Pulmonary Infection Caused by Mycobacterium neoaurum: The First Case in Korea
A 25-yr-old woman visited the Department of Pulmonology and Allergy Clinic at a university hospital to evaluate her cough and whitish sputum that began a month ago. She didn't have any history of chronic illnesses and was not on any medication. To test for common conditions, such as asthma and bronchitis, the physician arranged for allergy and induction tests, and all of the results were negative. Her initial blood cell counts were as follows: hemoglobin, 15.3 g/dL; white blood cells, 8.64×109/L (neutrophils, 66.1%; lymphocytes, 20.5%; monocytes, 10.2%; eosinophils, 2.4%; and basophils, 0.8%); and platelets, 353×109/L. There was no prominent change in the routine chemistry tests, except for mildly elevated hepatic enzyme levels (AST, 43 IU/L; and ALT, 65 IU/L). An acid-fast bacillus (AFB) smear based on auramine fluorescent stain showed trace result (1-2 AFB/300 fields) according to the Centers for Disease Control and Prevention (CDC) report system. High resolution computed tomography (HRCT) scans were arranged to further investigate the presence of pulmonary mycobacteria infection. A HRCT scan displayed a well-marginated centrilobular nodule with branching in the left upper lobe, apicoposterior segment, indicating the presence of pulmonary tuberculosis in the left upper lobe. The physician performed bronchoscopy, which revealed no specific endobronchial lesions, but cultures of bronchial washing specimens for AFB were positive. An endobronchial washing specimen culture was negative for other bacterial or fungal pathogens. On the basis of these results, the patient was thought to have a mycobacterial lung infection and was given rifampicin, ethambutol, and pyrazinamide.\nAFB was identified by an auramine fluorescent stain and further confirmed by Ziehl-Neelsen-stained smears from colonies grown on Mycobacteria Growth Indicator Tube (MGIT; Becton Dickinson, Sparks, MD, USA) liquid medium. On blood agar plates, we observed yellow colonies within 4 days after inoculation and incubation at 42℃. The results of the nitrate reduction test were negative. Cultural characteristics indicated that the organism belonged to a group of rapidly growing mycobacteria.\nThe American Thoracic Society (ATS) has suggested diagnostic criteria of nontuberculous mycobacterial lung disease, which require clinical, radiographic, and microbiologic evidence []. In our case, the pulmonary symptoms of the patient and HRCT scan findings suffice clinical and radiologic criteria. In terms of the microbiologic criteria, we obtained positive culture results for NTM from at least one bronchial washing specimen without any evidence of an M. tuberculosis complex infection.\nTo further identify the mycobacteria, reverse hybridization assays (GenoType Mycobacterium CM/AS, Hain Lifescience, Nehren, Germany) were performed. The test results were consistent with Mycobacterium celatum, but cultural characteristics and clinical features were inconsistent with this species. Therefore, full-length 16S rRNA gene sequencing was performed by Macrogen (Seoul, Korea) using the following detailed methods:\nUsing bacterial colonies grown on blood agar plates, bacterial genomic DNA samples were extracted using InstaGene Matrix (BIO-RAD, Hercules, CA, USA). The following primers were used for PCR: forward primer 27F, 5'-AGAGTTTGATCMTGGCTCAG-3'; and backward primer 1492R, 5'-TACGGYTACCTTGTTACGACTT-3'. This primer pair amplified a 1,492-bp fragment of the 16S rRNA gene of the Escherichia coli 16S rRNA gene. The PCR reaction was performed with 20 ng of genomic DNA as a template in a 30 µL reaction mixture using EF-Taq-DNA polymerase (SolGent, Daejeon, Korea). The following PCR program was used: activation of Taq polymerase at 95℃ for 2 min; 35 cycles at 95℃, 55℃, and 72℃ for 1 min; and a 10-min step at 72℃.\nThe amplification products were purified via a multiscreen filter plate (Millipore Corp., Billerica, MA, USA). Sequencing reactions were performed using the PRISM BigDye Terminator v3.1 Cycle Sequencing Kit. The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA, USA). The mixture was incubated at 95℃ for 5 min, placed on ice for 5 min, and then analyzed using an ABI Prism 3730XL DNA analyzer (Applied Biosystems).\nAfter sequencing, the raw sequence file (.abi) was processed using Lasergene SeqMan Pro 7.1 (DNASTAR, Madison, WI, USA) to get trimmed nucleotide sequences. We compared the obtained sequences with those deposited in GenBank (). The sequence of the 16S rRNA gene was consistent with that of M. neoaurum ATCC25796 (FJ172307.1) based on having a sequence similarity of 99.8%.\nA phylogenetic tree was generated using the Neighbor-Joining method, which is based on nucleotide sequences of the clinical isolate with those of other Mycobacteria reference strain sequences prepared from the National Center for Biotechnology Information (NCBI) database using Mega 5.0 software [] ().\nThe drug susceptibility test was performed at the Korean Institute of Tuberculosis using first-line and second-line drugs, and the minimal inhibitory concentrations (MIC) were determined using the broth microdilution method in Muller-Hinton media. MICs were interpreted based on the broth microdilution interpretive criteria for rapidly growing mycobacteria [] ().\nAfter 1 month of rifampicin, ethambutol, and pyrazinamide treatment, the patient was placed on a clarithromycin regimen for 4 months, and her symptoms improved.