|Year : 2016 | Volume
| Issue : 2 | Page : 241-244
Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis
Fanourios Kontos1, Dimitrios Nikitas Mavromanolakis2, Marina Chari Zande2, Zoe Georgios Gitti2
1 Department of the Clinical Microbiology Laboratory, Medical School of Athens, Attikon University Hospital, Athens, Greece
2 Department of the Clinical Microbiology Laboratory, University Hospital of Heraklion, Crete, Greece
|Date of Submission||19-Apr-2015|
|Date of Acceptance||24-Sep-2015|
|Date of Web Publication||14-Apr-2016|
Department of the Clinical Microbiology Laboratory, Medical School of Athens, Attikon University Hospital, Athens
Source of Support: None, Conflict of Interest: None
Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid.
Keywords: Mycobacterium kumamotonense, nontuberculous mycobacteria, pulmonary infection
|How to cite this article:|
Kontos F, Mavromanolakis DN, Zande MC, Gitti ZG. Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis. Indian J Med Microbiol 2016;34:241-4
|How to cite this URL:|
Kontos F, Mavromanolakis DN, Zande MC, Gitti ZG. Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis. Indian J Med Microbiol [serial online] 2016 [cited 2019 Sep 22];34:241-4. Available from: http://www.ijmm.org/text.asp?2016/34/2/241/180356
| ~ Introduction|| |
Nontuberculous mycobacteria (NTM) are environmental bacteria that incidentally cause opportunistic infections in humans. Human diseases due to NTM include pulmonary disease, lymphadenitis, cutaneous disease and disseminated disease. Among these, pulmonary disease is the most common manifestation.  To determine the clinical relevance of NTM, the statement of the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) offers an important support to guide clinicians.  At present, more than 160 distinct Mycobacterium species have been validly published ( http://www.bacterio.net/mycobacterium.htlm ). The clinical relevance of isolated NTM differs strongly by species, with a spectrum ranging from species which should nearly always be considered pathogenic to typical saprophytes.  Optimal treatment regimens also differ by species with major differences between slowly and rapidly growing NTM. Because of these differences in clinical relevance and specific treatment regimens, accurate species identification is of utmost importance.
In the last 8 years, approximately 50 new NTM species were described. 
The introduction of molecular techniques facilitates the identification of novel NTM species, the clinical relevance of which is under constant evaluation. Mycobacterium kumamotonense is a novel, slowly growing NTM and was described in 2006  belongs to Mycobacterium terrae complex and is most closely related to M. terrae species.  Along with human specimens,  M. kumamotonense have been isolated from fish and water specimens. We report, for the first time in Greece the case of a pulmonary infection by the novel species M. kumamotonense in an immunocompetent patient with latent tuberculosis.
| ~ Case Report|| |
A 32-year-old English immunocompetent woman, working summertime in Crete, was presented to University Hospital of Crete for further investigation of a positive tuberculin skin test (Mantoux test) (40 mm). Chest computed tomography scan revealed a nodular lesion in the right upper lobe and enlarged hilar lymph nodes. Further examinations such as haemodiagram, blood count and biochemical tests were in normal range with the only exception of lactate dehydrogenase (LDH), which was elevated. No other risk factor for particular NTM lung disease was found.
Apart from cultures for common bacteria, three sputum samples were sent to our laboratory for mycobacterial culture. Samples were treated according to standard procedures. Lowenstein-Jensen (LJ, bioMeriex, Marcy l'Etoile, France) slants were used for solid culture and vials of the Bact-Alert three-dimension (3D) automated system (bioMeriex, Marcy l'Etoile, France) was used for liquid culture; both incubated at 37°C. Acid-fast staining was positive (5-10 bacilli/field) for one of the three sputum specimens, while acid-fast bacilli were isolated from two specimens after 2 weeks incubation in the Bact-Alert 3D system, followed by a positive LJ culture 20 days later.
For identification of the recovered isolates, the following commercial kits based on DNA strip technology for the molecular identification of mycobacteria were used: GenoType mycobacterium common mycobacteria (CM) which identifies 22 of the most frequently isolated species of NTM and the M. tuberculosis complex, and GenoType additional species (AS, Hain-Lifescience, Nehren, Germany) which identifies an additional 13 less common species. The banding patterns obtained for GenoType CM was genus-specific (bands 1, 2, 3, 10) while the banding patterns obtained for GenoType AS (bands 1, 2, 3, 6, 12, 14) was specific for Mycobacterium celatum.
For further molecular, identification of the strain (AT-249) a region of 839 bp of 16S rRNA gene and a fragment of 440 bp of the 65-kDa heat shock protein (hsp65) gene were sequenced in an automated DNA sequencer (3730 DNA analyser, Applied Biosystems) using the Big Dye terminator sequencing kit (Applied Biosystems) and the previously described primers. , The sequences were aligned with the sequences of GenBank (www.ncbi.nlm.nih.gov) databases. The sequence of 16S rRNA and hsp65 genes show 99.9% (838/839 identities) and 99.4% similarity (358/360 identities), with the respective sequences of the type strain of M. kumamotonense (strain CST7274) [Figure 1]. In addition, the 16S rRNA sequence show 99.6% (477/479 identities) similarity with M. kumamotonense sqvs FI-10008 and FI-08055 (GenBank accession number JN57117-9), and 99.4% (476/479 identities) similarity with sqv FI-07082 (JN571177)  [Figure 2]. According to sequencing results, the strain identified as M. kumamotonense. The 16S rRNA and hsp65 sequences have been deposited in GenBank with accession numbers HQ332524 and HQ332525, respectively.
|Figure 1: Phylogenetic relationships of strain Mycobacterium kumamotonense AT-249 among other Mycobacterium species based on 16S rRNA sequences. The tree was constructed using the neighbour-joining method and Kimura's two-parameters substitution model and was evaluated by bootstrap analysis based on 1000 replicons; values <50% are not shown. Mycobacterium tuberculosis ATCC 27294 was used as out group. Bar indicate 0.005 substitutions per nucleotide position|
Click here to view
|Figure 2: Alignments of the 16S rRNA sequences of Mycobacterium kumamotonense strain AT-249, reference strain GTC2729 and sqvs FI-08055, FI-07082 and FI-10008. Nucleotide positions are according to the 16S rRNA gene sequence of Escherichia coli. The hypervariable region A begins at nucleotide position 124. Only base pairs that differ from Mycobacterium kumamotonense GTC 2729 are shown|
Click here to view
The polymerase chain reaction (PCR) product of hsp65 was further used for restriction fragment length polymorphism analysis. The PCR product digested with HaeIII (New England Biolabs) and BsteII (New England Biolabs) and the restriction mixture was run on 3.5% metaphor agarose gel (Camprex Bioscience, Rockland, Inc.) at 100 V. BstEII digestion produced two fragments of 325, 1115 bp and HaeIII digestion produced four major fragments of 128,123 60 and 40 bp (data not shown).
The minimum inhibitory concentration (MICs) in μg/ml of drugs selected for their activity on slowly growing mycobacteria were determined using commercially available microdilution plates (SLOMYCOI, TREK Diagnostic systems) following the Clinical and Laboratory Standards Institute recommendations.  The antibiotics tested and the MIC obtained (in parenthesis) were: Amikacin (8 μg/ml), rifabutin (<0.25 μg/ml), ciprofloxacin (2 μg/ml), clarithromycin (0.5 μg/ml), ethambutol (2 μg/ml), ethionamide (>20 μg/m), isoniazid (>8 μg/ml), linezolid (4 μg/ml), moxifloxacin (1 μg/ml), rifampin (1 μg/ml) and streptomycin (32 μg/ml). The strain considered as susceptible in vitro to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol, linezolid, and resistant to isoniazid, ethionamide and streptomycin.
Unfortunately, we could not trace the patient from whom the M. kumamotonense strain was isolated. As the species isolation and identification were performed long after sputum sample collection and the patient traveled away Greece immediately, his follow-up was not successful.
| ~ Discussion|| |
We report for the first time in Greece the isolation of the novel species M. kumamotonense in an immunocompetent patient with latent tuberculosis. To determine the clinical relevance of NTM, the statement of the ATS/IDSA offers an important support to guide clinicians  consist of the following criteria: (1) Pulmonary symptoms, nodular or cavitary opacities on chest radiograph, or a HRCT scan that show multifocal bronchiectasis with multiple small nodules, (2) appropriate exclusion of other diagnoses, such as tuberculosis and (3) positive culture results from at least two separate expectorated sputum samples. Our case met these diagnostic criteria with a nodular lesion in the right upper lobe and enlarged hilar lymph nodes on HRCT scan, positive culture results from two separate sputum specimens, and negative results for M. tuberculosis. According to ATS/IDSA criteria, the recovered M. kumamotonense strain was considered as clinically relevant.
There is limited information in the literature about the distribution and clinical relevance of M. kumamotonense. This species were described in 2006, on the basis of a single strain isolated from a sputum sample.  No clinical information is available about the patient from whose sputum the original strain was isolated. In a survey of 150 strains belonging to M. terrae complex Tortoli et al. analysed 31 M. kumamotonense strains and concluded that M. kumamotonense along with M. arupense, are the species most frequently isolated and a large degree of sequence variability is present in all of the investigated housekeeping genes of the studied M. kumamotonense strains.  Three M. kumamotonense strains have recovered from pulmonary specimens in Spain and another four strains in Canada.  Unfortunately, there is no clinical information about all these recovered strains. Finally, only one case of generalised lymphadenopathy due to M. kumamotonense in an HIV-positive patient has been reported; the patient was treated with an antituberculosis regimen for 10 months.  The same paper also reported the misidentification of the strain as a member of the M. tuberculosis complex by commercial probes (Accuprobe and INNO-LiPA Mycobacteria). In our case, the use of the commercial identification test Genotype Mycobacterium AS misidentified the strain as M. celatum. The sequencing analysis of the hsp65 and 16S rDNA genes led to the correct identification of the strain as M. kumamotonense.
The information about the susceptibility/resistance of this species is limited. Tortoli  suggested that M. kumamotonense is susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin and moxifloxacin. Our results are, in concordance with this suggestion. 
| ~ Conclusion|| |
We report, for the first time in Greece, the isolation of the novel species M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense should be listed in the aetiologies causing pulmonary infection. This report should increase the awareness for the ubiquity to this species and raise, the index of suspicion for the detection of the pathogen, particularly in a patient with latent TB. In addition, more attention should be given to the use of currently available commercial assays for the correct identification of NTM. The introduction of more advanced molecular diagnostic methods as sequencing analysis of the 16S rRNA and hsp65 genes improved the ability to identify less CM species as M. kumamotonense.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
van Ingen J, Bendien SA, de Lange WC, Hoefsloot W, Dekhuijzen PN, Boeree MJ, et al.
Clinical relevance of non-tuberculous mycobacteria isolated in the Nijmegen-Arnhem region, The Netherlands. Thorax 2009;64:502-6.
Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al.
An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367-416.
Tortoli E. Microbiological features and clinical relevance of new species of the genus Mycobacterium
. Clin Microbiol Rev 2014;27:727-52.
Masaki T, Ohkusu K, Hata H, Fujiwara N, Lihara H, Yamada-Noda M, et al
. Mycobacterium kumamotonense
sp. nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense
in Japan: Novel slowing growing, nonchromogenic clinical isolates related to Mycobacterium terrae
complex. Microbiol Immunol 2006;50:889-97.
Tortoli E, Gitti Z, Klenk HP, Lauria S, Mannino R, Mantegani P, et al.
Survey of 150 strains belonging to the Mycobacterium terrae
complex and description of Mycobacterium engbaekii
sp. nov. Mycobacterium heraklionense
sp. nov. and Mycobacterium longobardum
sp. nov. Int J Syst Evol Microbiol 2013;63(Pt 2):401-11.
Brunello F, Ligozzi M, Cristelli E, Bonora S, Tortoli E, Fontana R. Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol 2001;39:2799-806.
Hiraishi A. Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett Appl Microbiol 1992;15:210-3.
CLSI. Susceptibility testing of Mycobacteria, Nocardiae and other Aerobic Actinomycetes; Approved Standard. 2 nd
ed. Wayne, PA: CLSI Document M24-A2, Clinical and Laboratory Standards Institute; 2011.
Hoefsloot W, van Ingen J, Andrejak C, Angeby K, Bauriaud R, Bemer P, et al.
The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: An NTM-NET collaborative study. Eur Respir J 2013;42:1604-13.
Rodríguez-Aranda A, Jimenez MS, Yubero J, Chaves F, Rubio-Garcia R, Palenque E, et al.
Misindentification of Mycobacterium kumamotonense
as M. tuberculosis. Emerg Infect Dis 2010;16:1178-80.
[Figure 1], [Figure 2]