JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Mar. 2000, p. 1094–1104 Vol. 38, No. 3

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Novel Diagnostic Algorithm for Identification of Mycobacteria UsingGenus-Specific Amplification of the 16S-23S rRNA Gene

Spacer and Restriction EndonucleasesANDREAS ROTH,1* UDO REISCHL,2 ANNA STREUBEL,1 LUDMILA NAUMANN,2

REINER M. KROPPENSTEDT,3 MARION HABICHT,1 MARGA FISCHER,1

AND HARALD MAUCH1

Institut fur Mikrobiologie und Immunologie, Lungenklinik Heckeshorn, 14109 Berlin,1 Institut fur MedizinischeMikrobiologie und Hygiene, Universitat Regensburg, 93053 Regensburg,2 and Deutsche Sammlung

von Mikroorganismen und Zellkulturen, 38124 Braunschweig,3 Germany

Received 11 August 1999/Returned for modification 22 October 1999/Accepted 8 December 1999

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restrictionfragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spaceras a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Amongnonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimationof variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns,of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results wassufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLPgenotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearlylaid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely relatedspecies (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that weresuccessfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growingmycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcino-genes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16SrDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the gooddiscriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identi-fication of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacersequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strainswith unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identificationof a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, thismethod can be used by both reference (or research) and routine laboratories.

The genus Mycobacterium is represented by a wide range ofspecies. They form a heterogenous group in terms of occur-rence in clinical or environmental material, complex pheno-typic and genetic data, and disease association (25, 33). Cur-rently, identification of mycobacteria grown in culture isachieved by standard culture and biochemical methods, andfor a few species, probes are commercially available (Mycobac-terium tuberculosis, Mycobacterium avium, Mycobacterium in-tracellulare, Mycobacterium gordonae, Mycobacterium kansasii,and Mycobacterium fortuitum) (10, 12). Determination of phe-notypic features is time-consuming, difficult to assimilate into aprecise diagnosis concerning closely related taxa, and not al-ways highly reproducible (26). The majority of clinically iso-lated nontuberculous bacteria, such as M. gordonae or rapidlygrowing species, are not pathogenic or are of doubtful clinicalrelevance (3, 7). On the other hand, a rise in incidence ofnontuberculous bacteria, including newly described species orsubspecific phylogenetic lineages of potential clinical signifi-cance, and the crucial role of the laboratory in establishing the

diagnosis demand methods that provide accurate results in amore timely fashion (7, 26). Therefore, efforts for rapid andaccurate molecular identification have been undertaken in re-cent years (13–17, 21–23, 26, 29, 30; J. L. Miller, trainingmanual, MIDI Inc., Newark, N.J., 1997). Today, sequencing ofthe 16S RNA gene (rDNA) is regarded as the most suitablemethod for identification of mycobacteria (14, 32). Even so,the high expense, together with a lack of clinical relevance formost species identified in routine laboratory practice, renderssequencing unacceptable for general use. Limitations of the16S RNA gene are evident because the number of polymor-phic sites within the genus Mycobacterium is rather low (13,22). Some species have the same sequence or a very highdegree of similarity (22). This leads to problems in develop-ment of simpler sequence analysis methods, such as restrictionfragment length polymorphism (RFLP) analysis or hybridiza-tion with probes (3, 6, 15). To meet this need, alternativegenetic targets have been studied (13, 16, 22, 27, 28). Of these,the hsp65 gene has so far been best investigated, and the datawere recently improved by inclusion of more species, especiallysome rapidly growing mycobacteria (e.g., Mycobacterium che-lonae and Mycobacterium abscessus) (5, 21). However, hsp65gene-based PCR-RFLP analysis has been impeded by difficul-ties, such as minor differences of band sizes between some

* Corresponding author. Mailing address: Institut fur Mikrobiolo-gie und Immunologie, Lungenklinik Heckeshorn-Zehlendorf, ZumHeckeshorn 33, D 14109 Berlin, Germany. Phone: 49-30-8002 2254.Fax: 49-30-8002 2299. E-mail: mikromau@zedat.fu-berlin.de.

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species and the occurrence of new patterns not previouslyreported (20, 34; B. A. Forbes, K. S. Hicks, and D. L. Kiska,Abstr. 9th Eur. Cong. Clin. Microbiol. Infect. Dis., Clin. Mi-crobiol. Infect. 5:(Suppl. 3), abstr. O37, p. 62, 1999). Moreover,all current molecular approaches to detect mycobacteria havethe common disadvantage that the primers used for amplifi-cation are not specific for mycobacteria. Thus, undesirableamplification of other gram-positive bacterial contaminants,such as corynebacteria, represents a potential issue of concernin most clinical settings.

In view of this, we aimed to investigate the 16S-23S ribo-somal DNA (rDNA) internal transcribed spacers of a largernumber of mycobacterial species for their suitability to estab-lish a PCR-RFLP-based identification scheme (16, 23). Thefirst goal was to develop and evaluate novel primers for genus-specific amplification of mycobacteria and, secondly, to estab-lish a reliable diagnostic algorithm for identification of a broadspectrum of mycobacterial species with one to three endo-nucleases. The interspecies discriminatory power and the de-gree of intraspecies divergence of patterns of such a newRFLP-based approach were investigated by using 678 myco-bacterial strains within 48 species.

MATERIALS AND METHODS

Bacterial strains, identification, and sequencing. The bacteria used in thisstudy comprised 811 strains listed in Table 1. They constituted 122 species within37 genera of 286 reference strains and 525 clinical isolates. Six Nocardia clinicalisolates were identified to the genus level only. Species other than mycobacteriawere chosen mostly from taxa of actinomycetes closely related to mycobacteria.Mycobacteria were represented by a total of 678 strains (179 reference strainsand 499 clinical isolates) of 48 defined species and 5 Mycobacterium spp. thatfailed to match either biochemical species patterns or known 16S rDNA signa-ture sequences (26).

All clinical isolates were identified to the species level by standard biochemicalmethods and/or AccuProbes (12, 25). Most of the reference strains and allclinical isolates—with the exception of M. avium, M. tuberculosis, 23 M. gordonaeisolates, and 14 Mycobacterium xenopi isolates—were sequenced in the variableregions A and B within the 16S RNA gene (14, 22, 25). To allow for a betterunderstanding, we sequenced the nearly 1.5-kbp 16S rDNA in a few strains withunique or discordant RFLP patterns (one clinical isolate each of M. kansasii,Mycobacterium phlei, and Mycobacterium triviale, and the reference strains My-cobacterium flavescens S526 and S318 and Mycobacterium parafortuitum DSM43526) and some of those with unsettled taxonomic status (strains M511, S245,S279, S369, and S504) using a method described elsewhere (24). To obtain more16S-23S spacer sequence data, a selection of strains were also sequenced withinthe 16S-23S spacer (22). A few DSM reference strains with apparently wrongdesignation according to the RFLP results were subsequently reclassified afterpartial 16S rDNA sequencing and analyses of fatty acids by gas chromatographyand of mycolic acids by high-performance liquid chromatography (17; Miller,1997).

PCR amplification. Chromosomal DNA was released from bacterial suspen-sions by sonication with glass beads according to methods described elsewhere(22). Amplification of a part of the 16S-23S spacer was performed with primersSp1 (59-ACC TCC TTT CTA AGG AGC ACC-39) (AAGGA corresponds to thebeginning of the spacer sequence) and Sp2 (59-GAT GCT CGC AAC CAC TATCCA-39) (positions 210 to 190 of the M. tuberculosis spacer sequence; EMBLaccession number L15623). The amplification was done with a 50-ml reactionmixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1%Triton X-100, 200 mM (each) deoxynucleoside triphosphate (dATP, dGTP,dCTP, and dUTP), 75 ng of each primer, 1 U of Thermus aquaticus DNApolymerase (all reagents were from Pharmacia Biotech, Freiburg, Germany), and5 ml of DNA. The thermal profile involved initial denaturation for 5 min at 96°Cand 38 cycles with the following steps: 1-min denaturation at 94°C, annealing at59°C, and extension at 72°C.

RFLP analysis. The amplified products were digested separately with 2 U ofrestriction enzyme HaeIII, CfoI, TaqI, MspI (Sigma, Diesenhofen, Germany),DdeI (Promega, Madison, Wis.), or AvaII and HinfI (Amersham, Braunschweig,Germany) according to the recommendations of the manufacturers and electro-phoresed in 4% Small agarose (Biozym, Oldendorf, Germany) in the presence ofethidium bromide at 65 V for 2.0 to 3.0 h. For restriction with HinfI, dUTP in thePCR mixture was replaced by dTTP. Fragment band sizes were estimated visu-ally by comparison with appropriate controls run in parallel (type strains of M.avium, M. intracellulare, and M. kansasii) and a 100-bp ladder. All restrictionfragment sizes of patterns shown for slowly growing mycobacteria rely on se-quence data (fragments smaller than 30 bp are not shown). Due to unavailablesequence data for most rapidly growing representatives, their RFLP fragment

sizes were estimated visually without computerized help and rounded to thenearest 5 bp.

Nucleotide sequence accession numbers. The 16S RNA gene sequences ofMycobacterium spp. strains S245 (MCRO 33; scrofulaceum), S318 (M. flavescens),and S369 (M. xenopi) were deposited in the GenBank database under the acces-sion numbers AF152559, AF174289, and AF174290, respectively. Cultures ofS245, S279, S369, S318, and S522 were deposited in the strain collection of theDeutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig,Germany under the numbers 44427, 44429, 44428, 44430, and 44431.

RESULTS

Specificity of primers and RFLP patterns. With the excep-tion of Gordonia terrae, none of the bacteria other than myco-bacteria were amplifiable. Amplification products of varyingsizes were obtained from all mycobacteria tested. Ampliconsizes varied from 200 bp (M. xenopi) to 330 bp (Mycobacteriumneoaurum). Mycobacterium nonchromogenicum, Mycobacte-rium terrae, M. triviale, and rapidly growing species showedfragments larger than 250 bp. HaeIII was selected as the first-line enzyme that, together with the knowledge about ampliconsizes, would produce the most discriminative RFLP patterns.Of 58 discernible HaeIII patterns, 49 (84%) were unique andthus indicative and sufficient for identification to the specieslevel. HaeIII species-specific patterns are highlighted in Fig. 1and 2. The HaeIII patterns of slowly growing mycobacteria, M.fortuitum, and Mycobacterium peregrinum are displayed in Fig.3 and 4. Except for the patterns shown in Fig. 3, lanes 3 and 4,6 and 7, 13 and 16, 18 and 19, or 24 and 25 exhibiting minordifferences of 3 to 9 bp, HaeIII restriction produced mostly twoto three DNA fragments whose sizes could be easily estimatedby visual inspection of the gels. Since primer-dimer formationwas never noticed, fragments as small as 30 bp were also usedfor classification of RFLP patterns.

The remaining nine HaeIII patterns that did not give a finalspecies assignment needed further analysis with additional en-donucleases. Therefore, all test organisms were subjected toCfoI digestion. Although CfoI patterns were not necessary forfinal identification of most species (in particular, rapidly grow-ing ones), as a practical routine, immediate restriction withboth HaeIII and CfoI may be advisable (and thus it is shown forall species) because the reliability of results for one enzyme isaugmented if confirmed by a second endonuclease analysis.Following the established algorithm for slowly growing myco-bacteria (Fig. 1), precise estimates of fragments after CfoIrestriction were not generally required. Rather, the distin-guishing feature of this enzyme was mostly confined to thequestion of whether the amplicon was cut or not. Owing to thehigh sequence similarity of some species such as M. avium, M.chelonae, M. kansasii, and Mycobacterium simiae to their near-est relatives, these species formed groups that needed furtheranalysis by DdeI, TaqI, or AvaII for accurate identification asshown in Fig. 1, 2, and 3. Four of these clusters (Fig. 3, lanes2, 7, 9, and 22) were readily resolved using DdeI, and threewere resolved using TaqI, but the latter enzyme could notdistinguish Mycobacterium genavense, Mycobacterium lentifla-vum, and Mycobacterium triplex. If necessary, these species,which have a high spacer sequence similarity of 95%, can beseparated by restriction with MspI: M. simiae and M. lentifla-vum strains were cut once (139 and 86 bp), and M. genavenseand M. triplex were cut twice (114, 86, and 25 and 86, 79, and60 bp). The endonucleases AvaII and HinfI may be of use inexceptional cases, notably, in research or reference facilities,for separation of M. kansasii V from Mycobacterium leprae(HinfI), or Mycobacterium porcinum from Mycobacterium far-cinogenes (AvaII). One isolate of this specific M. kansasii ge-notype and the M. porcinum type strain (Table 1) tested werecut, with resulting fragments of 116 and 105 bp and 215 and 85

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TABLE 1. Reference strains and clinical isolates of bacteria used in the present study

Genus Species No. ofstrains Reference strain(s)a No. of clinical

isolates

Actinomadura madurae 1 DSM 43067T

Actinomadura pelletieri 1 DSM 43383T

Actinomyces israelii 3 DSM 43320T, S293 1Actinomyces meyeri 1 1Actinomyces naeslundii 2 DSM 43013T 1Actinomyces neuii 1 DSM 8577T

Actinomyces viscosus 2 DSM 43327T 1Amycolatopsis orientalis 2 DSM 40040T, 43387Arcanobacterium haemolyticum 3 DSM 20595T, S333 1Arcanobacterium pyogenes 1 DSM 20630T

Prevotella buccae 1 1Prevotella disiens 1 1Bacteroides fragilis 1 DSM 2151T

Cellulomonas cellulans 1 DSM 43879T

Cellulomonas turbata 2 DSM 20577T, S330Clostridium glycolicum 1 1Clostridium perfringens 1 1Corynebacterium diphtheriae 2 DSM 44123T 1Corynebacterium jeikeium 2 DSM 7171T 1Corynebacterium pseudodiphthericum 1 1Corynebacterium renale 1 DSM 20688T

Corynebacterium striatum 2 DSM 20668T 1Corynebacterium urealyticum 2 DSM 7109T 1Corynebacterium xerosis 5 DSM 20743T, 20170 3Dermatophilus congolensis 1 DSM 44180T

Dietzia maris 1 DSM 43672T

Escherichia coli 3 DSM 1103, 5923, ATCC 9637Enterococcus faecalis 1 DSM 2570Fusobacterium nucleatum 1 DSM 20482T

Gordonia aichensis 1 DSM 43978T

Gordonia amarae 2 DSM 43392T, 43391Gordonia bronchialis 1 DSM 43247T

Gordonia hirsuta 1 DSM 44140T

Gordonia hydrophobica 1 DSM 44015T

Gordonia rubropertinctus 2 DSM 43197T, 10347Gordonia sputi 2 DSM 43896T, 43979Gordonia terrae 5 DSM 43249T, 46040, 43342, 43568, 43569Haemophilus influenzae 1 ATCC 49247Lactobacillus rhamnosus 1 S331Listeria ivanovii 1 S302Listeria monocytogenes 3 S303, 305, 306Mycobacterium abscessus 9 DSM 44196T, 43492, 43493, S322–324 3Mycobacterium asiaticum 3 DSM 44056, 44292, 44297Mycobacterium aurum 2 DSM 43999T, S283Mycobacterium avium 111 DSM 44156T, DSM 44133T 109Mycobacterium bohemicum 2 DSM 44277T 1Mycobacterium celatum 15 DSM 44243T, S274, 275 12Mycobacterium chelonae 14 DSM 43804T, 43217, 43276, 43483, 43484, 43487, 43488–43490, 46626, S268 3Mycobacterium chlorophenolicum 1 DSM 43826T

Mycobacterium conspicuum 1 DSM 44136T

Mycobacterium duvalii 1 DSM 44244T

Mycobacterium farcinogenes 20 DSM 43637T, M9, 15, 16, 39, 52, 57, 191, 217, 269, 274, 275, 281, 285, 612,687, 785, N710, 715, 725

Mycobacterium flavescens 7 DSM 43991T, S318, 523–527Mycobacterium fortuitum 26 DSM 46621T, 44220T, M205, 368, 390, N723, S113, S485, 487–493 11Mycobacterium gastri 5 DSM 43505T, 43506, S227–229Mycobacterium genavense 11 11Mycobacterium gordonae 48 DSM 44160T 47Mycobacterium haemophilum 3 3Mycobacterium hassiacum 1 DSM 44199T

Mycobacterium hodleri 1 DSM 44183T

Mycobacterium interjectum 1 DSM 44064T

Mycobacterium intermedium 4 DSM 44049T 3Mycobacterium intracellulare 34 DSM 43223T, S138, 347 31Mycobacterium kansasii 52 DSM 44162T 51Mycobacterium lentiflavum 5 DSM 44195T, 44194, S136, 360 1Mycobacterium malmoense 11 DSM 44163T, S217 9Mycobacterium marinum 16 DSM 44344T, 43518, 43519, 43824, 44345, S287 10Mycobacterium mucogenicum 1 DSM 44124Mycobacterium neoaurum 1 DSM 44074T

Mycobacterium nonchromogenicum 4 DSM 44164T, S264–266Mycobacterium obuense 1 DSM 44075T

Continued on following page

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bp, respectively. M. farcinogenes isolates were not cut, nor wasM. leprae according to the database sequence. Finally, AvaII isshown in Fig. 1 for optional application in connection with M.avium, M. kansasii IV, and Mycobacterium bohemicum becausethese showed HaeIII patterns with the highest degree of sim-

ilarity. The first was digested by AvaII (144 and 75 bp); thelatter two taxons were not. Mycobacterium marinum and My-cobacterium ulcerans possess identical spacer sequences (1, 22).Therefore, these organisms could not be separated by thismethod.

TABLE 1—Continued

Genus Species No. ofstrains Reference strain(s)a No. of clinical

isolates

Mycobacterium parafortuitum 4 DSM 43528T, 43526, 43527, S528Mycobacterium peregrinum 17 DSM 43271T, M418–420, S254, 486, 494–496 8Mycobacterium phlei 5 DSM 43239T, 43214, 44018 2Mycobacterium porcinum 1 DSM 44242T

Mycobacterium rhodesiae 1 DSM 44223T

Mycobacterium scrofulaceum 11 DSM 43992T, 43226, 43512, 43513, S343, 244 5Mycobacterium senegalense 10 DSM 43656T, 43658, M266, 555, N714, 717–718, 721, 728, S114Mycobacterium shimoidei 3 DSM 44152T, S234 1Mycobacterium simiae 15 DSM 44165T, S137, 140, 141, 146, 148, 149 8Mycobacterium smegmatis 2 DSM 43756T, 43299Mycobacterium sp. 2 M511, 516Mycobacterium sp. (gastri)b 10 DSM 43221, 43507, S230–233, R230–233Mycobacterium sp. (malmoense) 2 S222, 279 2Mycobacterium sp. (scrofulaceum) 5 S245, 313–314, 316, R39 5Mycobacterium sp. (xenopi) 2 S369, 504 2Mycobacterium szulgai 8 DSM 44166T, S97 6Mycobacterium terrae 9 DSM 43227T, S280–281, 353 5Mycobacterium triplex 2 S139 1Mycobacterium triviale 2 DSM 44153T 1Mycobacterium tuberculosis 90 DSM 44156 89Mycobacterium ulcerans 3 ATCC 19423T, S219 1Mycobacterium vaccae 4 DSM 43292T, 43229, 43514, S345Mycobacterium xenopi 59 DSM 43995T 58Nocardia asteroides 12 DSM 43757T, 43003–43005, 43208, 43244, 43289, S308, 328, ATCC 23824 2Nocardia brasiliensis 2 DSM 43758T, 43009Nocardia brevicatena 1 DSM 43024T

Nocardia carnea 2 DSM 43397T, 40840Nocardia corynebacterioides 1 DSM 20151T

Nocardia farcinica 4 DSM 43665T, 43666, S304, 327Nocardia nova 1 ATCC 33726Nocardia otitidiscaviarum 3 DSM 43242T, 43010, 43398Nocardia pseudobrasiliensis 1 DSM 44290T

Nocardia seriolae 1 DSM 44129T

Nocardia sp. 6 6Nocardioides albus 1 DSM 43109T

Nocardiopsis dassonvillei 1 DSM 43111T

Peptostreptococcus anaerobius 1 DSM 2949Pseudomonas aeruginosa 1 DSM 1117Pseudonocardia autotrophica 2 DSM 535T, 43082Rhodococcus equi 2 DSM 20307T, S307Rhodococcus rhodochrous 1 DSM 43241T

Rhodococcus ruber 1 DSM 43338T

Rothia dentocariosa 3 DSM 43762T, S325, 326Saccharomonospora glauca 1 DSM 43769T

Saccharomonospora viridis 1 DSM 43017T

Saccharopolyspora rectivirgula 1 DSM 43747T

Saccharothrix aerocolonigenes 1 DSM 40034T

Skermania piniformis 1 DSM 43998T

Staphylococcus aureus 2 DSM 2569, 1104Streptococcus pneumoniae 2 ATCC 6303 1Streptomyces albus 1 DSM 40313T

Streptomyces somaliensis 1 DSM 40738T

Thermoactinomyces vulgaris 1 DSM 43016T

Tsukamurella paurometabolum 3 DSM 20162T, 44119, S329Tsukamurella pulmonis 1 DSM 44142T

Tsukamurella tyrosinosolvens 1 DSM 44234T

Turicella otitidis 1 DSM 8821T

Total 811 525

a ATCC, American Type Culture Collection, Manassas, Va.; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; M andN, strain collection, Department of Microbiology, The Medical School, University of Newcastle, Newcastle upon Tyne, United Kingdom; R, strain collection, Institutfur Mikrobiologie und Hygiene, Universitatsklinikum Regensburg, Regensburg, Germany; S, strain collection, Institut fur Mikrobiologie und Immunologie, Kranken-haus Zehlendorf, Berlin, Germany.

b Provisionally termed Mycobacterium sp. on the basis of phenotypic and genotypic data (for details, see the text).

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According to the spacer RFLP method, four referencestrains deposited as M. fortuitum (DSM 43276 and 46626),Nocardia farcinica (DSM 43231), and Mycobacterium thermore-sistibile (DSM 43644) were diagnosed as M. chelonae (the first

two isolates), Mycobacterium senegalense, and M. phlei, respec-tively. Partial 16S rDNA sequencing and analysis of fatty andmycolic acids were in full agreement with these findings, andthese strains were thereafter reclassified (the names used in

FIG. 1. Algorithm of RFLP patterns of 28 slowly growing mycobacterial species and 4 Mycobacterium spp. of uncertain taxonomic status from PCR-amplified16S-23S rDNA spacer sequences (547 strains). PCR products and restriction fragments are designated by molecular sizes in base pairs. HaeIII species-specific patternsare highlighted by boxes. CfoI patterns A to D are as follows: A, 126 to 144 and 91 to 96 (digest size varies depending on the PCR product size); B, 129 to 146 and83; C, 126, 63, and 30; D, 160 and 62. DdeI patterns A-E are as follows: A, 120 and 90; B, 120 and 80; C, 120 and 70; D, 120 and 100; E, 214. TaqI pattern A is 155and 70; 0, no restriction. Type strains were assigned to genotype I if more than one pattern occurred in a species. Genotypes Ia and Ib or IIa and IIb indicate that thestrains are genetically very similar but new RFLP genotypes have occurred after loss or acquisition of one HaeIII restriction site due to allelic microheterogeneity. M.leprae and M. kansasii III RFLP patterns were deduced from nucleotide sequence accession no. X56657 (EMBL) and the M. kansasii genotype III sequence publishedby Alcaide et al. (1). For details concerning AvaII, HinfI, and MspI patterns and descriptions of Mycobacterium spp., see the text.

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Table 1 are those after reclassification). Of five G. terraestrains, three showed a 315-bp and two showed a 330-bp PCRproduct, and the respective HaeIII RFLP patterns were 200,170, and 130 and 185 and 160 bp, respectively.

Taxonomically uncertain strains. Two rapidly photochro-mogenic strains deposited as Mycobacterium sp. (referencestrains M511 and M516) had a unique RFLP pattern com-pared to other rapidly growing mycobacteria. Data on theexact phenotype were not available, but complete 16S rDNA

sequencing revealed three substitutions compared to Mycobac-terium smegmatis: ACA 3 ATA, TAG 3 TGG, and TTA 3TGA at positions 137, 162, and 1075 of the reference sequence(EMBL X52922). Four groups comprising slowly growingstrains with uncertain taxonomic status were formed and ten-tatively named Mycobacterium sp. (Table 1). Although thisnomenclature has no taxonomic standing (the names of themost closely related species are provisionally added in paren-theses), phenotypic and genotypic data together with the find-

FIG. 2. Algorithm of RFLP patterns of 21 rapidly growing mycobacterial species and one rapidly growing Mycobacterium sp. of unknown taxonomic status fromPCR-amplified 16S-23S rDNA spacer sequences. Details are given in the legend to Fig. 1.

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ing that all showed unique spacer RFLP patterns obviated theneed to separate these groups from established species. Thefollowing is a detailed description of the results.

(i) Mycobacterium sp. (gastri). Ten reference strains origi-nally deposited as either M. kansasii or Mycobacterium gastriclustered in one RFLP genotype (Fig. 3, lane 21). All thesestrains were sequenced in the spacer except for strains S230 to

S233, which had previously been characterized as M. gastrispacer genotype Mga B (22). This revealed that the four M.kansasii strains and two M. gastri strains (DSM 43221 and43507) were attributable to spacer M. kansasii genotype IVdescribed by Alcaide et al. (1) (Table 2 gathers available datatogether with the findings of this study). The spacer genotypesequences IV and Mga B have a similarity value of 99.9% due

FIG. 3. Gel electrophoresis and HaeIII RFLP patterns of slowly growing mycobacteria from PCR-amplified 16S-23S rDNA spacer sequences (the upper panelshows PCR products without restriction). The molecular sizes of the fragments are given in Fig. 1. The patterns are displayed in order of increasing size of the biggestfragment. M, molecular size marker (100-bp ladder). MAIS, M. avium-M. intracellulare-M. scrofulaceum.

FIG. 4. Gel electrophoresis and HaeIII RFLP patterns of M. fortuitum (lanes 1 to 8) and M. peregrinum (lanes 9 to 11) from PCR-amplified 16S-23S rDNA spacersequences (the upper panel shows PCR products without restriction). The patterns are described in the legend to Fig. 2. M, molecular size marker (100-bp ladder).

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to a 1-nucleotide substitution at position 223 (ACT 3 AAT),and sequences Mga B and Mga A (M. gastri type strain se-quence) display a similarity value of 98% (4-nucleotide differ-ence). Careful reassessment of biochemical tests showed thatthese strains were incapable of hydrolyzing Tween 80 (bothM. gastri and M. kansasii hydrolyze Tween), while tests clearlypositive for M. kansasii (nitrate reduction, catalase, and pho-tochromogenicity) were weakly positive (delayed colony pig-ment formation). Thus, these strains were assumed to be anindeterminate (or new) taxon near to the species M. gastri, asevidenced by their 16S-23S rDNA sequences. In contrast, allM. kansasii strains hydrolyzed Tween, and their spacer se-quences showed sufficient diversity to emerge as three distinctsubgroups (dendrogram not shown): sequence genotypes (se-quevars) I together with II, and III together with Mka B andMka C as separate clusters, while type V showed the highestdegree of similarity to M. kansasii type IV (90%). Resolutionof 16S rDNA sequences within these entities was poor (Table2). We found and thus confirmed minor variants in the variableregion B recently described by Richter et al. (20). Full-length16S rDNA sequencing of one strain of concern because of itsnegative AccuProbe result (strain S522 with spacer genotypeMka C) flawed the probability that this strain could be anunrecognized new species, since it showed complete identitywith the M. kansasii type strain sequence downstream fromvariable region B.

(ii) Mycobacterium sp. (malmoense). Two clinical scotochro-mogenic isolates phenotypically resembling Mycobacteriummalmoense exhibited a 16S rDNA sequence with nine substi-tutions compared to that of M. malmoense: CCC CGA3 CCACTT, GGG3 GTG, ACG3 ATG, TGG3 TAG, CCT TGT3 CCC CGT, and TCG3 TTG at positions 141, 159, 220, 601,1062, and 1403 of the reference sequence (EMBL X52930).These strains could represent subspecies of M. malmoense and,interestingly, they emerged as a distinct RFLP genotype in thevicinity of M. malmoense.

(iii) Mycobacterium sp. (scrofulaceum). Similarly to the form-er case, five clinical isolates phenotypically very closely relatedto Mycobacterium scrofulaceum (the only physiological differ-ence was a lack of growth at 25°C) showed an RFLP genotypenear to but distinguishable from that of M. scrofulaceum, andin good correlation with this, possessed a distinctive 16S rDNAsequence. The latter was identical to the MCRO 33 sequencepublished previously (26), which typically shows identity with

M. scrofulaceum in variable region A and identity with M. si-miae in region B (deletion of 12 nucleotides).

(iv) Mycobacterium sp. (xenopi). Two strains, S369 and S504,isolated from the sputa of two patients with lung disease, dis-played identical complete 16S rDNA sequences, with the high-est similarity to that of the M. xenopi type strain (97%). Amissing arylsulfatase activity (2 weeks) and negative nicotin-amidase and pyrazinamidase were reactions in discordancewith M. xenopi. The RFLP results were somewhat differentfrom those of M. xenopi, exhibiting a unique HaeIII pattern.

Intraspecies stability of spacer sequences. Intraspecies spacersequence polymorphisms seemed to be more frequent in rap-idly growing mycobacteria than in slowly growing species. Infact, many of the rapidly growing representatives for whichmultiple strains within a species were studied presented morethan one RFLP pattern. As expected from the known variabil-ity found in the 16S rDNA, M. fortuitum was associated with aconsiderable variability leading to eight different HaeIII pat-terns (Fig. 4). They all shared a 108- to 110-bp band, and someof them showed typical PCR products of two different sizes dueto interoperon variability. These features, together with theCfoI result, gave the correct species identification in all cases.M. fortuitum subsp. acetamidolyticum (DSM 44220) was as-signed to RFLP genotype II, and one strain with a 16S signa-ture of M. fortuitum biovariant 3 was assigned to RFLP geno-type VII.

The occurrence of RFLP genotypes different from typestrain genotypes in M. flavescens and M. parafortuitum refer-ence strains (Fig. 2) was found to be associated with hithertounknown sequence polymorphisms in the 16S RNA gene. M.flavescens II (S526) and III (S318) had identical 16S sequences,but they differed by as many as 23 nucleotides from the typestrain sequence, which raises the question of the species integ-rity of these reference strains. M. parafortuitum RFLP geno-type II (DSM 43526) displayed six base substitutions comparedto the type strain 16S rDNA sequence: five differences in thesignature sequence of variable region A (AAT AGG ATCACT GGC TTC ATG GTC) and one mismatch (GAA 3GGA) at position 882 of the reference sequence (EMBLX93183). Full-length 16S rDNA sequences of M. phlei and M.triviale RFLP genotypes II (one strain each) showed 100%identity with the respective type strain sequences.

Of slowly growing mycobacteria, only M. kansasii (as noted),the M. terrae complex, and, to a lesser extent, M. intracellulare

TABLE 2. Spacer genotyping results for M. kansasii and M. gastrii compared to 16S RNA and hsp65 gene

Species

Results

16S-23S spacer16S rDNA

sequence variants(nt 461–469)b

AccuProbe(new version)

hsp65 genec

RFLPgeno-type

No. ofstrains

analyzed

Sequencesequevara

No. ofstrains

analyzedEMBL no. Geno-

type

Restrictionfragments (bp)

BstEII HaeIII

M. kansasii I 23 I/Mka A 6 X97632/L42262 M. kansasii/gastriCGG GTT CTC

Positive I 231, 212 127, 103, 78

II 15 II 7 CGG GTT GTC Positive II 231, 133, 79 127, 103 (70)IV 9 Mka B 2 L42263 CGG GTT TTC Positive III 231, 133, 79 127, 94, 69V 1 Mka C 1 L42264 CGG GTT TCC Negative VI 231, 133, 79 127, 103, 69VI 4 V 1 M. kansasii/gastri Positive V 325, 125 140, 100, 80

Mycobacterium sp.(gastri)

10 IV 6 M. kansasii/gastri Positive IV 231, 118, 79 127, 112, 69Mga B 4 Y14182 M. kansasii/gastri Positive Unknown Unknown

M. gastri 5 Mga A 4 X97633 M. kansasii/gastri Negative 231, 133, 79 127, 103, 69

a Genotypes I to IV and Mga A and B in accordance with references 1 and 22, respectively. Sequevar Mka B and C sequences were deposited in the EMBL databasein 1995 (unpublished data) and were later confirmed by Richter et al. (20). Sequence sequevar III (1) is not shown because the genotype was not found in this study.

b Refers to partial 16S rDNA sequencing within the variable region B only. Underlined bases indicate positions of substitutions.c According to references 1, 18, and 20. Boldface numbers indicate patterns that are identical. The HaeIII 70-bp fragment was not present in all isolates (1).

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and M. scrofulaceum were characterized by a genetic hetero-geneity leading to more than one RFLP pattern in a species.Intraspecies sequence variations for many slowly growing my-cobacteria, such as the M. avium complex, M. simiae, M. gor-donae (the last deposited as EMBL accession numbers L42258to 42261), and M. xenopi have been described (8, 22). Despitethis, allelic heterogeneities had no detrimental impact on theclearly arranged RFLP algorithm. Even so, we sequenced aselection of strains by way of example to obtain a better esti-mate of the occurrence of sequence diversity within the spacer.Species were chosen in which a diversity might be expectedbecause sequence variations occur both in the 16S rDNA andthe hsp65 gene. The results are shown in an alignment withpublished sequences in Fig. 5. The reproducibility (and thusthe degree of stability) of spacer sequences was confirmedbecause the sequences found were in full agreement with pub-lished data (8, 9), albeit some new sequevars were found (MacJ to L and Mgo E and F). Of 81 M. avium strains examined, 44and 37 fell into the Mav A and B sequevars, respectively. Thespacer sequences of six M. celatum isolates were all identical.The positions of restriction sites that generate species-specificor subspecific RFLP genotypes are located in the more con-served stem-loop regions. For example, in the case of M. in-tracellulare these contain distinct sequence motifs found insubspecific groups related to either Min or Mac sequevars,which in turn have led to the formation of two RFLP geno-types. These clusters consist of a larger number of sequevars,which are characterized by a high rate of substitutions in vari-able regions, such as the antitermination elements (position130 to 160) or within helices 2, 5, and 6. The last two lie beyond

the part of the spacer amplified by primers Sp1 and Sp2, and asingle sporadic mutation with generation of a new restrictionsite within helix 2 was observed only once (Mac K with RFLPgenotype M. intracellulare IIb). Although substitutions in thestem regions can be expected to occur rarely, we found sub-stitutions at the transition from helix 3 to the stem sequence(position 76 [Fig. 5]). Hence, M. kansasii II and M. scrofula-ceum were split into two RFLP genotypes attributable to thesame mutational event. Ultimately, the high number of strainsstudied within some species associated with only one RFLPgenotype despite sequence microheterogeneities, such as M.gordonae or M. xenopi, provide firm evidence that the degree ofstability of the RFLP patterns is very high.

DISCUSSION

We sought to establish a new molecular method for identi-fication of mycobacteria that on the one hand would be capa-ble of identifying all taxons to the species level with highaccuracy and reliability and on the other hand would be simpleenough for application even in routine laboratories. In view ofthis, emphasis was laid on inclusion of a broad spectrum ofspecies and, even more important, on examination of a largernumber of reference and clinical isolates within a species. First,this was important in order to determine the reliability of newprimers chosen within a genetic target with a tendency to showmore frequent sequence rearrangements due to a higher evo-lutionary rate (11, 22). Second, the occurrence of additionalRFLP patterns due to sequence polymorphisms not presentlyrecognized due to unavailable clinical isolates may later seri-

FIG. 5. Sequence stability and microheterogeneity of 16S-23S rDNA spacer sequences in conserved and more variable regions with relevance for the cleaving actionof HaeIII (GGCC) and CfoI (GCGC). Sequences not found in this study but published elsewhere are included (4, 8, 9). The respective sequevar designations are shownin brackets, and the number of strains sequenced for this study are shown in parentheses. Sequevars combined in one line exhibit base substitutions located in otherregions of the spacer that are not displayed. Of Mav A to E and Min A to C, only Mav A or B and Min A were found. The sequevar Mgo B was not found amongseven M. gordonae isolates examined.

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ously compromise the devised diagnostic algorithm or evenmake evaluation of additional enzymes necessary. Besides thegenus specifity of the primers shown, the size variations of thePCR amplicons described are particularly useful because theyprovide a simple means for distinguishing rapidly growing fromslowly growing species at first glance, since the latter produceamplicons larger than 250 bp. Furthermore, this valuable newfeature of the method can prove helpful in unequivocally rec-ognizing mixed cultures. For example, a culture containing aslowly growing relevant pathogen like M. malmoense can easilybe recognized when overgrowth by M. fortuitum or M. terraeoccurs because, irrespective of mixed patterns, the latter ex-hibit PCR products far larger than 220 bp.

Concerning intraspecies stability of RFLP patterns, we canstate that the spacer-based method was successfully evaluatedwith respect to expanded groups of strains within slowly grow-ing species, such as M. tuberculosis, M. avium, M. xenopi, or M.gordonae. These are frequently found in a routine laboratorysetting, and we were indeed satisfied to see that, with a fewexceptions, all of these strains in a species were associated withonly one RFLP pattern. This is in contrast to results obtainedusing the hsp65 gene-based RFLP method, which exhibits agreater number of RFLP genotypes within one species (e.g., sixpatterns for M. gordonae) (5, 29). The finding of distinct M.kansasii subgroups accurately defined by unique spacer RFLPgenotypes is in perfect correlation with previous reports (1, 18,20). Of note, the hsp65 gene RFLP method is unable to dis-tinguish the clinically relevant subspecies M. kansasii II andMka C from the nonpathogenic M. gastri (Table 2). By con-trast, the use of the spacer is flawed by the sequence identity ofM. marinum and M. ulcerans, but this represents a minor prob-lem from a clinical point of view, since these species appearunder completely different epidemiological circumstances (7).

The sequence variability of rapid growers was considerable.We can expect that additional spacer RFLP patterns will befound when more strains are analyzed. This may be particularlytrue for the M. terrae-M. nonchromogenicum complex or rap-idly growing species such as M. neoaurum, which exhibitedspecies-specific results, although the small number of strainsused probably underestimates their true genetic heterogeneity(32). Hence, we can state that data on most rapid growers arestill insufficient and remain to be improved in further studies.Similar observations have been made concerning the hsp65gene as a genetic target (21). It could be interesting to validatethe biological significance of spacer RFLP genotypes in com-parison to type strains. Since 16S rDNA sequencing data forrapidly growing mycobacteria are still very incomplete (21, 26),it appears mandatory to look for the possibility that additionalRFLP genotypes found may represent unknown infrasubspe-cific 16S rDNA genotypes. Evidence for this was shown herefor M. flavescens and M. parafortuitum, but further investiga-tions of the exact phenotypes are certainly warranted becausethese reference strains were not reassessed by biochemicaltests in this study. Besides phylogenetic or taxonomic consid-erations, such RFLP subgroups may reflect clinically, physio-logically, or epidemiologically significant subdivisions, as hasbeen proposed for M. chelonae (19) or the M. avium complex(4, 8, 9). Some additional RFLP genotypes in a species may nothave recognizable phenotypic or genotypic correlates in eitherthe physiological tests usually performed or in their complete16S rDNAs, respectively, due to the higher phylogenetic res-olution of 16S-23S spacer sequences. This was nicely shown by16S rDNA sequencing of M. phlei and M. triviale RFLP geno-types II.

The high similarity of M. avium to M. bohemicum and M.kansasii IV represents an undesirable shortcoming of the

method, since M. avium is the most frequently isolated mem-ber of the genus Mycobacterium. If gel electrophoresis wasperformed carefully, the above-mentioned similar patternswere not confused by technical staff in our laboratory (Fig. 3,compare lanes 6 and 7), and ultimately, application of a thirdenzyme resulted in a definite correct assignment in all cases. Inaddition, M. bohemicum and M. kansasii RFLP genotype IV(sequevar Mka B) are very rare in clinical specimens (1, 20). Apossible failing of the method in this case can be disregarded inmost laboratories that use GenProbes for identification of M.tuberculosis and the M. avium complex. However, if RFLP isused as the sole procedure for identification, investigatorsshould remain vigilant for this pattern by letting gels run longerin comparisons to M. avium as the proposed internal standard.In this context, a conclusion that deserves mention is thatjudicious inclusion of closely related species is crucial for acomplete assessment of the reliability and discriminatorypower of these methods. In fact, the necessity to apply addi-tional enzymes in a few groups was only recognized becausecare was taken to study, if possible, closely related mycobac-teria as well (i.e., M. simiae together with M. lentiflavum and M.triplex). Apparently, in contrast to the hsp65 gene, the diversityof spacer sequences is not high enough at a species level in allphylogenetic groups to allow separation by only two digests.Nevertheless, we believe that this disadvantage is compensatedfor by the overall simplicity of the scheme for the majority ofother species and the large amount of information yieldedafter HaeIII digestion by itself. RFLP results for species suchas M. lentiflavum-M. triplex, M. bohemicum (which is closelyrelated to M. avium), Mycobacterium interjectum-Mycobacte-rium intermedium, M. farcinogenes, or Mycobacterium obuensehave not yet been reported for the hsp65 gene (5, 27–30). Thisissue must keep us alert to the fact that the hsp65 data have yetto be perfected.

A major result that emerges from our study is the fact thatthe method presented reveals the potential to be used in my-cobacterial taxonomy. It is interesting that species closely re-lated to each other clustered in the same or similar HaeIIIpatterns. Examples are the M. avium complex together with M.scrofulaceum and M. bohemicum, M. simiae and relatives, M.fortuitum and M. senegalense, and similar HaeIII patternsfound for M. terrae and M. nonchromogenicum. By contrast,one included taxon that probably represents a new species(termed Mycobacterium sp. xenopi), as indicated by a low 16SrDNA similarity of only 97% with M. xenopi, clearly possesseda distinct HaeIII pattern. Similarly, HaeIII patterns rather dif-ferent from the type strain pattern found in six M. flavescensreference strains suggested a genetic disintegrity of this group,a finding that was later confirmed by 16S sequencing. Organ-isms provisionally denoted subspecies, for example, Mycobac-terium sp. malmoense, by contrast emerged as unique identifi-able entities but shared the same HaeIII pattern with the mostclosely related species. Irrespective of the taxonomical validityof these observations, they are a reflection of a high degree ofspacer sequence conservation and reinforce the previously dis-cussed view that 16S-23S rDNA spacer sequence analysis con-stitutes an adjunct to mycobacterial phylogeny (8, 9, 22). Thisstudy provides additional evidence that spacer sequence anal-ysis results are in good correlation with 16S rDNA data. Validdescriptions of new species or subspecies were not addressedin this study, but the M. kansasii-M. gastri and M. flavescenscases illustrate the usefulness of our method in identifyingnovel taxons before more accurate but labor-intensive compar-ative sequencing investigations (or those involving numericaltaxonomy) are initiated. The problem of pigmented M. gastristrains was addressed by Anz and Schroder as early as 1970 (2),

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and this debate was taken up again as the genetic heterogene-ity of M. kansasii was recognized later (1, 20). The somewhatconfusing nomenclature of different studies is gathered in Ta-ble 2, and it can be acknowledged that spacer-based methodsare superior to all other genetic approaches, including the 16SrDNA method, which cannot differentiate deeply enough toaccount for the genotypes found, and the AccuProbe method,which misses one M. kansasii subgroup while the taxonomicallyindeterminate subgroup with the spacer genotype IV producespositive results. The latter finding has no impact on clinicalreports because so far these strains have only been isolated inenvironmental samples (1).

In conclusion, 16S-23S rDNA PCR-RFLP is a promisingnew method with consistent advantages over the previouslyused hsp65 gene-based method for reliable and easy identifi-cation of mycobacteria. Compared to new but technically de-manding (and thus still cost prohibitive) techniques, such asthe development of DNA probe arrays (31), this method hasthe advantage of being both simple and extensive in its diag-nostic spectrum and cost-effective at the same time. Despiteallelic diversity, good intraspecies stabilities of recognizableRFLP genotypes were demonstrated. More significant poly-morphisms on a subspecies level do not preclude the use of thismethod; rather, finding correlations of specific genetic sub-types to medically relevant linkages represents an issue worthyof further investigation.

REFERENCES

1. Alcaide, F., I. Richter, C. Bernasconi, B. Springer, C. Hagenau, R. Schulze-Robbecke, E. Tortoli, R. Martin, E. Bottger, and A. Telenti. 1997. Hetero-geneity and clonality among isolates of Mycobacterium kansasii: implicationsfor epidemiological and pathogenicity studies. J. Clin. Microbiol. 35:1959–1964.

2. Anz, W., and K.-H. Schroder. 1970. Photochromogenic strains of Mycobac-terium gastri? Zentrabl. Bakteriol. 214:553–554.

3. De Beenhouwer, H., Z. Liang, P. de Rijk, C. van Eekeren, and F. Portaels.1995. Detection and identification of mycobacteria by DNA amplificationand oligonucleotide-specific capture plate hybridization. J. Clin. Microbiol.33:2994–2998.

4. De Smet, A. L., I. N. Brown, M. Yates, and J. Ivanyi. 1995. Ribosomalinternal transcribed spacers are identical among Mycobacterium avium-intra-cellulare complex isolates from AIDS patients, but vary among isolates fromelderly pulmonary disease patients. Microbiology 141:2739–2747.

5. Devallois, A., K. S. Goh, and N. Rastogi. 1997. Rapid identification ofmycobacteria to species level by PCR-restriction fragment length polymor-phism analysis of the hsp65 gene and proposition of an algorithm to differ-entiate 34 mycobacterial species. J. Clin. Microbiol. 35:2969–2973.

6. Emler, S., B. Ninet, P. Rohner, R. Auckenthaler, D. Jager, and B. Hirschel.1995. Molecular basis for cross-reactivity between a strain of Mycobacteriumterrae and DNA probes for Mycobacterium tuberculosis complex. Eur. J.Microbiol. Infect. Dis. 14:627–629.

7. Falkinham, J. O. 1996. Epidemiology of infection by nontuberculous myco-bacteria. Clin. Microbiol. Rev. 9:177–215.

8. Frothingham, R., and K. H. Wilson. 1993. Sequence-based differentiation ofstrains in the Mycobacterium avium complex. J. Bacteriol. 175:2818–2825.

9. Frothingham, R., and K. H. Wilson. 1994. Molecular phylogeny of theMycobacterium avium complex demonstrates clinically meaningful divisions.J. Infect. Dis. 169:305–312.

10. Good, R. C. 1985. Opportunistic pathogens in the genus Mycobacterium.Annu. Rev. Microbiol. 39:347–369.

11. Gurtler, V., and V. A. Stanisch. 1996. New approaches to typing and iden-tification of bacteria using the 16S-23S rDNA spacer region. Microbiology142:3–16.

12. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology—aguide for the level III laboratory. U.S. Department of Health and HumanServices publication (CDC) 86-8230. Centers for Disease Control, Atlanta,Ga.

13. Kim, B.-J., S.-H. Lee, M.-A. Lyu, S.-J. Kim, G.-H. Bai, S.-J. Kim, G.-T. Chae,E.-C. Kim, C.-Y. Cha, and Y.-H. Kook. 1999. Identification of mycobacterialspecies by comparative sequence analysis of the RNA polymerase gene(rpoB). J. Clin. Microbiol. 37:1714–1720.

14. Kirschner, P., B. Springer, U. Vogel, A. Meier, A. Wrede, M. Kiekenbeck,

F. C. Bange, and E. C. Bottger. 1993. Genotypic identification of mycobac-teria by nucleic acid sequence determination: report of a 2-year experiencein a clinical laboratory. J. Clin. Microbiol. 31:2882–2889.

15. Kox, L. F. F., J. van Leeuwen, S. Knijper, H. M. Jansen, and A. H. Kolk.1995. PCR assay based on DNA coding for 16S rRNA for detection andidentification of mycobacteria in clinical samples. J. Clin. Microbiol. 33:3225–3233.

16. Lappayawichit, P., S. Rienthong, D. Rienthong, C. Chuchottaworn, A.Chaiprasert, W. Panbangred, H. Saringcarinkul, and P. Palittapongarnpim.1996. Differentiation of Mycobacterium species by restriction enzyme analysisof amplified 16S-23S ribosomal DNA spacer sequences. Tubercle Lung Dis.77:257–263.

17. Muller, K.-D., E. N. Schmid, and R. M. Kroppenstedt. 1998. Improvedidentification of mycobacteria by using the Microbial Identification System incombination with additional trimethylsulfonium hydroxide pyrolysis. J. Clin.Microbiol. 36:2477–2480.

18. Picardeau, M., G. Prod’Hom, L. Raskine, M. P. LePennec, and V. Vincent.1997. Genotypic characterization of five subspecies of M. kansasii. J. Clin.Microbiol. 35:25–32.

19. Portaeles, F., P. de Rijk, G. Jannes, R. Lemans, W. Mijs, L. Riqouts, and R.Rossau. 1996. The 16S-23S rRNA spacer, a useful tool for taxonomical andepidemiological studies of the M. chelonae complex. Tubercle Lung Dis.77(Suppl. 2):17–18.

20. Richter, E., S. Niemann, S. Rusch-Gerdes, and S. Hoffner. 1999. Identifica-tion of M. kansasii by using a DNA probe (AccuProbe) and moleculartechniques. J. Clin. Microbiol. 37:964–970.

21. Ringuet, H., C. Akoua-Koffi, S. Honore, A. Varnerot, V. Vincent, P. Berche,J. L. Gaillard, and C. Pierre-Audigier. 1999. hsp65 sequencing for identifi-cation of rapidly growing mycobacteria. J. Clin. Microbiol. 37:852–857.

22. Roth, A., M. Fischer, H. E. Hamid, W. Ludwig, S. Michalke, and H. Mauch.1998. Differentiation of phylogenetically related slowly growing mycobacte-ria based on 16S-23S rRNA gene internal transcribed spacer sequences.J. Clin. Microbiol. 36:139–147.

23. Sanguinetti, M., B. Pasteraro, F. Ardito, S. Zanetti, A. Cingolani, L. Sechi,A. De Luca, L. Ortona, and G. Fadda. 1998. Routine use of PCR-reversecross-blot hybridization assay for rapid identification of Mycobacterium spe-cies growing in liquid media. J. Clin. Microbiol. 36:1530–1533.

24. Schroder, K.-H., L. Naumann, R. M. Kroppenstedt, and U. Reischl. 1997.Mycobacterium hassiacum sp. nov., a new rapidly growing thermophilic my-cobacterium. Int. J. Syst. Bacteriol. 47:86–91.

25. Shinnick, T. M., and R. C. Good. 1994. Mycobacterial taxonomy. Eur. J. Clin.Microbiol. Infect. Dis. 13:884–901.

26. Springer, B., L. Stockman, K. Teschner, G. D. Roberts, and E. C. Bottger.1996. Two-laboratory collaborative study on identification of mycobacteria:molecular versus phenotypic methods. J. Clin. Microbiol. 34:296–303.

27. Steingrube, A. V., J. L. Gibson, B. A. Brown, Y. Zhang, R. W. Wilson, M.Rajagopalan, and R. J. Wallace. 1995. PCR amplification and restrictionendonuclease analysis of a 65-kilodalton heat shock protein gene sequencefor taxonomic separation of rapidly growing mycobacteria. J. Clin. Microbiol.33:149–153.

28. Steingrube, V. A., R. W. Wilson, B. A. Brown, K. C. Jost, Z. Blacklock, J. L.Gibson, and R. J. Wallace. 1997. Rapid identification of clinically significantspecies and taxa of aerobic actinomycetes, including Actinomadura, Gor-donia, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, byDNA amplification and restriction endonuclease analysis. J. Clin. Microbiol.35:817–822.

29. Taylor, T. B., C. Patterson, Y. Hlae, and W. W. Safranek. 1997. Routine useof PCR-restriction fragment length polymorphism analysis for identificationof mycobacteria growing in liquid media. J. Clin. Microbiol. 35:79–85.

30. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Bottger, and T. Bodmer.1993. Rapid identification of mycobacteria to the species level by polymerasechain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175–178.

31. Troesch, A., H. Nguyen, C. G. Miyada, S. Desvarenne, T. R. Gingeras, P. M.Kaplan, P. Cros, and C. Mabilat. 1999. Mycobacterium species identificationand rifampin resistance testing with high-density DNA probe arrays. J. Clin.Microbiol. 37:49–55.

32. Torkko, P., M. Suutari, S. Suomalainen, L. Paulin, L. Larsson, and M.-L.Katila. 1998. Separation among species of Mycobacterium terrae complex bylipid analysis: comparison with biochemical tests and 16S rRNA sequencing.J. Clin. Microbiol. 36:499–505.

33. Wallace, R. J. 1994. Recent changes in taxonomy and disease manifestationsof the rapidly growing mycobacteria. Eur. J. Clin. Microbiol. Infect. Dis.13:953–960.

34. Wilson, R. W., V. A. Steingrube, B. A. Brown, and R. J. Wallace. 1998.Clinical application of PCR-restriction enzyme pattern analysis for rapididentification of aerobic actinomycete isolates. J. Clin. Microbiol. 36:148–152.

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