Probable disseminated Mycobacterium abscessus subspecies bolletii infection in a patient with idiopathic CD4+ T lymphocytopenia: a case report

ABSTRACT: INTRODUCTION: Rapidly growing mycobacteria are opportunistic pathogens in patients with underlying riskfactors. Mycobacterium abscessus subsp. bolletii is a newly recognized member of rapidlygrowing mycobacteria, isolated from respiratory tract and cutaneous infections. CASE PRESENTATION: We describe a case of chronic disseminated infection caused by M. abscessus subsp. bolletiiin a 38-year-old Sri Lankan man with idiopathic CD4+ T lymphocytopenia. Idiopathic CD4+T lymphocytopenia is a rare cause of immunodysfunction that, similar to humanimmunodeficiency virus infection. M. abscessus subsp. bolletii infection was diagnosed byculture isolation from two sputum samples. CONCLUSIONS: To the best of our knowledge this is the first report of M. abscessus subsp. bolletiidisseminated infection in a patient affected by idiopathic CD4+ T lymphocytopenia. Incontrast to previous reports, the isolate of M. abscessus subsp. bolletii presented intermediateresistance to clarithromycin and was susceptible to cefoxitin and imipenem.     

Annotated Genome Sequence of Mycobacterium massiliense Strain M154, Belonging to the Recently Created Taxon Mycobacterium abscessus subsp. bolletii comb. nov

Mycobacterium massiliense has recently been proposed as a member of Mycobacterium abscessus subsp. bolletii comb. nov. Strain M154, a clinical isolate from the bronchoalveolar lavage fluid of a Malaysian patient presenting with lower respiratory tract infection, was subjected to shotgun DNA sequencing with the Illumina sequencing technology to obtain whole-genome sequence data for comparison with other genetically related strains within the M. abscessus species complex.     

Draft Genome Sequence of Mycobacterium bolletii Strain M24, a Rapidly Growing Mycobacterium of Contentious Taxonomic Status

The whole-genome sequence of Mycobacterium bolletii M24, isolated from the bronchoalveolar lavage fluid of a Malaysian patient, is reported here. The circular chromosome of 5,507,730 bp helped to clarify the taxonomic position of this organism within the M. abscessus complex and revealed the presence of proteins potentially important for pathogenicity in a human host.     

Genomic Analysis of Mycobacterium massiliense Strain M115, an Isolate from Human Sputum

We report the draft genome sequence of a clinical isolate, strain M115, identified as Mycobacterium massiliense, a member of the newly created taxon of Mycobacterium abscessus subspecies bolletii comb. nov.     

Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov.

We studied the taxonomic positions of the rapidly growing organism Mycobacterium fortuitum and phenotypically related organisms. We confirmed that "Mycobacterium peregrinum" ATCC 14467T (T = type strain) is genetically independent of M. fortuitum ATCC 6841T by using various DNA hybridization conditions. Strains that were genetically identified as "M. peregrinum" were phenotypically differentiated from M. fortuitum ATCC 6841T. Thus, we propose that "M. peregrinum" should be revived as an independent species, Mycobacterium peregrinum sp. nov., nom. rev. The type strain is strain ATCC 14467. M. fortuitum subsp. acetamidolyticum ATCC 35931T exhibited a high level of DNA relatedness to M. fortuitum ATCC 6841T. The hybridized DNAs maintained stable heteroduplexity at high stringency; thus, we confirmed that M. fortuitum subsp. acetamidolyticum is identical to M. fortuitum ATCC 6841T. We found that M. chelonae subsp. abscessus ATCC 19977T is genetically different from M. chelonae subsp. chelonae NCTC 946T on the basis of the results of quantitative hybridization even under optimal conditions. There was no reason to maintain this organism as a subspecies of M. chelonae. Thus, we propose that M. chelonae subsp. abscessus should be elevated to species status as Mycobacterium abscessus (Kubica et al.) comb. nov. The type strain is strain ATCC 19977.     

Genome sequence of the Mycobacterium abscessus strain M93

Mycobacterium abscessus is a rapid-growing species of nontuberculous mycobacteria that is frequently associated with opportunistic infections in humans. We report herein the draft genome sequence of M. abscessus strain M93.     

Numerical classification of rapidly growing nonphotochromogenic mycobacteria

Numerical analysis of 211 strains of rapidly growing, nonphotochromogenic mycobacteria was carried out by using 116 characters. New species reported after 1981 and not yet compared with known species by numerical classification were included. At the level of 90% of the matching coefficient, the following species could be differentiated from each other and were shown as distinct clusters: Mycobacterium pulveris, M. moriokaense, M. chitae, M. diernhoferi, M. porcinum, M. fortuitum, M. chelonae subspecies chelonae, M. chelonae subspecies abscessus, M. smegmatis, M. agri, M. fallax, M. parafortuitum, M. fortuitum subspecies acetamidolyticum. Four taxa, M. fortuitum, M. porcinum, M. chelonae subsp. chelonae, and M. chelonae subsp. abscessus, were regarded as distinct. However, these were combined into one large cluster at a level of 90% of the matching coefficient and, therefore, were regarded as forming one series or complex. M. fortuitum subsp. acetamidolyticum was reported as a subspecies of M. fortuitum, but this was differentiated clearly from the other species including M. fortuitum.     

脓肿分枝杆菌经Toll样受体2介导的JNK/ERK信号通路诱导巨噬细胞表达肿瘤坏死因子α和白细胞介素8

为探讨脓肿分枝杆菌脓肿亚种和马赛亚种经Toll样受体2(Toll-like receptor 2,TLR2)介导的c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)诱导THP-1巨噬细胞内肿瘤坏死因子α(tumor necrosis factor α,TNF-α)和白细胞介素8(interleukin 8,IL-8)表达的相关分子机制,本研究将脓肿分枝杆菌脓肿亚种和马赛亚种感染THP-1巨噬细胞,细菌与巨噬细胞最佳感染之比为感染复数(multiplicity of infection,MOI)＝3,用荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测THP-1巨噬细胞感染两细菌亚种6 h后的胞内TNF-α和IL-8 mRNA水平,以及分别阻断TLR2、JNK 和ERK信号蛋白后TNF-α和IL-8 mRNA水平的变化。结果显示,脓肿分枝杆菌脓肿亚种和马赛亚种作用于THP-1巨噬细胞6 h后,均可诱导细胞内TNF-α和IL-8 mRNA水平显著上调,差异有统计学意义(P＜0.05);分别阻断TLR2、JNK和ERK信号蛋白,脓肿亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平出现明显抑制,差异有统计学意义(P＜0.05);分别阻断TLR2和JNK信号蛋白,马赛亚种感染THP-1巨噬细胞后胞内TNF-α和IL-8 mRNA上调水平均出现明显抑制,差异有统计学意义(P＜0.05);而阻断ERK信号蛋白后,马赛亚种组仅见IL-8 mRNA水平明显抑制,差异有统计学意义(P＜0.05),而TNF-α mRNA水平未见明显变化,差异无统计学意义(P＞0.05)。本研究提示,脓肿分枝杆菌脓肿亚种和马赛亚种均可作用于TLR2,诱导THP-1细胞内TNF-α和IL-8 mRNA水平上调,脓肿亚种可经JNK和ERK信号蛋白诱导TNF-α mRNA上调,马赛亚种可经JNK信号蛋白诱导TNF-α mRNA上调;脓肿亚种和马赛亚种诱导IL-8 mRNA上调可能与JNK和ERK信号蛋白相关。     

Comparison of the Pathogenicity for Mice of Mycobacterium fortuitum and Mycobacterium abscessus

The pathogenicity for mice of 12 strains of Mycobacterium abscessus was compared with that for 8 strains of M. fortuitum. Both species caused lesions in kidneys and produced "spinning disease" resulting from inner ear infections. No major differences in pathogenicity of these two species were demonstrated. Strain to strain variation was marked, especially with M. abscessus. For example, 1.6 x 10(6) organisms of strain 11188 of M. abscessus produced death in four of five animals within 42 days, whereas strain 380 of M. abscessus failed to produce any deaths within 42 days. In the case of M. fortuitum, the greatest mortality observed was one of five animals, yet the incidence of spinning disease and kidney disease occurred earlier postinfection than in mice infected with M. abscessus. Histologically, abscess formation by a strain of M. abscessus was greater than by a strain of M. fortuitum, but this difference cannot be interpreted as a species difference.     

Whole-genome sequence of the emerging pathogen Mycobacterium abscessus strain 47J26

Mycobacterium abscessus is a rapidly growing environmental mycobacterium commonly found in soil and water which is often also associated with infections in humans, particularly of the lung. We report herein the draft genome sequence of M. abscessus strain 47J26.     

Evaluation of antimicrobial susceptibilities of rapidly growing mycobacteria by Sensititre RAPMYCO panel

This study used Sensititre RAPMYCO to test the activities of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, sulfamehoxazole, tigecycline and tobramycin against 25 clinical isolates of rapidly growing mycobacteria (RGM), including the common disease producing species Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium peregrinum. Analysis of the four different RGM species showed that isolates of M. fortuitum and M. peregrinum were more susceptible than M. abscessus and M. chelonae. Different antimicrobials showed a variable sensitivity in all strains. Therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before the treatment of infections due to RGM.     

Nontuberculous mycobacteria isolated from pulmonary specimens between 2004 and 2009: causative agent or not?

Nontuberculous mycobacteria were identified from 45891 samples of 19553 patients with a prediagnosis of pulmonary tuberculosis between November 2004 and January 2009. Among 10041 (21.9%) culture positive samples, 208 (2.1%) pulmonary samples recovered from 77 individual patients were differentiated as mycobacteria other than tuberculosis (MOTT). Proportion of mycobacteria evaluated as causative agent for clinical infection were found as 0.16% (n = 31), mostly M. avium complex, M. abscessus and M. kansasii. Additionally, M. fortuitum-peregrinum complex, M. simiae, M. szulgai / intermedium and M. scrofulaceum were found as causative agent in 2, 2, 2 and 1 patient, respectively. Identification of infections caused by environmental or opportunistic pathogen mycobacteria is required in rapid and accurate diagnosis, infection control and treatment planning of infections caused by M. tuberculosis complex and/or MOTT.     

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HβD2 gene expression and IL-8 release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.     

Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Drosophila melanogaster model for Mycobacterium abscessus infection

Mycobacterium abscessus is a human pathogen that is responsible for a broad spectrum of tissue infections and disseminated infections in immunodeficient patients. This pathogen is one of the most resistant organisms to chemotherapeutic agents. Therefore, we tested the hypothesis that the fruit fly, Drosophila melanogaster, is a genetically tractable model host for M. abscessus. In this context, we infected D. melanogaster with M. abscessus. This M. abscessus infection results in dissemination in the fly body, followed by death, which is accompanied by severe indirect flight muscle and brain damage. Our data show that M. abscessus can grow and replicate in D. melanogaster w¹¹¹⁸ and that it elicited a humoral immune response, especially of the Toll antimicrobial peptide pathway. To the best of our knowledge, this is the first report that mycobacteria induce the production of antimicrobial peptides in D. melanogaster.     

脓肿分支枝杆菌embB基因耐乙胺丁醇的分析

目的 分析脓肿分支枝杆菌的embB基因,以探讨其耐乙胺丁醇的分子机制.方法 用16S rRNA基因序列分析法鉴定5株脓肿分枝杆菌临床株,测定乙胺丁醇对临床株及标准株( ATCC 19977)的最低抑菌浓度(MIC).PCR扩增embB基因的全序列,将所测序列进行生物信息学分析.结果 乙胺丁醇对5株脓肿分支杆菌标准株和临床株的MIC均为128μg/mL.,属高度耐药.从脓肿分支枝杆菌的标准株和临床株均扩增出约3200 bp片段,与GenBank中脓肿分支枝杆菌标准株的embB基因大小一致.5株临床株与标准株比较,其核苷酸序列存在9个点突变,在突变位点所编码的氨基酸序列中,仅第18位、87位、770位密码子编码与标准株不同的氨基酸.6株脓肿分支枝杆菌的embB基因与对EMB敏感的结核分支杆菌标准株(H37RV)的相应基因序列比较,第303-305位密码子的核苷酸序列存在差异,但仅第303、304位核苷酸编码的氨基酸序列不同,第306位密码子的核苷酸序列无差异.结论 脓肿分支杆菌对乙胺丁醇的耐药并非embB基因的突变所致,为embB基因天然存在结构的不同,属于天然耐药.结构的差异与第306位密码子无关,可能与第303、304密码子有关.     

A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.     

Mycobactins as chemotaxonomic characters for some rapidly growing mycobacteria

Thirty-nine strains of rapidly growing mycobacteria were examined for the production of mycobactins (lipid-soluble, iron-binding compounds) when grown under conditions of iron-limitation on solidified medium. Different growth conditions had little effect on the structure of individual mycobactins, indicating them to be strongly conserved molecules showing intra-species consistency and thus suitable for use as chemotaxonomic characters of high discriminatory power. Strains of Mycobacterium aurum, M. chitae, M. chelonae subsp. abscessus, 'M. diernhoferi', M. duvalii, M. flavescens, M. fortuitum, M. gadium, 'M. gallinarum', M. neoaurum, M. parafortuitum, 'M. peregrinum', M. phlei, M. smegmatis, M. thermoresistible and M. vaccae formed mycobactins which were readily isolated and characterized by a combination of thin-layer and high-performance liquid chromatography. All strains of M. komossense and 'M. kanazawa' failed to produce a mycobactin; some strains of M. aurum, M. chelonae, M. parafortuitum, M. thermoresistible and M. vaccae were similarly negative. Mycobacteria of the M. fortuitum complex (M. fortuitum, M. chelonae and 'M. peregrinum') formed distinctive mycobactins, as did those in the M. parafortuitum complex (M. aurum, M. neoaurum, 'M. diernhoferi', M. vaccae and M. parafortuitum). The mycobactin from 'M. gallinarum' was different from those of the related species M. flavescens, for which four distinct mycobactin patterns were recorded. For routine examination of mycobactins in a diagnostic laboratory with limited resources, thin-layer chromatography used alone offers a simple but adequate means of characterization and final identification of the producing mycobacterium. High-performance liquid chromatography is only needed in those few instances where a high degree of discrimination is required.