a novel Mycobacterium from cervical lymphadenitis

Surgery

Abstract: A slow-growing mycobacterium was isolated from a cervical lymph node of an adolescent male. This isolate produced small, smooth, scotochromogenic colonies after 6 weeks of incubation at 25'C and 30'C (but not at 37'C or 43'C). The results of 16S--RNA gene sequencing and high-performance liquid chromatography suggest that this isolate belongs to a hitherto unrecognised pathogenic species.

Introduction

Members of the genus Mycobacterium are recognised as causative agents of cervical lymphadenitis, particularly in children. Mycobacterium scrofulaceum was once the most frequently isolated species, but members of the Mycobacterium avium complex now predominate in Western countries [1]. Over the last few years, mycobacterial laboratories have introduced improved identification methods, such as 16S--RNA gene sequencing and high-performance liquid chromatography (HPLC), resulting in the detection of cases of lymphadenitis due to novel or unusual mycobacteria [2-6]. This report describes a case of lymphadenitis caused by another novel mycobacterium.

Case report

A healthy 15-year-old male from a rural community in South Australia presented with a 3-week history of a cervical swelling. Ultrasonography showed a nonhomogenous, lobulated mass (5 x 2 x 3 cm) consistent with a matted group of enlarged lymph nodes at the apex of the left anterior triangle. Other investigations, including chest radiograph and serological investigations for Epstein-Bar- virus and cat scratch disease, showed no abnormalities. The mass was surgically excised, the wound healed satisfactorily, and the patient has remained well more than 12 months after surgery.

Histological examination of the operative specimen revealed a necrotising granulomatous reaction, but no acid-fast bacilli were seen. Cultures were performed at 30'C and 35 °C in radiometric broths (Bactec 1213; Becton Dickinson, USA) and on ferric ammonium citrate- and pyruvate-supplemented L6wenstein-Jensen media. Isolate identification was performed using conventional methods [7], commercial DNA probes (Accuprobe; Gen-Probe, USA), HPLC [8, 9], and 16SrRNA gene sequencing [9], as described previously. The 16S--RNA gene sequences were aligned and compared using the Clustal W computer package [10].

Results and discussion

A mycobacterium was detected after 3 weeks of incubation at 30'C in a radiometric broth inoculated with a portion of the operative specimen. When subcultured on L6wenstein-Jensen medium, the isolate produced smooth, scotochromogenic colonies less than 1 mm in diameter after 6 weeks of incubation at 25'C and 30'C (no growth was observed at 37'C or 43'C). Conventional biochemical tests revealed Tween 80 hydrolysis at 10 days and low semiquantitative catalase, but negative results for nitrate reduction, tellurite reduction, urease, arysulfatase (3 and 14 days), pyrazinamidase activity, and growth on L6wenstein-Jensen medium containing 5% sodium chloride. Growth was not affected by the presence or absence of 1.5% ferric ammonium citrate.

The isolate (B76676) failed to hybridise with the commercial probes for Mycobacterium gordonae or the Mycobacterium tuberculosis and Mycobacterium avium complexes. High-performance liquid chromatography produced an unusual mycolic acid profile with three high peaks clustering around the low-molecular-weight internal standard (Figure 1). The same pattern was obtained when subcultures of the organism were retested over a 12-month period. Mycobacterium avium was the closest match in our HPLC database of more than 30 species, but the profile resembled that of Mycobacterium conspicutom when compared with a larger mycobacterial HPLC library (E. Tortoli, personal communication).

Approximately_ 91% (i.e. 1411 nucleotides) of the mycobacterial 16S-rRNA gene was also sequenced (GenBank nucleotide accession number AF016407) and found to be unique (Figure 2). Phylogenetic analysis positioned B76676 among the slow-growing mycobacteria but distant from the species with similar phenotypic characteristics (ie. Mycobacterium gordonae, Mycobacterium interjectum, Mycobacterium conspicuum. or Mycobacterium avium). The 16S-rRNA gene sequence of B76676 has a long helix 18 containing an additional two nucleotides, which is characteristic of organisms clustering with the Mycobacterium terraeMycobacterium nonchromogenicum complex [11]. The sequences of B76676 and Mycobacterium cookii [12], the nearest phylogenetic neighbour, diverged by 1.3% (i.e. 16 substitutions and 2 deletions).

Isolate B76676 was cultured from the surgical specimen of a patient with cervical lymphadenitis but not from any other samples processed in our laboratory. Microscopy failed to detect acid-fast bacilli in the lymph node, but this is not surprising because acid-fast bacilli are seen on histological examination in less than 50% of cases of mycobacterial lymphadenitis [13, 14]. Hence, isolate B76676 is probably not a contaminant but rather the cause of the granulomatous reaction in this lymph node.

The mycobacteria associated with cervical lymphadenitis (e.g. Mycobacterium avium complex, Mycobacterium scrofulaceum, Mycobacterium malmoense) have been isolated from soil and water [6, 14]. These environmental mycobacteria are opportunistic pathogens that probably cause cervical lymphadenitis by colonising the oropharynx and then, in a small minority of patients, infecting the draining lymph nodes [l, 14]. The source of isolate B76676 has not been investigated, but this organism is presumably another environmental mycobacteria that can occasionally cause disease by a similar process.

Phylogenetic analysis placed isolate B76676 on a distinct branch in the Mycobacterium terrae- Mycobacterium nonchromogenicum complex, with Mycobacterium cookii being the closest recognised species. Kazda et. al. [12] provided a full description of Mycobacterium cookii after studying 17 strains, all of which had been isolated from sphagnum vegetation and pond water in New Zealand. Like isolate B76676, Mycobacterium cookii is a scotochromogen that produces detectable growth at 22 °C and 31 °C (but not at 37 °C, 42'C, or 45'C) after 4 weeks. These two mycobacteria also share negative results for nitrate reductase, urease, and pyrazinamidase activity. However, unlike isolate 1376676, Mycobacterium cookii produces arylsulfatase, and only 6% of strains hydrolyse Tween 80. Incidentally, the patient from whom 1376676 was isolated has never travelled to New Zealand.

Wayne et. al. [15] could not specify a fixed Hamming distance that reliably defined a new mycobacterial species, but they suggested that more than ten nucleotide differences within the 16S-rRNA molecule probably indicated a distinct species. The 16 substitutions and two deletions that separate 1376676 and Mycobacterium cookii, its closest phylogenetic neighbour, exceed this threshold for defining a distinct species. Isolate 1376676 and Mycobacterium cookii also have disparate biochemical reactions, and the HPLC profile of 1376676 is unique. While additional phenotypic and genomic studies are required to fully characterise 1376676 [16], the data presented in this paper suggest that this isolate from a case of cervical lymphadenitis represents a novel mycobacterial species.

Acknowledgements

The authors thank Drs. G. Crowe and W. McLarty for referring the patient; Dr. T. Simon for reviewing the histological sections; R. Ratcliff, S. Kralj, and N. Sangster for their technical support; and Dr. E. Tortoli for his advice about the HPLC results.

References

1. Wolinsky E: Mycobacterial lymphadenitis in children: a prospective study of 105 nontuberculous cases with long-term follow-up. Clinical Infectious Diseases (1995) 20:954-963
2. Liberek V, Soravia C, Ninet B, Hirschel B, Siegrist CA: Cervical lymphadenitis caused by Mycobacterium genavense in a child. Pediatric Infectious Disease Journal (1996) 15:269-270
3. Springer B, Kirschner P, Rost-Meyer G, Schr6der K-H, Kroppenstedt RM, B6ttger EC: Mycobacterium interjectum, a new species isolated from a patient with chronic lymphadenitis. Journal of Clinical Microbiology (1993) 31:3083-3089
4. Hasse G, Kentrup H, Skopnik H, Springer B, B6ttger EC: Mycobacterium lentiflavum: an etiologic agent of cervical lymphadenitis. Clinical Infectious Diseases (1997) 25:1245-1246

Fig. 2

Figure 2 Phylogenetic tree of isolate B76676 and related mycobacteria. The Clustal W multiple sequence alignment package [10] was used to align and compare approximately 1350 base pairs from the 16S-rRNA sequences of the clinical isolate B76676 and closely related slow-growing mycobacteria. The alignment corresponds to Escherichia coli positions 111 to 1474. A rooted tree has been produced, assuming Mycobacterium tuberculosis is the outlier. The GenBank accession numbers of the mycobacterial sequences are shown, as are the branch lengths

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Isolation of a Novel Mycobacterium from an Adolescent with Cervical Lymphadenitis
A. Goodwin, R. Lumb, M. Patkin, I. Bastian

A. Goodwin, R. Lumb, 1. Bastian (®)
Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, PO Box 14, Rundle Mall, Adelaide, South Australia 5001, Australia
M. Patkin
Whyalla Hospital, Whyalla, South Australia 5600, Australia

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