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IJID 1912 1–6 International Journal of Infectious Diseases xxx (2014) e1–e6
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid 1 2 3
Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient
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Liu, Gui-Rong Wang, Xia Yu, Qian Liang, Jing Mu, Yuan-Yuan Shang, Ying Ling, Hai-Qing Zhang, Su-Hua Zheng *,1, Hai-Rong Huang *
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National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on Drug-Resistant Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, People’s Republic of China
4
Q1 Guan
A R T I C L E I N F O
Article history: Received 24 September 2013 Received in revised form 24 January 2014 Accepted 24 January 2014 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Mycobacterium parascrofulaceum Pathogenicity Bacteriological characteristics
S U M M A R Y
Background: A Mycobacterium parascrofulaceum strain was isolated from a pneumonia patient—the first such reported case from China. The bacteriological characteristics of the strain were determined. Methods: Species identification was performed by homologue gene sequence comparison, then a series of biochemical tests was conducted to elucidate the bacteriological characteristics. Drug susceptibility and pathogenicity to mice of the strain were tested. Results: The clinical M. parascrofulaceum strain presented a very similar phenotypic profile to that of Mycobacterium scrofulaceum. The M. parascrofulaceum strain was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin in vitro. At week 2 post-infection, the lung tissues of mice demonstrated a local inflammatory response denoted by peri-bronchiolar inflammatory infiltrates. At weeks 4 and 8, the lung tissues showed peri-bronchiolar inflammatory infiltrates with large aggregates of lymphocytes and part of the tissue showed granulomatous lesions; there was no appreciable necrosis. The colony-forming units (CFU) count of infected lung and spleen increased gradually during the 8 weeks of the experiment. Conclusions: The M. parascrofulaceum strain isolated in China was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin. The mycobacteria were capable of proliferating in mice and could lead to pathological changes in the lungs of the mice. ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/3.0/).
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1. Introduction
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The most common mycobacterial infection in the world is caused by Mycobacterium tuberculosis, however there are increasing reports of non-tuberculous mycobacterial (NTM) infection,1 and about 150 NTM species have been reported to date.2 NTM are environmental pathogens distributed via water, wet soil, house dust, dairy products, cold-blooded animals, vegetation, and human feces, amongst other means.3 As the clinical presentation of NTM infection is often difficult to differentiate from that of M. tuberculosis complex, the accurate
* Corresponding authors. Tel.: +86 10 89509159; fax: +86 10 89509359. E-mail addresses: [email protected] (S.-H. Zheng), [email protected] (H.-R. Huang). 1 Tel.: +86 10 89509371.
and timely identification of NTM in the laboratory is a critical step before appropriate treatments can be implemented.4 Mycobacterium parascrofulaceum, a newly defined species of NTM belonging to group II,4 was first reported by Turenne et al.5 in 2004. A total nine cases of infection with this bacterium have been reported to date,6–11 including one Chinese case of cutaneous infection in 2012. Similar to many other NTM species, M. parascrofulaceum is an opportunistic pathogen,5 and the most likely candidates for infection are hosts that have underlying co-morbidities or immune paresis. The previous literature has focused on the clinical presentation of the cases, but has not included a detailed bacteriological characterization and description of the isolates. An M. parascrofulaceum strain was isolated from a pneumonia patient presenting in 2008, but although the genus was known, the species was only recently identified by our team. This case might be the first pneumonia case with clinical M. parascrofulaceum
http://dx.doi.org/10.1016/j.ijid.2014.01.023 1201-9712/ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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infection reported from China. In this study, we aimed to determine the bacteriological characteristics of the M. parascrofulaceum strain.
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2. Methods
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2.1. Patient and strain information
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The strain was acquired from the sputum specimen of a 47-year-old male patient with pneumonia in an epidemiological study in China in 2008. Written informed consent for participation was obtained from all the patients in the epidemiological study. A total of 4069 clinical isolates were enrolled in the survey, and among them, 140 were defined as NTM stains; only one M. parascrofulaceum was eventually identified. The patient lived in Fujian Province (located in the south-east of China, close to the ocean) and worked at a government institution. He had a cough and expectoration for 3 weeks before consulting a doctor. The patient had no fever, no chest pain, and no sweating. His X-ray showed signs of pneumonia, and acid-fast staining of all three consecutive sputum samples obtained showed acid-fast bacilli (AFB). The patient had been diagnosed with pulmonary tuberculosis 2 years previously and had been cured following 6 months of first-line anti-tuberculosis drug regimen treatment. Whether the patient had complications like diabetes or other immune suppression diseases was not known. Unfortunately, we could not trace the patient from whom the M. parascrofulaceum strain was isolated. As the species identification was performed long after sputum sample collection, patient follow-up was not successful. A Capilia Test (TAUNS, Numazu, Japan) was performed to rule out co-infection with M. tuberculosis (data not shown), and given that M. parascrofulaceum is not among the list of the usual species for contamination when conducting culture, and three consecutive sputum samples from our patient were all AFB-positive (from which the M. parascrofulaceum strain was cultured), we suggest that the M. parascrofulaceum may have been the causative pathogen of pneumonia in our patient with AFB smear-positive sputa.
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2.2. Species identification by homologous gene sequence comparison
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The 16S rRNA,12,13 16–23S rRNA gene internal transcribed spacer (ITS),14,15 and hsp65 sequences16 were used for species identification, with tests performed as described previously. The PCR products were sent to a commercial laboratory for sequencing and the sequences were analyzed using BLAST at http:// www.ncbi.nlm.nih.gov.
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2.3. Phenotypic properties
80 A series of standard biochemical tests for Mycobacterium 81 Q2 characterization were performed, as described by Kent,17 including 82 a growth test, nitrate reductase assay, arylsulfatase assay, tellurite 83 reductase assay, Tween hydrolysis assay, urease assay, and 84 media growth test with 50 mg/ml para-aminobenzoic acid 85 (PNB), 10 mg/ml thiophene carboxylic acid (TCH), 5% NaCl, or 86 MacConkey agar. Quality control was performed using reference 87 strains Mycobacterium scrofulaceum ATCC 19981, Mycobacterium 88 simiae ATCC 25275, Mycobacterium intracellulare ATCC 13950, and 89 Mycobacterium avium ATCC 25291.
by microplate Alamar blue assay.18,19 The antimicrobial reagents employed were all purchased from Sigma Aldrich (St. Louis, MO, USA): rifampin, ethambutol, isoniazid, amikacin, sulfamethoxazole, ofloxacin, para-aminosalicylic acid, rifapentine, capreomycin, cefoxitin, imipenem, minocycline, doxycycline, clarithromycin, azithromycin, meropenem, trimethoprim, rifabutin, linezolid, streptomycin, ethionamide, moxifloxacin, and levofloxacin. The reference strain Mycobacterium tuberculosis H37Rv ATCC 27294 was used as control.
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2.5. Cell wall mycolic acid composition analysis
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The composition and structure analysis of the cell wall mycolic acids was performed by high-performance liquid chromatography (HPLC) in accordance with the instructions of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.,). Briefly, Q3 the mycolic acids were cleaved from the cells by saponification before being extracted with chloroform, and were then derivatized to fluorescence-absorbing bromophenacyl esters and applied to the HPLC (Agilent 1200). The reference strains M. intracellulare ATCC13950, M. avium ATCC 25291, M. scrofulaceum ATCC 19981, and M. simiae ATCC 25273 were used as controls.
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2.6. Pathogenicity analysis of the strain in mice
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Infection with M. parascrofulaceum was evaluated among 6- to 8-week-old female C57BL/6 mice. The mice were kept in sterile isolators in the biohazard animal unit of a biosafety level 3 (BSL-3) laboratory. A total of 72 mice were divided evenly into six groups. The mice were challenged with 0.1 ml of M. parascrofulaceum suspension containing approximately 1 107 bacilli. The M. tuberculosis laboratory strain H37Rv was used as a control strain, while phosphate-buffered saline (PBS) was used as negative control. Groups 1, 3, and 5 were inoculated via the tail vein with M. parascrofulaceum, H37Rv, and PBS, respectively. Groups 2, 4, and 6 were injected intraperitoneally with M. parascrofulaceum, H37Rv, and PBS, respectively. Bacterial loads in the lungs and spleens of the infected mice, as well as the control mice, were evaluated at weeks 2, 4, and 8 postchallenge. At every time point, four mice were sacrificed and the left lungs and half of the spleens were homogenized in 1 ml PBS containing 0.04% Tween 20. Ten-fold serial dilutions of homogenates were plated in duplicate onto Middlebrook 7H11 agar Q4 plates supplemented with 10% OADC and incubated at 37 8C for 1 Q5 month. The colony counts were expressed in log10. The right lung and half of the spleen from each mouse were fixed with 10% formalin in PBS and then subjected to standard processing and paraffin embedding. Multiple 4-mm-thick tissue sections were stained with hematoxylin and eosin (H&E) for the histological/ morphometric analysis. The histological characteristics of exudative and proliferative reactions were classified based on the criteria described previously.20
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2.7. Statistical analysis
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The Student’s t-test was used to assess statistical significance between groups of mice; p < 0.05 was considered statistically significant.
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3. Results
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2.4. Drug susceptibility testing (DST)
3.1. Species identification
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Susceptibility testing was performed using the serial two-fold broth microdilution method, and the minimum inhibitory concentration (MIC) of 23 drugs for the strain was determined
Analysis of the 16S rRNA, ITS, and hsp65 sequences of the isolate using BLAST against sequences from the GenBank database gave 99.64%, 99.06%, and 99.83% similarity, respectively, with that of
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Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Table 1 Phenotypic characteristics of the Mycobacterium parascrofulaceum clinical strain in comparison with other slowly growing Mycobacterium speciesa Characteristic Growth at 25 8C 37 8C Visible growth in 7 days Morphology Pigments produced Nitrate reductase Arylsulfatase 3 days 14 days Tellurite reductase Tween hydrolysis 32 >40 32 >128 32 >32 >16 >16 8 >160 >64
0.5 2 1 64 128 0.5 1 2 0.1 1 1 2 2 5 2.5 5 8 2 8 1 1 19 1
MIC, minimum inhibitory concentration; MABA, microplate Alamar blue assay; WHO, World Health Organization; TB, tuberculosis; CLSI, Clinical and Laboratory Standards Institute. a The WHO recommended critical concentrations of these antimicrobial drugs for TB. b The CLSI recommended critical concentrations for rapidly growing mycobacteria. c The critical concentrations for TB proposed by Brown et al.,21 Watt et al.22, and Hugonnet et al.23
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Figure 1. Mycolic acid patterns of Mycobacterium parascrofulaceum compared with those of MAIS complex (M. avium, M. intracellulare, and M. scrofulaceum) obtained by HPLC analysis using Sherlock software. The blue line represents the pattern of M. parascrofulaceum, while the red one represents the pattern of MAIS.
3.6. Lung and spleen histopathology in C57BL/6 mice
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10 0
A
M. par ascr of ul aceum H37Rv
2weeks
4weeks
M. parascrofulaceum has shown similarities to several known NTM species using single identification methods, but it is clearly a unique species as determined by a polyphasic approach. Tortoli et al. reported that about 10% of M. scrofulaceum isolates were
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8weeks
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B
4 3 2 1 0
8weeks
C
2weeks
4. Discussion
10 6 CFU/half of lung
10 6 CFU/half of spleen
10 6 CFU/half of spleen
After fixation, the tissues of the mice were sectioned and stained with H&E and microscopic examination was performed. With regard to histopathological examination, infection by Q6 M. parascrofulaceum and H37Rv were indistinguishable. Two weeks post-challenge, the lung tissues demonstrated a local inflammatory response denoted by peri-bronchiolar inflammatory infiltrates. At weeks 4 and 8 post-infection, lung tissues showed
peri-bronchiolar inflammatory infiltrates with large aggregates of lymphocytes and part of the tissue showed granulomatous lesions; no appreciable necrosis was apparent (Figure 3). The histopathological features of tissue from animals infected with either M. parascrofulaceum or H37RV were indistinguishable. In spite of the severe pathological changes observed in the lungs of the infected mice, all the animals maintained viability for up to 8 weeks of observation.
10 6 CFU/half of lung
with M. parascrofulaceum showed CFUs similar to those of mice infected with H37Rv at 2 and 4 weeks post-infection, but at 8 weeks post-infection the bacterial burden was lower in the M. parascrofulaceum-infected mice (p < 0.05) (Figure 2). Results were similar in mice infected either via the tail vein or intraperitoneally.
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2weeks
4weeks
8weeks
D
6 4 2 0 2weeks
4weeks
8weeks 7
Figure 2. Growth curve of tested bacilli in the lungs and spleens of C57BL/6 mice. The C57BL/6 mice were inoculated with 1 10 bacilli either through intraperitoneal (A, B) or tail vein (C, D) route. Data are presented as the mean and standard deviation at each time-point. *Statistically significant (p < 0.05) when compared with mice infected with H37Rv strain.
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Figure 3. Histological pictures of the lungs in C57BL/6 mice by H&E staining at week 4 post-infection. (A) Negative control mice. (B) M. parascrofulaceum-infected mice, through tail vein route. (C) H37Rv-infected mice, through tail vein route. (D) M. parascrofulaceum-infected mice, through intraperitoneal route. (E) H37Rv-infected mice, through intraperitoneal route. Original magnification: 40.
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actually M. parascrofulaceum strains.7 In our assay, the M. parascrofulaceum strain was identified as Mycobacterium-MAIS complex by the mycolic acid analysis method. Therefore, we suggest the only reliable way to identify M. parascrofulaceum is by sequence comparison of highly conserved genes. Genetically, M. parascrofulaceum is most related to M. simiae, whereas it presents a similar phenotypic profile to M. scrofulaceum.5 In our assays, the M. parascrofulaceum strain had almost the same phenotypic profile as the M. scrofulaceum type strain when considering all the biochemical tests, except for the tellurite reductase assay and the 14-day arylsulfatase assay. Although its phenotypic profile was also similar to those of M. intracellulare and M. avium, the urease, tellurite reductase, and arylsulfatase assays were helpful in differentiating them. The prognosis of M. parascrofulaceum infection remains unclear. Although anti-mycobacterial drugs have been used historically in the few clinical cases reported, most of the patients died before a reasonable evaluation of the treatment could be made. In three known M. parascrofulaceum infection cases in which the patient survived, one case improved by administering clarithromycin, ethambutol, and rifampin,5 the second did not show recurrence after a surgical debridement of the pathological lesions in the lung and lymph node without any chemotherapy,8 and the third slowly improved by administering clarithromycin, amikacin, and ethambutol.9 Turenne et al. showed that M. parascrofulaceum isolates were sensitive to rifampin, clarithromycin, amikacin, and sulfamethoxazole in vitro,5 and Teruya et al. suggested that rifampin, clarithromycin, and levofloxacin could be effective for M. parascrofulaceum infection,8 while Shojaei et al. showed that the organism was susceptible to amikacin, clarithromycin, rifampin, doxycycline, sulfamethoxazole, streptomycin, and imipenem in vitro.10 In this study, we tested the sensitivity of the organism to 23 agents including the commonly used anti-tuberculosis drugs and some general antibiotics that are frequently used in the treatment of NTM disease. The results showed that this isolate was susceptible to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin in vitro. The other M. parascrofulaceum strain isolated from a cutaneous infection in China was reported to be sensitive to clarithromycin and moxifloxacin, which is consistent with our findings. In our test, the MIC of the strain to rifampin was 2 mg/ml, and we define the strain as resistant according to the critical concentration recommended by the WHO for M. tuberculosis. However, as the MIC is so close to the critical concentration, administering rifampin might also be effective in clinical treatment. The levofloxacin MIC was similarly close to the critical concentration. The discrepancy in DST between our isolate and those reported in the literature may be due to strain differences. According to the previous reports and our results, rifamycin agents, clarithromycin, azithromycin, moxifloxacin, and levofloxacin may be the key drugs for the treatment of M. parascrofulaceum infection. Owing to its relatively recent recognition as a novel species, not much is known about the pathogenicity of M. parascrofulaceum. Our mouse infection model results at least suggest that
M. parascrofulaceum is capable of proliferating in mice and could lead to pathological changes in the lungs but not in the spleens of the mice. In conclusion, an M. parascrofulaceum strain isolated from a Chinese pneumonia patient was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin, was capable of proliferating in mice, and could lead to pathological changes in the lungs of the mice.
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Acknowledgements
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This work was supported by research funding from the Infectious Diseases Special Project, Ministry of Health of China (2012ZX10003002-009) and Beijing Natural Science Foundation grants (7132049). All the mycobacterial strains used in this project were acquired from the ‘Beijing bio-bank of clinical resources on tuberculosis’. Ethical approval: This study was approved by the ethics committee of Beijing Chest Hospital. The protocol of the mouse model experiment was approved by the Animal Ethics Committee of Beijing Chest Hospital. Conflict of interest: The authors declare no conflicts of interest, real or perceived, financial or non-financial.
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References
305
1. Rosenzweig DY. Nontuberculous mycobacterial disease in the immunocompetent adult. Semin Respir Infect 1996;11:252–61. 2. Tortoli E. The new mycobacteria: an update. FEMS Immunol Med Microbiol 2006;48:159–78. 3. Cook JL. Nontuberculous mycobacteria: opportunistic environmental pathogens for predisposed hosts. Br Med Bull 2010;96:45–59. 4. 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. 5. Turenne CY, Cook VJ, Burdz TV, Pauls RJ, Thibert L, Wolfe JN, et al. Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae. Int J Syst Evol Microbiol 2004;54: 1543–51. 6. Santos R, Fernandes J, Fernandes N, Oliveira F, Cadete M. Mycobacterium parascrofulaceum in acidic hot springs in Yellowstone National Park. Appl Environ Microbiol 2007;73:5071–3. 7. Tortoli E, Chianura L, Fabbro L, Mariottini A, Martin-Casabona N, Mazzarelli G, et al. Infections due to the newly described species Mycobacterium parascrofulaceum. J Clin Microbiol 2005;43:4286–7. 8. Teruya H, Tateyama M, Hibiya K, Tamaki Y, Haranaga S, Nakamura H, et al. Pulmonary Mycobacterium parascrofulaceum infection as an immune reconstitution inflammatory syndrome in an AIDS patient. Intern Med 2010;49: 1817–21. 9. Hibiya K, Tateyama M, Teruya H, Nakamura H, Tasato D, Kazumi Y, et al. Immunopathological characteristics of immune reconstitution inflammatory syndrome caused by Mycobacterium parascrofulaceum infection in a patient with AIDS. Pathol Res Pract 2011;207:262–70. 10. Shojaei H, Hashemi A, Heidarieh P, Daei-Naser A. Chronic pelvic pain due to Mycobacterium parascrofulaceum in an Iranian patient: first report of isolation and molecular characterization from Asia. Braz J Infect Dis 2011;15:186–7. 11. Zong W, Zhang X, Wang H, Xu XL, Wang Q, Tian W, et al. The first case of cutaneous infection with Mycobacterium parascrofulaceum. Ther Clin Risk Manag 2012;8:353–8. 12. Rogall T, Wolters J, Flohr T, Bottger EC. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol 1990;40:323–30.
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G. Liu et al. / International Journal of Infectious Diseases xxx (2014) e1–e6 Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 2001;39:3637–48. Turenne C, Chedore P, Wolfe J, Jamieson F, Broukhanski G, May K, et al. Mycobacterium lacus sp. nov., a novel slowly growing, non-chromogenic clinical isolate. Int J Syst Evol Microbiol 2002;52:2135–40. Harmsen D, Rothganger J, Frosch M, Albert J. RIDOM: Ribosomal Differentiation of Medical Micro-organisms Database. Nucleic Acids Res 2002;30:416–7. Kim BJ, Lee KH, Park BN, Kim SJ, Bai GH, Kook YH. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 2005;55:1649–56. Kent PT, G.P. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: Department of Health and Human Services, Centers for Disease Control; 1985. Brown-Elliott BA, Crist CJ, Mann LB, Wilson RW, Wallace RJ. In vitro activity of linezolid against slowly growing nontuberculous mycobacteria. Antimicrob Agents Chemother 2003;47:1736–8.
19. National Committee for Clinical Laboratory Standards. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes. Approved Standard M24-A. Wayne, PA: NCCLS; 2003. 20. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 2003;16:463–6. 21. Brown BA, Wallace Jr RJ, Onyi G. Activities of the glycylcyclines N,N-dimethylglycylamido-minocycline and N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline against Nocardia spp. and tetracycline-resistant isolates of rapidly growing mycobacteria. Antimicrob Agents Chemother 1996;40:874–8. 22. Watt B, Rayner A, Harris G. Comparative activity of azithromycin against clinical isolates of mycobacteria. J Antimicrob Chemother 1996;38:539–42. 23. Hugonnet JE, Tremblay LW, Boshoff HI, Barry 3rd CE, Blanchard JS. Meropenem–clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 2009;323:1215–8.
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IJID 1912 1–6 International Journal of Infectious Diseases xxx (2014) e1–e6
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid 1 2 3
Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient
5
Liu, Gui-Rong Wang, Xia Yu, Qian Liang, Jing Mu, Yuan-Yuan Shang, Ying Ling, Hai-Qing Zhang, Su-Hua Zheng *,1, Hai-Rong Huang *
6 7
National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on Drug-Resistant Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, People’s Republic of China
4
Q1 Guan
A R T I C L E I N F O
Article history: Received 24 September 2013 Received in revised form 24 January 2014 Accepted 24 January 2014 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Mycobacterium parascrofulaceum Pathogenicity Bacteriological characteristics
S U M M A R Y
Background: A Mycobacterium parascrofulaceum strain was isolated from a pneumonia patient—the first such reported case from China. The bacteriological characteristics of the strain were determined. Methods: Species identification was performed by homologue gene sequence comparison, then a series of biochemical tests was conducted to elucidate the bacteriological characteristics. Drug susceptibility and pathogenicity to mice of the strain were tested. Results: The clinical M. parascrofulaceum strain presented a very similar phenotypic profile to that of Mycobacterium scrofulaceum. The M. parascrofulaceum strain was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin in vitro. At week 2 post-infection, the lung tissues of mice demonstrated a local inflammatory response denoted by peri-bronchiolar inflammatory infiltrates. At weeks 4 and 8, the lung tissues showed peri-bronchiolar inflammatory infiltrates with large aggregates of lymphocytes and part of the tissue showed granulomatous lesions; there was no appreciable necrosis. The colony-forming units (CFU) count of infected lung and spleen increased gradually during the 8 weeks of the experiment. Conclusions: The M. parascrofulaceum strain isolated in China was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin. The mycobacteria were capable of proliferating in mice and could lead to pathological changes in the lungs of the mice. ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/3.0/).
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1. Introduction
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The most common mycobacterial infection in the world is caused by Mycobacterium tuberculosis, however there are increasing reports of non-tuberculous mycobacterial (NTM) infection,1 and about 150 NTM species have been reported to date.2 NTM are environmental pathogens distributed via water, wet soil, house dust, dairy products, cold-blooded animals, vegetation, and human feces, amongst other means.3 As the clinical presentation of NTM infection is often difficult to differentiate from that of M. tuberculosis complex, the accurate
* Corresponding authors. Tel.: +86 10 89509159; fax: +86 10 89509359. E-mail addresses: [email protected] (S.-H. Zheng), [email protected] (H.-R. Huang). 1 Tel.: +86 10 89509371.
and timely identification of NTM in the laboratory is a critical step before appropriate treatments can be implemented.4 Mycobacterium parascrofulaceum, a newly defined species of NTM belonging to group II,4 was first reported by Turenne et al.5 in 2004. A total nine cases of infection with this bacterium have been reported to date,6–11 including one Chinese case of cutaneous infection in 2012. Similar to many other NTM species, M. parascrofulaceum is an opportunistic pathogen,5 and the most likely candidates for infection are hosts that have underlying co-morbidities or immune paresis. The previous literature has focused on the clinical presentation of the cases, but has not included a detailed bacteriological characterization and description of the isolates. An M. parascrofulaceum strain was isolated from a pneumonia patient presenting in 2008, but although the genus was known, the species was only recently identified by our team. This case might be the first pneumonia case with clinical M. parascrofulaceum
http://dx.doi.org/10.1016/j.ijid.2014.01.023 1201-9712/ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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infection reported from China. In this study, we aimed to determine the bacteriological characteristics of the M. parascrofulaceum strain.
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2. Methods
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2.1. Patient and strain information
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The strain was acquired from the sputum specimen of a 47-year-old male patient with pneumonia in an epidemiological study in China in 2008. Written informed consent for participation was obtained from all the patients in the epidemiological study. A total of 4069 clinical isolates were enrolled in the survey, and among them, 140 were defined as NTM stains; only one M. parascrofulaceum was eventually identified. The patient lived in Fujian Province (located in the south-east of China, close to the ocean) and worked at a government institution. He had a cough and expectoration for 3 weeks before consulting a doctor. The patient had no fever, no chest pain, and no sweating. His X-ray showed signs of pneumonia, and acid-fast staining of all three consecutive sputum samples obtained showed acid-fast bacilli (AFB). The patient had been diagnosed with pulmonary tuberculosis 2 years previously and had been cured following 6 months of first-line anti-tuberculosis drug regimen treatment. Whether the patient had complications like diabetes or other immune suppression diseases was not known. Unfortunately, we could not trace the patient from whom the M. parascrofulaceum strain was isolated. As the species identification was performed long after sputum sample collection, patient follow-up was not successful. A Capilia Test (TAUNS, Numazu, Japan) was performed to rule out co-infection with M. tuberculosis (data not shown), and given that M. parascrofulaceum is not among the list of the usual species for contamination when conducting culture, and three consecutive sputum samples from our patient were all AFB-positive (from which the M. parascrofulaceum strain was cultured), we suggest that the M. parascrofulaceum may have been the causative pathogen of pneumonia in our patient with AFB smear-positive sputa.
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2.2. Species identification by homologous gene sequence comparison
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The 16S rRNA,12,13 16–23S rRNA gene internal transcribed spacer (ITS),14,15 and hsp65 sequences16 were used for species identification, with tests performed as described previously. The PCR products were sent to a commercial laboratory for sequencing and the sequences were analyzed using BLAST at http:// www.ncbi.nlm.nih.gov.
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2.3. Phenotypic properties
80 A series of standard biochemical tests for Mycobacterium 81 Q2 characterization were performed, as described by Kent,17 including 82 a growth test, nitrate reductase assay, arylsulfatase assay, tellurite 83 reductase assay, Tween hydrolysis assay, urease assay, and 84 media growth test with 50 mg/ml para-aminobenzoic acid 85 (PNB), 10 mg/ml thiophene carboxylic acid (TCH), 5% NaCl, or 86 MacConkey agar. Quality control was performed using reference 87 strains Mycobacterium scrofulaceum ATCC 19981, Mycobacterium 88 simiae ATCC 25275, Mycobacterium intracellulare ATCC 13950, and 89 Mycobacterium avium ATCC 25291.
by microplate Alamar blue assay.18,19 The antimicrobial reagents employed were all purchased from Sigma Aldrich (St. Louis, MO, USA): rifampin, ethambutol, isoniazid, amikacin, sulfamethoxazole, ofloxacin, para-aminosalicylic acid, rifapentine, capreomycin, cefoxitin, imipenem, minocycline, doxycycline, clarithromycin, azithromycin, meropenem, trimethoprim, rifabutin, linezolid, streptomycin, ethionamide, moxifloxacin, and levofloxacin. The reference strain Mycobacterium tuberculosis H37Rv ATCC 27294 was used as control.
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2.5. Cell wall mycolic acid composition analysis
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The composition and structure analysis of the cell wall mycolic acids was performed by high-performance liquid chromatography (HPLC) in accordance with the instructions of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.,). Briefly, Q3 the mycolic acids were cleaved from the cells by saponification before being extracted with chloroform, and were then derivatized to fluorescence-absorbing bromophenacyl esters and applied to the HPLC (Agilent 1200). The reference strains M. intracellulare ATCC13950, M. avium ATCC 25291, M. scrofulaceum ATCC 19981, and M. simiae ATCC 25273 were used as controls.
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2.6. Pathogenicity analysis of the strain in mice
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Infection with M. parascrofulaceum was evaluated among 6- to 8-week-old female C57BL/6 mice. The mice were kept in sterile isolators in the biohazard animal unit of a biosafety level 3 (BSL-3) laboratory. A total of 72 mice were divided evenly into six groups. The mice were challenged with 0.1 ml of M. parascrofulaceum suspension containing approximately 1 107 bacilli. The M. tuberculosis laboratory strain H37Rv was used as a control strain, while phosphate-buffered saline (PBS) was used as negative control. Groups 1, 3, and 5 were inoculated via the tail vein with M. parascrofulaceum, H37Rv, and PBS, respectively. Groups 2, 4, and 6 were injected intraperitoneally with M. parascrofulaceum, H37Rv, and PBS, respectively. Bacterial loads in the lungs and spleens of the infected mice, as well as the control mice, were evaluated at weeks 2, 4, and 8 postchallenge. At every time point, four mice were sacrificed and the left lungs and half of the spleens were homogenized in 1 ml PBS containing 0.04% Tween 20. Ten-fold serial dilutions of homogenates were plated in duplicate onto Middlebrook 7H11 agar Q4 plates supplemented with 10% OADC and incubated at 37 8C for 1 Q5 month. The colony counts were expressed in log10. The right lung and half of the spleen from each mouse were fixed with 10% formalin in PBS and then subjected to standard processing and paraffin embedding. Multiple 4-mm-thick tissue sections were stained with hematoxylin and eosin (H&E) for the histological/ morphometric analysis. The histological characteristics of exudative and proliferative reactions were classified based on the criteria described previously.20
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2.7. Statistical analysis
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The Student’s t-test was used to assess statistical significance between groups of mice; p < 0.05 was considered statistically significant.
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3. Results
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2.4. Drug susceptibility testing (DST)
3.1. Species identification
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Susceptibility testing was performed using the serial two-fold broth microdilution method, and the minimum inhibitory concentration (MIC) of 23 drugs for the strain was determined
Analysis of the 16S rRNA, ITS, and hsp65 sequences of the isolate using BLAST against sequences from the GenBank database gave 99.64%, 99.06%, and 99.83% similarity, respectively, with that of
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Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Table 1 Phenotypic characteristics of the Mycobacterium parascrofulaceum clinical strain in comparison with other slowly growing Mycobacterium speciesa Characteristic Growth at 25 8C 37 8C Visible growth in 7 days Morphology Pigments produced Nitrate reductase Arylsulfatase 3 days 14 days Tellurite reductase Tween hydrolysis 32 >40 32 >128 32 >32 >16 >16 8 >160 >64
0.5 2 1 64 128 0.5 1 2 0.1 1 1 2 2 5 2.5 5 8 2 8 1 1 19 1
MIC, minimum inhibitory concentration; MABA, microplate Alamar blue assay; WHO, World Health Organization; TB, tuberculosis; CLSI, Clinical and Laboratory Standards Institute. a The WHO recommended critical concentrations of these antimicrobial drugs for TB. b The CLSI recommended critical concentrations for rapidly growing mycobacteria. c The critical concentrations for TB proposed by Brown et al.,21 Watt et al.22, and Hugonnet et al.23
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Figure 1. Mycolic acid patterns of Mycobacterium parascrofulaceum compared with those of MAIS complex (M. avium, M. intracellulare, and M. scrofulaceum) obtained by HPLC analysis using Sherlock software. The blue line represents the pattern of M. parascrofulaceum, while the red one represents the pattern of MAIS.
3.6. Lung and spleen histopathology in C57BL/6 mice
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10 0
A
M. par ascr of ul aceum H37Rv
2weeks
4weeks
M. parascrofulaceum has shown similarities to several known NTM species using single identification methods, but it is clearly a unique species as determined by a polyphasic approach. Tortoli et al. reported that about 10% of M. scrofulaceum isolates were
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B
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C
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4. Discussion
10 6 CFU/half of lung
10 6 CFU/half of spleen
10 6 CFU/half of spleen
After fixation, the tissues of the mice were sectioned and stained with H&E and microscopic examination was performed. With regard to histopathological examination, infection by Q6 M. parascrofulaceum and H37Rv were indistinguishable. Two weeks post-challenge, the lung tissues demonstrated a local inflammatory response denoted by peri-bronchiolar inflammatory infiltrates. At weeks 4 and 8 post-infection, lung tissues showed
peri-bronchiolar inflammatory infiltrates with large aggregates of lymphocytes and part of the tissue showed granulomatous lesions; no appreciable necrosis was apparent (Figure 3). The histopathological features of tissue from animals infected with either M. parascrofulaceum or H37RV were indistinguishable. In spite of the severe pathological changes observed in the lungs of the infected mice, all the animals maintained viability for up to 8 weeks of observation.
10 6 CFU/half of lung
with M. parascrofulaceum showed CFUs similar to those of mice infected with H37Rv at 2 and 4 weeks post-infection, but at 8 weeks post-infection the bacterial burden was lower in the M. parascrofulaceum-infected mice (p < 0.05) (Figure 2). Results were similar in mice infected either via the tail vein or intraperitoneally.
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2weeks
4weeks
8weeks
D
6 4 2 0 2weeks
4weeks
8weeks 7
Figure 2. Growth curve of tested bacilli in the lungs and spleens of C57BL/6 mice. The C57BL/6 mice were inoculated with 1 10 bacilli either through intraperitoneal (A, B) or tail vein (C, D) route. Data are presented as the mean and standard deviation at each time-point. *Statistically significant (p < 0.05) when compared with mice infected with H37Rv strain.
Please cite this article in press as: Liu G, et al. Bacteriological characterization of a Mycobacterium parascrofulaceum strain isolated from a Chinese pneumonia patient. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2014.01.023
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Figure 3. Histological pictures of the lungs in C57BL/6 mice by H&E staining at week 4 post-infection. (A) Negative control mice. (B) M. parascrofulaceum-infected mice, through tail vein route. (C) H37Rv-infected mice, through tail vein route. (D) M. parascrofulaceum-infected mice, through intraperitoneal route. (E) H37Rv-infected mice, through intraperitoneal route. Original magnification: 40.
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actually M. parascrofulaceum strains.7 In our assay, the M. parascrofulaceum strain was identified as Mycobacterium-MAIS complex by the mycolic acid analysis method. Therefore, we suggest the only reliable way to identify M. parascrofulaceum is by sequence comparison of highly conserved genes. Genetically, M. parascrofulaceum is most related to M. simiae, whereas it presents a similar phenotypic profile to M. scrofulaceum.5 In our assays, the M. parascrofulaceum strain had almost the same phenotypic profile as the M. scrofulaceum type strain when considering all the biochemical tests, except for the tellurite reductase assay and the 14-day arylsulfatase assay. Although its phenotypic profile was also similar to those of M. intracellulare and M. avium, the urease, tellurite reductase, and arylsulfatase assays were helpful in differentiating them. The prognosis of M. parascrofulaceum infection remains unclear. Although anti-mycobacterial drugs have been used historically in the few clinical cases reported, most of the patients died before a reasonable evaluation of the treatment could be made. In three known M. parascrofulaceum infection cases in which the patient survived, one case improved by administering clarithromycin, ethambutol, and rifampin,5 the second did not show recurrence after a surgical debridement of the pathological lesions in the lung and lymph node without any chemotherapy,8 and the third slowly improved by administering clarithromycin, amikacin, and ethambutol.9 Turenne et al. showed that M. parascrofulaceum isolates were sensitive to rifampin, clarithromycin, amikacin, and sulfamethoxazole in vitro,5 and Teruya et al. suggested that rifampin, clarithromycin, and levofloxacin could be effective for M. parascrofulaceum infection,8 while Shojaei et al. showed that the organism was susceptible to amikacin, clarithromycin, rifampin, doxycycline, sulfamethoxazole, streptomycin, and imipenem in vitro.10 In this study, we tested the sensitivity of the organism to 23 agents including the commonly used anti-tuberculosis drugs and some general antibiotics that are frequently used in the treatment of NTM disease. The results showed that this isolate was susceptible to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin in vitro. The other M. parascrofulaceum strain isolated from a cutaneous infection in China was reported to be sensitive to clarithromycin and moxifloxacin, which is consistent with our findings. In our test, the MIC of the strain to rifampin was 2 mg/ml, and we define the strain as resistant according to the critical concentration recommended by the WHO for M. tuberculosis. However, as the MIC is so close to the critical concentration, administering rifampin might also be effective in clinical treatment. The levofloxacin MIC was similarly close to the critical concentration. The discrepancy in DST between our isolate and those reported in the literature may be due to strain differences. According to the previous reports and our results, rifamycin agents, clarithromycin, azithromycin, moxifloxacin, and levofloxacin may be the key drugs for the treatment of M. parascrofulaceum infection. Owing to its relatively recent recognition as a novel species, not much is known about the pathogenicity of M. parascrofulaceum. Our mouse infection model results at least suggest that
M. parascrofulaceum is capable of proliferating in mice and could lead to pathological changes in the lungs but not in the spleens of the mice. In conclusion, an M. parascrofulaceum strain isolated from a Chinese pneumonia patient was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, cefoxitin, and moxifloxacin, was capable of proliferating in mice, and could lead to pathological changes in the lungs of the mice.
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Acknowledgements
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This work was supported by research funding from the Infectious Diseases Special Project, Ministry of Health of China (2012ZX10003002-009) and Beijing Natural Science Foundation grants (7132049). All the mycobacterial strains used in this project were acquired from the ‘Beijing bio-bank of clinical resources on tuberculosis’. Ethical approval: This study was approved by the ethics committee of Beijing Chest Hospital. The protocol of the mouse model experiment was approved by the Animal Ethics Committee of Beijing Chest Hospital. Conflict of interest: The authors declare no conflicts of interest, real or perceived, financial or non-financial.
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References
305
1. Rosenzweig DY. Nontuberculous mycobacterial disease in the immunocompetent adult. Semin Respir Infect 1996;11:252–61. 2. Tortoli E. The new mycobacteria: an update. FEMS Immunol Med Microbiol 2006;48:159–78. 3. Cook JL. Nontuberculous mycobacteria: opportunistic environmental pathogens for predisposed hosts. Br Med Bull 2010;96:45–59. 4. 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. 5. Turenne CY, Cook VJ, Burdz TV, Pauls RJ, Thibert L, Wolfe JN, et al. Mycobacterium parascrofulaceum sp. nov., novel slowly growing, scotochromogenic clinical isolates related to Mycobacterium simiae. Int J Syst Evol Microbiol 2004;54: 1543–51. 6. Santos R, Fernandes J, Fernandes N, Oliveira F, Cadete M. Mycobacterium parascrofulaceum in acidic hot springs in Yellowstone National Park. Appl Environ Microbiol 2007;73:5071–3. 7. Tortoli E, Chianura L, Fabbro L, Mariottini A, Martin-Casabona N, Mazzarelli G, et al. Infections due to the newly described species Mycobacterium parascrofulaceum. J Clin Microbiol 2005;43:4286–7. 8. Teruya H, Tateyama M, Hibiya K, Tamaki Y, Haranaga S, Nakamura H, et al. Pulmonary Mycobacterium parascrofulaceum infection as an immune reconstitution inflammatory syndrome in an AIDS patient. Intern Med 2010;49: 1817–21. 9. Hibiya K, Tateyama M, Teruya H, Nakamura H, Tasato D, Kazumi Y, et al. Immunopathological characteristics of immune reconstitution inflammatory syndrome caused by Mycobacterium parascrofulaceum infection in a patient with AIDS. Pathol Res Pract 2011;207:262–70. 10. Shojaei H, Hashemi A, Heidarieh P, Daei-Naser A. Chronic pelvic pain due to Mycobacterium parascrofulaceum in an Iranian patient: first report of isolation and molecular characterization from Asia. Braz J Infect Dis 2011;15:186–7. 11. Zong W, Zhang X, Wang H, Xu XL, Wang Q, Tian W, et al. The first case of cutaneous infection with Mycobacterium parascrofulaceum. Ther Clin Risk Manag 2012;8:353–8. 12. Rogall T, Wolters J, Flohr T, Bottger EC. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol 1990;40:323–30.
306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341
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G. Liu et al. / International Journal of Infectious Diseases xxx (2014) e1–e6 Turenne CY, Tschetter L, Wolfe J, Kabani A. Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 2001;39:3637–48. Turenne C, Chedore P, Wolfe J, Jamieson F, Broukhanski G, May K, et al. Mycobacterium lacus sp. nov., a novel slowly growing, non-chromogenic clinical isolate. Int J Syst Evol Microbiol 2002;52:2135–40. Harmsen D, Rothganger J, Frosch M, Albert J. RIDOM: Ribosomal Differentiation of Medical Micro-organisms Database. Nucleic Acids Res 2002;30:416–7. Kim BJ, Lee KH, Park BN, Kim SJ, Bai GH, Kook YH. Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65). Int J Syst Evol Microbiol 2005;55:1649–56. Kent PT, G.P. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: Department of Health and Human Services, Centers for Disease Control; 1985. Brown-Elliott BA, Crist CJ, Mann LB, Wilson RW, Wallace RJ. In vitro activity of linezolid against slowly growing nontuberculous mycobacteria. Antimicrob Agents Chemother 2003;47:1736–8.
19. National Committee for Clinical Laboratory Standards. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes. Approved Standard M24-A. Wayne, PA: NCCLS; 2003. 20. Smith I. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin Microbiol Rev 2003;16:463–6. 21. Brown BA, Wallace Jr RJ, Onyi G. Activities of the glycylcyclines N,N-dimethylglycylamido-minocycline and N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline against Nocardia spp. and tetracycline-resistant isolates of rapidly growing mycobacteria. Antimicrob Agents Chemother 1996;40:874–8. 22. Watt B, Rayner A, Harris G. Comparative activity of azithromycin against clinical isolates of mycobacteria. J Antimicrob Chemother 1996;38:539–42. 23. Hugonnet JE, Tremblay LW, Boshoff HI, Barry 3rd CE, Blanchard JS. Meropenem–clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science 2009;323:1215–8.
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