A New Agent of Mycobacterial Lymphadenitis in Children: Mycobacterium heidelbergense sp. nov.

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1997, p Vol. 35, No /97/$ Copyright 1997, American Society for Microbiology A New Agent of Mycobacterial Lymphadenitis in Children: Mycobacterium heidelbergense sp. nov. WALTER H. HAAS, 1 * W. RAY BUTLER, 2 PHILLIP KIRSCHNER, 3 BONNIE B. PLIKAYTIS, 2 MARIE B. COYLE, 4 BEATE AMTHOR, 1 ARNOLD G. STEIGERWALT, 5 DON J. BRENNER, 5 MAX SALFINGER, 6 JACK T. CRAWFORD, 2 ERIC C. BÖTTGER, 3 AND HANS J. BREMER 1 Department of General Pediatrics, Children s Hospital, Heidelberg University, Heidelberg, 1 and Institute for Medical Microbiology, Medizinische Hochschule Hannover, Hannover, 3 Germany; Tuberculosis/Mycobacteriology Branch, Division of AIDS, STD, and Tuberculosis Laboratory Research, 2 and Emerging Bacterial and Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, 5 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333; Harborview Medical Center, University of Washington, Seattle, Washington ; and Wadsworth Center, New York State Department of Health, Albany, New York Received 27 May 1997/Returned for modification 12 July 1997/Accepted 28 August 1997 Nontuberculous mycobacterial lymphadenitis presents an increasing clinical problem in immunocompetent young children. A slowly growing, nonphotochromogenic mycobacterium was recovered twice (isolates 2553/91 and 2554/91) from the lymphatic tissue of a child with recurrent cervical lymphadenitis. It could be differentiated biochemically from described Mycobacterium species, although it most closely resembled Mycobacterium malmoense by thin-layer chromatography and high-performance liquid chromatography of mycolic acids. A striking characteristic of the isolate was its high degree of susceptibility to antituberculous drugs in vitro, including isoniazid. Direct determination of the 16S rrna gene sequence revealed a unique sequence and positioned the strain phylogenetically on a branch separate from M. malmoense within a group of slowly growing mycobacteria that show a high degree of similarity to M. simiae at the 16S rrna gene level. Despite 99.6% sequence identity with M. simiae at the 16S rrna gene level, DNA-DNA hybridization studies (hydroxyapatite method) demonstrated DNA relatedness of less than 40%. We conclude that this organism is a new species for which we propose the name M. heidelbergense. A culture of the type strain, strain 2554/91, has been deposited in the American Type Culture Collection as strain ATCC Cervical lymphadenitis caused by nontuberculous mycobacteria was first described in The causative organism was named Mycobacterium scrofulaceum, after the clinical condition (16). With the decline of tuberculosis in industrialized countries, nontuberculous lymphadenitis has increasingly been diagnosed in immunocompetent young children (5, 10, 21). Today, the most frequently isolated species is M. avium. However, a growing number of emerging mycobacterial pathogens, including previously unrecognized species, have been isolated (4, 11, 13). Rapid recognition and detailed characterization of the causative organism are obligatory for the institution of appropriate treatment regimens, especially in patients with a complicated clinical course. The introduction of comparative 16S rrna gene sequencing has made a major contribution to the distinctive characterization of phenotypically similar organisms and recognition of new Mycobacterium species (1, 18, 19, 25). We have previously reported the isolation of a slowly growing, nonphotochromogenic mycobacterium from a child presenting with cervical lymphadenitis (7). Direct 16S rrna gene sequencing revealed that the isolate may represent a previously unrecognized mycobacterium. Here we describe the taxonomic characterization of this organism and suggest that it represents a new Mycobacterium species, and we propose the name M. heidelbergense. * Corresponding author. Mailing address: Department of General Pediatrics, University Children s Hospital, Im Neuenheimer Feld 150, Heidelberg, Germany. Phone: Fax: ic1@ix.urz.uni-heidelberg.de. MATERIALS AND METHODS Bacterial strains. Isolates 2554/91 T and 2553/91 were cultured from primarily sterile tissue of cervical lymph nodes microscopically positive for acid-fast bacilli. Direct microscopy was carried out with thick smears of the lymphatic tissue after staining by the Kinyoun method (23). The specimens were processed by standard procedures (17) and were inoculated into Middlebrook 7H12 medium and Löwenstein-Jensen (L-J) medium for primary isolation. Mycobacterial growth was detected radiometrically with the BACTEC system (BACTEC; Becton Microbiology Instrument Systems, Sparks, Md.). The type strain was deposited in the American Type Culture Collection as strain ATCC Phenotypic analysis. Colony morphology and pigmentation were determined after 4 weeks of incubation on L-J slants. The ability to grow at different temperatures (25, 30, 33, 37, 45, and 52 C) was tested on L-J medium, L-J medium enriched with ferric ammonium citrate, American Trudeau Society (ATS) medium (9), and Middlebrook 7H11 agar. The following biochemical tests were performed as described previously (9, 17): resistance to 5% NaCl; iron uptake; tellurite reduction; Tween hydrolysis; niacin production; nitrate reduction; and enzymatic activities, which included catalase (semiquantitative catalase and heatstable catalase at 68 C), urease, pyrazinamidase, arylsulfatase, and acid phosphatase activities (8). Antimicrobial susceptibilities were determined by the proportion method (9) for isoniazid (1 and 10 g/ml), rifampin (1 g/ml), pyrazinamide (25 g/ml), streptomycin (2 and 10 g/ml), ethambutol (5 g/ml), ethionamide (10 g/ml), rifabutin (2 g/ml), and cycloserine (30 g/ml). Mycolic acids and whole-cell fatty acids were analyzed by thin-layer chromatography (TLC) (12), gas-liquid chromatography (GLC) (22), and high-performance liquid chromatography (HPLC) (2) as described previously. For GLC, an aliquot of 30 to 50 mg of mycobacterial cells was inoculated into the saponification reagent (45 g of NaOH, 150 ml of methanol, 150 ml of deionized distilled water), and the mixture was heated in a boiling water bath for 30 min. Fatty acids were methylated by adding 6 N HCl methanol (13:11, vol/vol), and the mixture was incubated at 80 C for 10 min. The cooled samples were mixed with 1.25-ml aliquots of hexane methyl-tert-butyl ether (1:1, vol/vol) for extraction of the methyl esters. The extracted organic phase was washed with NaOH solution (12.0 g in 1,000 ml), and two-thirds of the upper layer was aspirated through Na 2 SO 4 for GLC. A Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard, Avondale, Pa.) equipped with a fused-silica capillary 3203

2 3204 HAAS ET AL. J. CLIN. MICROBIOL. column and a flame ionization detector was used for the analysis. The samples were injected with a 100:1 split ratio at 250 C. The initial column temperature of 170 C was increased to 270 C at a rate of 5 C min 1. Hydrogen, nitrogen, and air were used as detector gases; the carrier gas was hydrogen. The fatty acid methyl esters were compared with authentic standards (Hewlett-Packard) and were analyzed with the Microbial Identification System (MIDI, Newark, Del.). To prepare samples for TLC, one loop of mycobacteria from a 4-week L-J culture was transferred into 2 ml of saponification solution (ethylene glycol monomethyl ether containing 5% [wt/vol] NaOH and 12% [vol/vol] H 2 O), and the mixture was heated to 110 C for 2 h. The samples were acidified with sulfuric acid, and the fatty acids were extracted twice with ether. After removal of traces of acid by washing with H 2 O, the remaining ether was evaporated at 40 C. Methylation was performed by heating the sample overnight at 80 C in 2 ml of methylation reagent consisting of sulfuric acid, toluene, and methanol (1:15:30, vol/vol/vol). After cooling to room temperature, the fatty acids were extracted with hexane and evaporated just to dryness under nitrogen. The methylated lipids were redissolved in 100 l of ethyl ether, and half of the sample was spotted onto two Silica Gel F-254 plates (E. Merck AG, Darmstadt, Germany). One plate was developed three times with petroleum ether; the other plate was developed once with dichloromethane. The mycolic acids were visualized by spraying with 0.01% ethanolic rhodamine. HPLC was performed with the mycolic acids extracted from whole cells. Briefly, the fatty acids were extracted and saponified with a 25% KOH solution in methanol for 1 h in an autoclave at 121 C. Mycolic acids were derivatized to their bromophenacyl esters at 85 C in chloroform containing potassium bicarbonate and bromophenacyl-8 reagent (Pierce Chemical Co., Rockford, Ill.). HCl, water, and methanol were added to the sample; after mixing, the chloroform layer was transferred to a fresh tube and its contents were evaporated to dryness for HPLC. HPLC was carried out with a System Gold chromatograph (Beckman Instruments, Inc., Berkeley, Calif.) with a C 18 reverse-phase column as described previously (2). Gradient elution with the solvents methanol and methylene chloride over a period of 10 min was used to separate the mycolic acids. Genotypic analysis. The 16S rrna gene in two overlapping fragments was amplified by using the PCR technique, and the sequence was determined as described previously (3). With the sequencing strategy used, we determined the nucleotides of isolate 2554/91 T at 1,446 contiguous sequence positions. The sequence obtained was aligned with selected 16S rrna gene sequences as described previously (19). For the phylogenetic analysis, regions of alignment uncertainty were omitted, i.e., positions 45 to 54, 178 to 181, and 748 to 796 of the sequence corresponding to Escherichia coli positions 83 to 95, 121 to 217, and 841 to 851, respectively, as indicated in Fig. 1. Pairwise distances were calculated by weighing nucleotide differences and insertions-deletions equally (Hamming distance). The phylogenetic tree was constructed by using the neighborliness method as described previously (19). Restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene was performed as described previously (15). Briefly, a 1,380-bp fragment was amplified by PCR with primers M1 and M2, which are highly conserved throughout the genus Mycobacterium. The fragment that was produced was digested with either BstNI (New England Biolabs, Inc., Beverly, Mass.) or XhoI (Bethesda Research Laboratories, Gaithersburg, Md.). The restriction fragments were separated on a 6% polyacrylamide gel and visualized by staining with 0.5 g of ethidium bromide per ml. The relatedness at the level of genomic DNA was determined by total DNA- DNA hybridization and determination of the change in melting temperature ( T m ) as described previously (1a). Briefly, cesium chloride-purified genomic DNA of each strain was labeled with [ 32 P]dCTP (1 Ci/0.1 ml) by nick translation (nick translation kit; Boehringer Mannheim, Mannheim, Germany). After removal of unincorporated label, the strands were separated by boiling, and residual double-stranded DNA was removed by adsorption to hydroxyapatite. About cpm of labeled, single-stranded DNA was analyzed for reassociation with an excess of 75 g of sonicated genomic DNA of the same strain and the strain used for comparison. The results were corrected for self-annealing of the label. Each experiment was performed in duplicate at optimum (65 C) and stringent (80 C) reassociation temperatures. To determine T m values, reassociated label was removed from the hydroxyapatite by raising the incubation temperature to 100 C in 5 C increments. Qualitative analysis of DNA-DNA relatedness between mycobacterial species was performed as described recently (24). Briefly, purified chromosomal DNAs of M. simiae ATCC T, M. intermedium 1669/91 T, M. interjectum 4185/92 T, M. lentiflavum 2186/92 T, M. genavense 2289 T, and isolate 2554/91 T were diluted to 50 g/ml, and 10 l was applied to an Immobilon-S nylon membrane (Millipore, Bedford, Mass.). Hybridization was carried out with 1.0 g of biotinylated whole chromosomal DNA of isolate 2554/91 T at 68 C under stringent conditions as described before (24). The amount of hybridized DNA was detected by chemiluminescence (Phototope detection kit; New England Biolabs) and was recorded by exposure of TML-1 diagnostic film (Kodack Co., Rochester, N.Y.) for 20 to 30 min. Nucleotide sequence accession number. The EMBL nucleotide sequence accession number for the 16S rrna gene sequence of strain 2554/91 T is AJ FIG S rrna gene sequence of strain 2554/91 T (total length, 1,446 nucleotides). The first and last nucleotides correspond to E. coli rrna positions 31 and 1498, respectively. Positions that were omitted for the phylogenetic analysis are underlined. RESULTS The cells of strain 2554/91 T grown on L-J medium were rod-shaped (0.5 to 0.8 m by 2.0 to 3.0 m), acid-fast coccobacilli. They were often polymorphic (up to 8.0 m in length), but no spores, capsules, or aerial hyphae were observed. Primary isolation of the organism on L-J medium failed, and cultures in BACTEC Middlebrook 7H12 liquid medium (BACTEC 12B; Becton Dickinson) were positive after 4 weeks (7). After incubation of subcultures on L-J medium at 35 C for 3 to 4 weeks, the isolate formed eugonic, nonpigmented, smooth, dome-shaped colonies. At 37 C growth was observed after 4 to 6 weeks, but no growth occurred at 25 or 45 C on L-J slants. On Middlebrook 7H11 agar, nonpigmented, smooth, dome-shaped, transparent colonies developed at incubation temperatures ranging between 25 and 45 C. Variable growth enhancement was observed on ATS medium and L-J medium containing ferric ammonium citrate. In Table 1 the enzymatic and biochemical characteristics of strain 2554/91 T are compared with the characteristics of selected nonphotochromogenic mycobacteria. Antimicrobial susceptibility testing showed an unusual susceptibility of strain 2554/91 T to antituberculous drugs, with resistance to pyrazinamide (25 g/ml) and cycloserine (30 g/ml) only. GLC with the Microbial Identification System demonstrated the presence of tuberculostearic acid and a distribution of short-chain fatty acid methyl esters similar to those found in M.

3 VOL. 35, 1997 MYCOBACTERIUM HEIDELBERGENSE SP. NOV TABLE 1. Biochemical analysis and antimicrobial susceptibility of strain 2554/91 Ta Characteristic Strain 2554/91 T M. malmoense M. avium M. shimoidei M. simiae M. triplex Pigmentation n n n n n/p n Growth at: 25 C / 30 C / 33 C 37 C NA 45 C / Susceptibility to: Isoniazid (1 g/ml) S R R R R R Streptomycin (2 g/ml) S R R NA R R Ethambutol (5 g/ml) S S S/R NA R S/R Rifampin (1 g/ml) S S/R S/R NA S/R R NaCl tolerance Catalase activity at: 22 C 68 C / / Tween hydrolysis Urease activity / Niacin production / Nitrate reduction Acid phosphatase activity NA Arylsulfatase activity (3 days) Pyrazinamidase activity / / / NA a n, nonchromogenic; p, photochromogenic; /, 50 to 85% of the isolates; /, 15 to 50% of the isolates; R, resistant; S, susceptible; NA, not available. malmoense (7). The mycolic acid profile of strain 2554/91 T by TLC demonstrated type I long nonoxygenated mycolates ( mycolates), type II short nonoxygenated mycolates ( -mycolates), and type IV ketomycolates (Fig. 2). The same mycolate pattern is found in M. malmoense, M. simiae, and M. intermedium. This result was in good agreement with the mycolic acid profile obtained by HPLC, which produced almost identical patterns for M. malmoense (27) and strain 2254/91 T (Fig. 3). In contrast, M. simiae and M. intermedium could clearly be differentiated from isolate 2254/91 T by a triple-mycolate pattern produced by HPLC of mycolic acids (6). This specific mycolic acid profile is shared by a number of mycobacteria closely related to M. simiae at the level of the 16S rrna gene, including M. triplex, a recently described species (6). Determination of a hypervariable region within the 16S rrna gene sequence (corresponding to E. coli positions 129 to 270), which is known to be specific for mycobacteria at the species level (18), revealed a sequence which was identical for both isolates (2553/91 and 2554/91 T ) but which differed from the sequence of the homologous region of described Mycobacterium species. Thus, the almost complete sequence of the 16S rrna gene of isolate 2554/91 T was determined (Fig. 1). Comparison of the sequence that was obtained with published (1, 6, 18, 19, 25, 26) and unpublished sequences of all slowly growing mycobacteria that have been described, including M. simiae, M. genavense, M. triplex, M. shimoidei, and more than a dozen different isolates of M. malmoense, demonstrated a unique sequence. A phylogenetic tree based on the 16S rrna gene sequences assigns the isolate to the same branch as M. simiae, positioned at the border between the fast growing and slowly growing mycobacteria (Fig. 4). A distance matrix with Hamming distances between 16S rrna gene sequences of selected mycobacterial species is given in Table 2. A comparison of isolate 2554/91 T with M. malmoense and M. simiae based on the sequence of the 65-kDa heat shock protein gene (hsp65) is presented in Fig. 5. All species could be separated unambiguously by the PCR-RFLP pattern of the amplified 65-kDa gene fragment. The type strain of M. haband, which has been demonstrated to be identical to M. simiae serovar 1 (14), exhibited the same pattern as M. simiae, reflecting the specificity of this method. The results of DNA-DNA hybridization studies of isolate 2554/91 T and M. simiae ATCC T showed DNA relatedness of 32 and 12% at optimum and stringent temperatures, respectively. The calculation of T m resulted in divergence values of between 11.0 and 13.5 C. Qualitative hybridization analysis of whole genomic DNA with the DNAs of other Mycobacterium species, in particular, close phylogenetic relatives, i.e., M. simiae, M. interjectum, M. intermedium, M. genavense, and M. lentiflavum, revealed no significant degree of crosshybridization (Fig. 6). DISCUSSION The biochemical analysis of strains 2554/91 T and 2553/91 could not assign the isolates to a described Mycobacterium

4 3206 HAAS ET AL. J. CLIN. MICROBIOL. FIG. 2. TLC patterns of strain 2554/91 T and selected mycobacteria. The solvents used were petroleum ether-ether (90:10, vol/vol) (A) and dichloromethane (B). The mycolic acid methyl esters were visualized by spraying the plates with 0.01% ethanolic rhodamine; the plates then were photographed under UV light. Lanes: 1, M. smegmatis ATCC 19420; 2, M. avium TMC 1475; 3, M. szulgai 954 Wolinsky/Marks; 4, M. tuberculosis TMC 111; 5, M. malmoense CDC#6764; 6, strain 2554/91 T ; and 7, M. simiae CDC# The mycolate designations are as described previously, as follows: type I, -mycolate; type II, -mycolate; type III, methoxymycolate; type IV, ketomycolate; type V, epoxymycolate; and type as follows: type VI, dicarboxylic mycolate. The - and -mycolates of M. malmoense and M. simiae as well as those of strain 2554/91 T are longer and migrate farther than the corresponding mycolates of the other species. species. The results of lipid analysis by TLC and HPLC and biochemical testing indicated that strains 2554/91 T and 2553/91 most closely resembled M. malmoense. The HPLC patterns of strains 2554/91 T and 2553/91 were clearly distinct from those of other M. simiae-related taxa, including M. simiae, M. interjectum, and M. triplex, which exhibit a triple-mycolate pattern. As demonstrated by GLC of whole-cell fatty acids, strain 2554/91 T contained dodecanoic acid, which is not present in M. malmoense (7). In contrast to M. malmoense, M. avium, M. simiae, and M. triplex, antimicrobial resistance testing of isolate 2554/ FIG. 4. Phylogenetic tree, based on 16S rrna gene sequences, illustrating the position of strain 2554/91 T. The tree was rooted by using Nocardia asteroides as the outgroup. The total length of the horizontal lines connecting two species corresponds to the Hamming distance between the 16S rrna gene sequences (which is equal to the equally weighted number of substitutions or deletions or insertions). The bar represents a Hamming distance of 10. M. intermed, M. intermedium. FIG. 3. Mycolic acid profile of strain 2554/91 T determined by HPLC. The last peak corresponds to the internal standard. 91 T showed a remarkable susceptibility to antituberculous agents, especially isoniazid and ethambutol (Table 1). Comparative sequence analysis of the unique 16S rrna gene sequence of 2554/91 T placed this isolate within a growing group of slowly growing Mycobacterium species which reside on a separate branch deeply rooted at the border between fast growing and slowly growing mycobacteria. The division between slowly growing and fast growing species based on the structure of the 16S rrna molecule is marked by length and sequence variation within helix 18 (positions 456 to 476 in E. coli). Generally, an extended helix is found among the members of the slowly growing species, while rapidly growing mycobacteria are characterized by a deletion of 14 bp, resulting in a short helix 18 (25). The closest phylogenetic relationship to strain 2554/91 T based on the 16S rrna gene sequence is found for M. simiae and M. triplex, with sequence similarities of 99.6 and 99.4% (Hamming distances of 5 and 8 per 1,442 nucleotides), respectively (Table 2 and Fig. 4). Comparison of the 16S rrna gene sequence allowed for a clear phylogenetic separation of 2554/91 T from M. malmoense, which possesses a long helix 18 and an overall similarity to 2554/91 T of 96.5%, as confirmed by sequence analysis of several isolates of M. malmoense. Like the 16S rrna gene, the hsp65 gene is highly conserved at the species level for the species analyzed so far (15). The differentiation of the analyzed species at an independent ge-

5 VOL. 35, 1997 MYCOBACTERIUM HEIDELBERGENSE SP. NOV TABLE 2. Hamming distances of selected mycobacterial 16S rrna gene sequences a with strain 2554/91 T Hamming distance Species M. avium M. celatum M. flavescens M. fortuitum M. gastri M. genavense M. gordonae M. interjectum M. intermedium M. intracellulare M. lentiflavum M. leprae M. malmoense M. marinum M. nonchromogenicum M. paratuberculosis M. scrofulaceum M. simiae M. smegmatis M. szulgai M. terrae M. triplex M. tuberculosis M. xenopi 2554/91 T M. avium M. celatum M. flavescens M. fortuitum M. gastri M. genavense M. gordonae M. interjectum M. intermedium M. intracellulare M. lentiflavum M. leprae M. malmoense M. marinum M. nonchromogenicum M. paratuberculosis M. scrofulaceum M. simiae M. smegmatis M. szulgai M. terrae M. triplex M. tuberculosis M. xenopi /91 T 0 a Sequences were obtained from previous reports (1, 6, 18, 19, 25, 26). netic locus underlines the results of 16S rrna gene sequencing. Because of the high degree of sequence similarity of their 16S rrna genes, DNA-DNA hybridization studies of isolate 2554/94 T and M. simiae were performed. In spite of the sequence identity of 99.6% at the 16S rrna gene level, the results of total genomic DNA hybridization showed a relatedness of less than 40% and a T m of more than 10 C. According to published criteria for the species definition, DNA relatedness of more than 70% and a T m of less than 5 C for members of the same species are required (28). Hybridization of labeled genomic DNA of M. intermedium, M. interjectum, M. genavense, and M. lentiflavum, which exhibit high degrees of similarity to M. simiae at the 16S rrna gene level, resulted in no significant hybridization with Mycobacterium sp. strain 2554/ 91 T. Although M. triplex was not included in this analysis owing to the fact that it has only recently been described, the results from HPLC analysis, biochemical characterization, and antimicrobial susceptibility testing rule against a possible identity of Mycobacterium sp. strain 2554/91 T and M. triplex. Our results demonstrate the ability of sequence determination of the hypervariable region within the 16S rrna gene to clearly discriminate between described mycobacterial species (18). However, for isolates with highly similar 16S rrna gene sequences, FIG. 5. PCR-RFLP analysis of the 65-kDa heat shock protein gene. DNA fragments were separated on a 6% polyacrylamide gel and were visualized under UV light by staining with 0.5 g of ethidium bromide per ml. Lanes: 1, M. malmoense; 2,M. simiae; 3,M. habanae; 4, strain 2554/91 T ;5,M. tuberculosis H37Rv; M, 100-bp DNA ladder; C, negative control. FIG. 6. Dot blot DNA-DNA hybridization of biotinylated whole genomic DNA of Mycobacterium sp. strain 2554/91 T with DNAs of M. simiae ATCC T, M. intermedium 1669/91 T, M. interjectum 4185/92 T, M. lentiflavum 2186/ 92 T, and M. genavense 2289 T.

6 3208 HAAS ET AL. J. CLIN. MICROBIOL. the absolute percent similarity might show only a weak correlation to the phylogenetic distance on the basis of total DNA relatedness. The isolation of isolates 2554/91 T and 2553/91 from the surgically obtained lymph node and fistula drainage on two separate occasions, as well as the granulomatous inflammation of the excised tissue with the detection of acid-fast bacteria by microscopy, confirmed the pathogenic role of the isolates. The child presented with bilateral, extensive disease that required repeated surgical intervention (7). Even though cervical lymphadenitis caused by nontuberculous mycobacteria usually takes an uncomplicated course after removal of all diseased lymphatic tissue, multiple interventions are required for a considerable number of patients, and children might suffer from the sequelae of surgery itself, with temporary or permanent paralysis of the facial nerve (29). Therefore, rapid identification of the causative agent to the species level by direct sequencing of the hypervariable region of the 16S rrna gene can add valuable information for designing the treatment regimen. This might especially be true for M. heidelbergense, which could easily be confused with M. malmoense by standard biochemical techniques but which exhibits an unusual pattern of susceptibility to antituberculous drugs in vitro. In fact, after the initial publication of the sequence of the hypervariable region of the 16S rrna gene of M. heidelbergense, six additional clinical isolates could be identified by the German Reference Center for Mycobacteria. These isolates were cultured from sputum (four patients), gastric aspirate (one patient), and urine (one patient). The ages of the patients ranged between 41 and 82 years, and none of the patients was known to be positive for human immunodeficiency virus (20). Taxonomic description of M. heidelbergense sp. nov. Mycobacterium heidelbergense (hei.del.ber.gen se. L. n. adj., referring to Heidelberg, Germany, the source of the strain on which the description of the species is based). The isolated organism caused cervical lymphadenitis with recurrent fistula formation in an immunocompetent child (7). The description is based on an isolate from an excised lymph node. The cells are acidalcohol-fast coccobacilli (0.5 to 0.8 m by 2.0 to 3.0 m). Spores, capsules, and aerial hyphae are absent. Dilute inocula on L-J medium incubated at 35 C yield visible growth within 3 to 4 weeks. Colonies are eugonic, smooth, dome-like, nonpigmented, and 0.5 to 1 mm in diameter. Optimal growth is on L-J medium at 33 to 35 C; growth occurs at 30, 33, and 37 C, but not at 25 or 45 C. Biochemical characteristics are listed in Table 1. M. heidelbergense is susceptible in vitro to isoniazid, rifampin, and ethambutol and is resistant to pyrazinamide and cycloserine. TLC reveals a simple mycolate profile with - and -mycolates and ketomycolates. The phylogenetic position of M. heidelbergense, based on the evaluation of 16S rrna gene sequences, is within the genus Mycobacterium on a branch rooted deeply from the base of the slowly growing mycobacteria (Fig. 4). Differentiation from closely related mycobacteria. M. heidelbergense can be differentiated biochemically from M. avium, M. shimoidei, and M. simiae by the combination of positive Tween 80 hydrolysis, urease activity, negative semiquantitative catalase test, and a lack of acid phosphatase activity. M. triplex can be differentiated by a positive nitrate reduction test, as well as by its characteristic HPLC profile, which shows a triple-mycolate pattern. Apparently, M. heidelbergense phenotypically resembles M. malmoense very closely. The wider range of susceptibility to antituberculous drugs, including isoniazid, and the lack of growth at 25 C on L-J medium may serve as markers for the differentiation of this species in the clinical mycobacteriology laboratory. Definitive identification is provided by sequence analysis of the hypervariable signature region and helix 18 within the 16S rrna gene (18) (Fig. 1). In addition, the hsp65 RFLP procedure can be used to differentiate M. heidelbergense from M. malmoense. ACKNOWLEDGMENTS This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (HA 1921/3-1,3-2), the Commission of the European Communities ( High resolution automated microbial identification ), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (FKZ 01 KA 9301), and the Niedersächsischer Verein zur Bekämpfung der Tuberkulose e.v. 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