 ...Mycobacterium sp. (pigmented rapid grower) Mo 875 ...Mycobacterium sp. (slow grower)

Isolates distinguished as aero-bic actinomycetes, other than Mycobacterium sp., based on the absence of

BstEII recognition sitesb,d

Mo 728#1 ...Corynebacterium aquaticum Mo 728#2 ...Actinomyces viscosus

N 1420 ...Unidentifiable; possible N. transvalensis Mo 780 ...Rhodococcus sp.

Mo 791 ...Gordona sp. Mo 798 ...N. asteroides complex Mo 802 ...Tsukamurella sp. As 121...Streptomyces sp. As 124...N. asteroides complex Mo 816 ...M. vaccae

Mo 820 ...Unidentifiable; possible Mycobacterium sp. (slow grower)

a_{Identification by biochemical tests, antimicrobial susceptibility patterns, and}

HPLC analysis.

b_{Work of Steingrube et al. (9, 11).}

c_{Total of 8.}

d_{Total of 11.}

on May 15, 2020 by guest

http://jcm.asm.org/

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,

iden-tification results based on PRA were generally achievable on a

2- to 5-day schedule.

DISCUSSION

PRA correctly identified 274 of 293 (93.5%) aerobic

actino-mycete clinical isolates, compared to traditional identification

methods that unequivocally identified 278 of those isolates

(94.9%). Comparison of the two identification methods

re-sulted 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

acti-nomycete that was not readily recognizable on isolation plates.

The specificity of PRA was further demonstrated with six

cul-tures 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

ac-complished with virtually 100% accuracy. Only very rarely

oc-curring 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

data-base has been well developed for species and taxa of

nonpig-mented 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

rap-idly growing mycobacteria remain to be developed. As this

study has progressed over the past 2 years, data for less

fre-quently 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

biovari-ant complex; M. fortuitum third biovar,

sorbitol-positive–My-cobacterium peregrinum Pip

_{complex (10); and the}

Mycobac-terium terrae-MycobacMycobac-terium nonchromogenicum complex each

represent two groups containing very closely related

mycobac-terial taxa that are not readily differentiated by either

tradi-tional methods or PRA (10, 13). Three isolates listed as M.

abscessus-M. chelonae complex could not be differentiated

fur-ther 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

data-base 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

pre-viously discussed (11), identification of Streptomyces isolates

beyond the genus level was considered to be clinically

irrele-vant 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

aer-obic actinomycete isolates is particularly important when

inva-sive 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

immunocompro-mised patients such as those with advanced human

immuno-deficiency 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

mo-lecular size standards and patterns produced by internal

con-trol 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

methodol-ogy 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

incorpo-rated 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.

TABLE 3. Timed protocol for aerobic actinomycete

clinical isolate identification by PRA

PRA

step no. PRA procedure required (h)Time

1 Cell harvest, suspension in buffer, heat kill, grind

in Mini-Beadbeater 1.5

2 Set up PCRs and program Temp-cycler 1

3 PCR amplification in Temp-cycler with 4°C

hold-ing mode Overnight

4 Preparation of four agarose gels (one for step 5

and three for step 8) 1

5 Gel loading and electrophoresis of PCR products

(amplicons) 1.75

6 Set up restriction endonuclease digests (three

restriction endonucleases and up to 36 digests) 1 7 Incubation of restriction endonuclease digests 1 8 Gel loading and electrophoresis of restriction

endonuclease digests (three gels) 2–2.5

9 Photograph gels, evaluate PRA patterns, and

identify clinical isolates 2.25

a_{Time required to analyze 12 clinical isolates per procedure.}

on May 15, 2020 by guest

http://jcm.asm.org/

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.

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 identi-fication of nocardias in a clinical laboratory. J. Appl. Bacteriol. 65:55–59. 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.

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 infec-tion: an unusual opportunistic pathogen in severely immunocompromised patients. J. Infect. Dis. 165:175–178.

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.

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.

8. Springer, B., E. C. Bo¨ttger, 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

muco-genicum sp. nov. Int. J. Syst. Bacteriol. 45:262–267.

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 amplifi-cation 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.

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.

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

Actinoma-dura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella

iso-lates, by DNA amplification and restriction endonuclease analysis. J. Clin. Microbiol. 35:817–822.

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

b-lactamases. Antimicrob. Agents Chemother. 37:1850–1855.

13. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bo¨ttger, 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.

14. Tsukamura, M. 1977. Extended numerical study of Nocardia. Int. J. Syst. Bacteriol. 27:311–323.

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.

16. Wallace, R. J., Jr. 1997. Presented at the 18th Annual Congress of the European Society of Mycobacteriology and the 2nd International Sympo-sium on Mycobacteria of Clinical Interest, Co´rdoba, 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

inva-sive disease. J. Clin. Microbiol. 33:1528–1533.

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. 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. Mi-crobiol. 29:2407–2411.

20. Wallace, R. J., Jr., L. C. Steele, G. Sumter, and J. M. Smith. 1988. Antimi-crobial susceptibility patterns of Nocardia asteroides. Antimicrob. Agents Chemother. 32:1776–1779.

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.

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 ami-noglycosides, including amikacin, and PCR-restriction fragment length poly-morphism analysis. J. Clin. Microbiol. 35:2235–2242.

on May 15, 2020 by guest

http://jcm.asm.org/