Microbiologically identified isolates of Mycobacterium heckeshornense in two patients

PURPOSE: To identify mycobacteria isolated from sputa of a 51-year-old female and a 72-year-old male patient with pneumoconiosis. OBJECT AND METHOD: Mycobacteria species were isolated from sputa of a 51-year-old female. The culture was always negative in spite of positive smears before the final isolation in 1988. A 72-year-old male patient suffered from pneumoconiosis and the acid-fast bacillus was isolated by routine sputum examination in 2003. These two strains of acid-fast bacilli were identified as Mycobacterium heckeshornense by partial sequencing of 16S rRNA and rpoB and conventional methods (biochemical and routine culture methods). RESULT: These two strains grew on 1% Ogawa's slant medium at 37 degrees C and 42 degrees C, but not at 28 degrees C. They formed yellowish colonies in the dark (Scotochromogen). They were classified as a slowly growing Mycobacteria. As it was difficult to distinguish M. heckeshornense from M. xenopi by conventional methods including growth rate, temperature range of mycobacterial growth, light coloration reaction, biochemical and biological tests, virulence using guinea pigs and drug susceptibility test were further explored. Finally two were identified as M. heckeshornense by summing of these results. CONCLUSION: Mycobacteria species that grow at 42 degrees C for four weeks, imply M. xenopi with a DDH method. It is essential to perform both sequencing of 16S rRNA and rpoB gene and a biochemical method for the purpose of distinguishing M. heckeshornense from M. xenopi.     

Molecular-genetic evidence for the relationship of Mycobacterium leprae to slow-growing pathogenic mycobacteria

A total of 1170 nucleotides of the 16S rRNA from Mycobacterium leprae were compared to the homologous regions of M. tuberculosis, M. bovis Vallée, M. avium, M. scrofulaceum, M. phlei, M. fortuitum and one representative each of the genera Corynebacterium, Nocardia, and Rhodococcus. Homology values were calculated and a phylogenetic tree was constructed from the evolutionary distance values. Despite differences in DNA G + C content and genome size, M. leprae is a true member of the slow-growing pathogenic mycobacteria, branching off intermediate to the other members of this subgroup. Slow- and fast-growing mycobacteria are phylogenetically well separated but constitute an individual branch of the actinomycetes proper. Significant structural variation of certain regions of the 16S rRNA may allow construction of M. leprae-specific probes used for rapid identification.     

Mycobacterium heckeshornense lung infection that was diagnosed as Mycobacterium xenopi disease by DNA-DNA hybridization (DDH)

The DNA sequencing analyses of the 16S rRNA gene, rpoB and hsp65 were conducted to characterize six strains that had been identified as Mycobacterium xenopi by DNA-DNA hybridization (DDH) for past ten years in our hospital. The results revealed Mycobacterium heckeshornense infection in one of the six cases. A 47-year-old man, who had been treated for pneumonia, had pulmonary nontuberculous mycobacterial disease. The sputa from the patient were culture positive for mycobacterium in three times. And it was diagnosed as M. xenopiby DDH method. Chest X-ray showed fibrocavitary lesion in right upper lobe was successfully treated with clarithromycin for four weeks.     

RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌的研究

目的建立RNA恒温扩增实时检测技术快速鉴定偶发分枝杆菌(RIARD-MF)的方法,评估其在鉴定分枝杆菌临床分离株中的应用效果。方法将偶发分枝杆菌的16SrRNA特异序列作为目的靶标进行检测,设计含T7启动子逆转录扩增引物和RNA探针,分别对5种非分枝杆菌细菌、20种分枝杆菌标准株和259株临床分枝杆菌分离株进行42℃恒温扩增实时检测,参考标准为PCR基因测序结果。结果RIARD-MF方法学检测灵敏度可达60CFU/mL。在25种细菌的RIARD-MF特异性检测中:只有偶发分枝杆菌检测结果为阳性,其余24种细菌均为阴性,与测序检测结果一致。在检测分枝杆菌临床分离菌株时,RIARD-MF鉴定偶发分枝杆菌5株,其余为非偶发分枝杆菌,与测序结果一致,检测灵敏度和特异度均为100%。结论 RIARD-MF鉴定偶发分枝杆菌具有较高的特异度、灵敏度和准确性,且检测快速,有望成为一种新的偶发分枝杆菌临床分离菌株鉴定方法。     

Comparison between relative analogy and homology of 16S rRNA partial sequences between Mycobacterium szulgai and Mycobacterium malmoense

A lot of nucleotide sequences of some genes, especially 16S rRNA gene, are registered in the public data-base. In order to identify clinical mycobacterial strains, 16S rRNA gene partial nucleotide sequences from 15 strains of Mycobacterium malmoense and 24 strains of Mycobacterium szulgai which are stored in the Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association were determined. Then homology of the sequences to the data of type strains submitted to public database were determined. Relative analogy to type strains by delta DDH was also measured. In the area of nucleotide position 51-588 corresponding accession number X52930 which is Mycobacterium malmoense type strain 16S rRNA gene sequence data, the homology of some partial sequences with Mycobacterium malmoense strains to data of accession number X52930 were lower than that with Mycobacterium szulgai type strain data, accession number X52926. In the area of nucleotide position 31-568 or nucleotide position 31-588, the homology of all nucleotide sequence data to collect species data of type strains were higher than the homology to another species data. Nucleotide position 38, 40, 47 and 49 might be differential nucleotides conserved between Mycobacterium malmoense strains and Mycobacterium szulgai one. These results suggest that homology of about 500 bp's 16S rRNA gene nucleotide sequence data may not be enough for differential identification. Nevertheless, database of RIDOM, Ribosomal differentiation of Medical Microorganisms, identified partial nucleotide sequences between nucleotide position 51-588 corresponding accession number X52930 correctly, though some were lower than 97% homology (data not shown). Therefore quality-controlled 16S rRNA gene nucleotide sequence database could be used for differential identification.     

Identification of 23 mycobacterial species by Invader assay with targeting 16S rRNA gene and ITS-1 region--comparison with DDH method in clinical isolates

PURPOSE AND METHOD: The Invader assay was developed to identify 23 mycobacterial species using probes derived from the species-specific region of the 16S rRNA gene and the 16S-23S rRNA internal transcribed spacer 1 (ITS-1) region, with minor modifications of our previous study. In the present study, we compared the identification capability between the Invader assay and DNA-DNA hybridization (DDH) method. DDH is commonly used to identify non-tuberculosis mycobacterium in Japan and 636 clinical mycobacterial strains cultured on Ogawa slants were tested. RESULTS: The Invader assay could identify 615 (96.7%) of the 636 strains. The results contained 14 M.lentiflavum, 3 M. parascrofulaceum and 1 M. intermedium, which were undetectable with DDH method. On the other hand, DDH method could identify 580 (91.2%) strains with duplicate assay. Of 628 strains except 8 strains identified as a few species by Invader assay, 551 (87.7%) strains were identified as the same species by two methods. Discordant results were mainly recognized for the identification of M. gordonae, M. avium, M. lentiflavum and M. intracellurare. The results of other methods targeting 16S rRNA indicated correctness of the Invader assay. CONCLUSION: These results indicate that Invader assay could identify more correctly than DDH method and could identify about 97% of clinically important mycobacterium.     

常见分枝杆菌不同株16S-23S rRNA转录间隔区序列分析

目的 分析常见分枝杆菌不同株之间的因型,为临床分离株的鉴定提供参考序列.方法 用16S-23S rRNA转录间隔区(internal transcribed spacer,ITS)序列分析法对94株由德国微生物和细胞保藏中心(DSMZ)引进的常见分枝杆菌进行种内不同株的基因型分析,并分别与12株国际标准株对比.通过种内不同株间同源性序列比对,绘制16S-23S rRNA ITS序列聚类分析树状谱,使用DNAStar的MegAlign软件计算株间相似性百分比.结果 成功完成上述共106株菌株的16S-23SrRNA ITS测序,发现除胞内分枝杆菌(4株)、鸟分枝杆菌(4株)、海分枝杆菌(6株)和马尔摩分枝杆菌(2株)各有一个基因型且与标准株相同外,其他分枝杆菌均可分为数个不同基因型.结论 16S-23S rRNA ITS序列分析可以根据种内各株的不同基因型将分枝杆菌鉴定到株,是分枝杆菌基因型分析的可靠方法.     

Mycobacterium shinshuense isolated from cutaneous ulcer lesion of right lower extremity in a 37-year-old woman

PURPOSE: Second clinical infection case of Mycobacterium shinshuense was presented, we tried the identification of M. shinshuense that is isolated from skin. OBJECT: Mycobacteria species isolated from cutaneous ulcer lesion of right lower extremity in a 37-year-old woman. METHOD: Identification by DNA-DNA Hybridization, 16S rRNA and rpoB method as genomic level and conventional method. RESULT: It did not grow on 1% Ogawa's slant medium at both 37 degrees C and 42 degrees C, but grew at 28 degrees C. It formed yellowish colonies in the dark. It was difficult to distinguish M. shinshuense from M. ulcerans and M. marinum by DNA-DNA hybridization (DDH) and DNA sequencing. To identify that it is M. shinshuense, growth rate, temperature range of mycobacterial growth, light coloration reaction, biochemical and biological tests, and drug susceptibility testing were further explored. Finally it was identified as M. shinshuense based on these CONSIDERATION: For Mycobacteria species which grow 2 weeks after inoculation at 28 degrees C, and which is identified as M. marinum by DDH method, it is necessary to identify with sequence and conventional method.     

Nontuberculous mycobacterial infections in Indian AIDS patients detected by a novel set of ESAT-6 polymerase chain reaction primers

Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities. To overcome this, we created a rapid PCR method for the species-specific diagnosis of Mycobacterium tuberculosis and its differentiation from other mycobacteria. A set of PCR primers targeting the gene encoding for early-secreted antigen-6 (ESAT-6) of the M. tuberculosis complex was designed and standardized on mycobacterial standard strains and on 75 recent isolates from AIDS patients and 70 isolates from HIV-negative patients seen at the hospital of the All India Institute of Medical Sciences, New Delhi, India. All 145 fresh mycobacterial isolates were identified using phenotypic methods and 16S rRNA PCR followed by sequencing of hypervariable region A. The ESAT-6 PCR detected all of the M. tuberculosis strains correctly (100% sensitivity), but none of the nontuberculous Mycobacterium spp. gave positive results (100% specific). Most nontuberculous mycobacteria were identified in patients with AIDS (24%) followed by those with tuberculous lymphadenitis (12.5%) and those with pulmonary tuberculosis whose treatment had failed (4.3%). The most common nontuberculous mycobacterial species isolated from AIDS patients was M. avium (6.6%), followed by M. fortuitum (5.7%), M. intracellulare and M. terrae (2.6% each). M. celatum, M. duvalii, M. austroafricanum, M. phlei and M. flavescence were also isolated from one patient each. The combination of genus-specific PCR primers with the novel ESAT-6 primer set could provide accurate and rapid diagnosis of mycobacteriosis.     

Species identification of neglected nontuberculous mycobacteria in a developing country

In developing countries where tuberculosis is still a health challenge, the prevalence of nontuberculous mycobacterial diseases is expected to rise as medical conditions that compromise the immune system become more widespread. In the current study, we aimed to determine the presence and diversity of nontuberculous mycobacteria (NTM) causing infections in Iranian patients. Sixty-seven clinical NTM isolates were identified using conventional and molecular methods, including PCR-restriction fragment length polymorphism analysis (PRA) and 16S rRNA sequencing. Out of 67 patients with confirmed mycobacterial infection, 29 had an associated immunosuppressive syndrome, including 9 who were HIV-infected. Forty-nine NTM isolates were identified using PRA, and the remaining 18 isolates were identified using 16S rRNA sequencing. We obtained the following results: Mycobacterium fortuitum, 30 isolates; M. kansasii, 12 isolates; M. gordonae, 8 isolates; M. porcinum, 3 isolates; M. conceptionense, 3 isolates; M. phlei, 2 isolates; and M. austroafricanum, M. avium, M. elephantis, M. intracellulare, M. lentiflavum, M. monacense, M. parascrofulaceum, and M. thermoresistibile, 1 isolate each; and 1 potentially novel mycobacterial species. With regard to the complexity of identification, it is recommended that laboratory diagnosis of NTM diseases be centralized by strengthening or setting up quality national and regional infrastructure.     

hsp65 and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定(英文)

目的探讨和评价hsp65and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定。方法收集经PNB/TCH鉴别培养基表型鉴定和16s rRNA基因测序鉴定为龟/脓肿分枝杆菌复合群的临床分离菌株,用hsp65and rpoBPCR-RFLP进行种/亚种鉴定。结果经表型鉴定为非结核分枝杆菌的27株临床菌株,16s rRNA基因测序分析与龟/脓肿分枝杆菌的同源性达到99.7%。经hsp65PCR-RFLP and rpoBPCR-RFLP鉴定18株为脓肿分枝杆菌(M.abscessus),4株为溃疡分枝杆菌(M.absecces),另5株表现为独特的指纹特征,可能是一个新的亚种。结论能够快速进行龟/脓肿分枝杆菌复合群种/亚种的鉴定。     

应用16SrRNA基因序列分析技术鉴定临床非典型细菌的研究

目的将16SrRNA基因序列分析技术应用于传统方法鉴定可靠率低或无法鉴定细菌的分类鉴定，以提高临床诊断的准确性。方法以本院微生物实验室保存的5株标准菌株验证实验方法的准确性后，收集实验事常规方法不能鉴定或鉴定可靠性低的临床分离菌15株（包括革兰阳性球菌，革兰阳性杆菌及革兰阴性杆菌），提取DNA模板，以通用引物PCR扩增16SrRNA目的片段后测序，将测序结果在GenBank数据库中比对分析以确定菌种。结果15株实验细菌中有14株（占93．3％）鉴定到种，包括鼻疽诺卡菌，大肠埃希菌，阿尔莱特葡萄球菌，脆弱拟杆菌，弗氏柠檬酸杆菌，短小芽孢杆菌，河流漫游球菌，极小短小杆菌，伴放线凝聚杆菌，毗邻颗粒球菌；1株菌鉴定到属（占6．7％），归属于垃状杆菌属。结论16SrRNA基因序列分析技术具有准确、快速和方法简便的特点，适用于对临床非典型蔚、少见菌以及新型细菌的鉴定，可作为细菌常规鉴定手段的必要补充。     

43株甘肃临床分离非结核分枝杆菌分类鉴定

目的了解甘肃当前临床流行的非结核分枝杆菌(NTM)的病原谱特点及其主要NTM种类,为指导临床诊疗、有效防治NTM肺病提供参考依据。方法收集2012年~2014年来源于甘肃省不同地区的875株临床分离分枝杆菌菌株,经PNB/TCH方法初步鉴定为疑似NTM菌株,采用16SrRNA基因测序技术进行菌种鉴定。结果 875株分枝杆菌临床分离菌株中分离出疑似NTM菌株46株。经16SrRNA基因序列分析鉴定,43株为NTM,3株为诺卡氏菌。43株NTM菌株分别为胞内分枝杆菌、堪萨斯分枝杆菌、塞内加尔分枝杆菌、鸟分枝杆菌、戈登氏分枝杆菌、楚尔盖分枝杆菌、罕见分枝杆菌、偶然分枝杆菌和脓肿分枝杆菌。其中,胞内分枝杆菌有31株,占总NTM菌株数的72.09%。结论甘肃省非结核分枝杆菌群以胞内分枝杆菌为主,应用16SrRNA基因序列分析鉴定方法能准确鉴定出NTM菌种,为临床诊治提供依据。     

某高级中学结核病爆发分离菌株的实验室鉴定

目的鉴定从某中学结核病爆发分离菌株的种属。方法对2006年5—8月,辽宁省某高级中学发生的10例痰检阳性病例中的3份痰标本分离的菌株经表型鉴定方法;传统生化方法;扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型;16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析进行鉴定。结果临床分离株经生化方法初步鉴定1份为结核分枝杆菌复合群和2份为非结核分枝杆菌,随后经表型鉴定和扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型,说明属于结核分枝杆菌复合群的菌株为结核分枝杆菌北京基因型现代株,MIRU基因型为223325173533。经16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析说明属于非结核分枝杆菌的菌株为猪分枝杆菌。结论本次从结核病爆发累及病人的标本中分离出结核分枝杆菌北京基因型现代株和猪分枝杆菌,猪分枝杆菌在暴发流行中的意义尚需进一步研究。     

分支杆菌临床分离株的16S-23S rRNA基因转录间隔区的序列分析

目的　建立鉴定分支杆菌分离株的 16S - 2 3SrDNA转录间隔区 (internaltranscribedspacer ,ITS)序列分析的方法 ,用该方法对临床分离株进行鉴定。方法　对从重庆某医院分离的 2 9株分支杆菌菌株分别进行了PCR扩增 ,得到它们的16S - 2 3SrDNAITS片段 ,并测定所得到片段的DNA序列。用DNA分析软件将所获得的序列与GenBank的分支杆菌序列相比较 ,计算种间相似性 ,并用序列构建分支杆菌菌株的系统发育树 ,对分离株进行鉴定。结果　在ITS序列分析中 ,有 10株的序列分别与结核分支杆菌复合群 (Mycobacteriumtuberculosiscomplex ,MTC)、戈登分支杆菌 ,脓肿分支杆菌的序列完全一致。依据完全一致的序列可以准确鉴定这些临床分离株为相应的种 ;4株 16SrDNA序列分析不能准确鉴定的分离株中其中 3株(M 4、M 5、M 12 )鉴定为戈登分支杆菌 ,1株 (M 2 3)鉴定为脓肿分支杆菌。部分分离株的的ITS序列在GenBank序列检索中无完全一致的匹配序列 ,这些不同的序列之间的相似程度为 2 7.1%～ 99.2 %。用临床分离株的ITS序列与其最相似的已知分支杆菌的ITS序列构建的系统发育树 ,菌株分群与 16SrDNA序列构建的系统发育树的分群基本一致。结论　分支杆菌的16S - 2 3SrDNAITS序列比 16SrDNA序列有更大的多样性 ,该序列分析可作为     

一株马鼻疽诺卡菌临床分离株的鉴定

目的应用基因测序分析进行诺卡菌（Nocardia farcinica）鉴定。方法将临床分离菌株BJ7菌株与诺卡菌标准参照菌株AY756551和结核分枝杆菌H37Rv接种L-J、PNB/TCH鉴别培养基。用生长良好的培养物提取全基因组DNA。设计引物,对rpoB、16S rDNA基因片段进行PCR扩增,对其PCR产物进行测序和网上同源性比对。结果 PNB/TCH鉴别培养基鉴定BJ7为非结核分枝杆菌,进化树中BJ7 16S rDNA序列亲缘关系与诺卡菌标准参照菌株AY756551比较相近,且16S rDNA和rpoBDNA序列与马鼻疽诺卡菌同源性极高,分别达到100%和99%。结论从结核病患者分离的菌株BJ7为马鼻疽诺卡菌。DNA测序分析能够快速、简便、准确地鉴定诺卡菌。     

福建省肺非结核分枝杆菌临床分离株的菌种鉴定

目的 对2005-2011年间,福建省福州市胸科医院分离获得的非结核分枝杆菌临床菌株进行菌种鉴定.方法 分别使用传统鉴别培养基法和多位点PCR、hsp65-PRA与rpoB-PRA,以及hsp65、rpoB、16s rRNA和ITS基因测序等分子生物学方法,对全部菌株进行菌种鉴定.结果 450株被初步鉴定为非结核分枝杆菌的临床分离菌株中,有45株被鉴定为结核分枝杆菌,其余405株被鉴定为23种不同的非结核分枝杆菌和1株支气管戈登菌.结论 在该地区有多种非结核分枝杆菌传播和流行,其中,6种非结核分枝杆菌和支气管戈登菌为在福建省内首次发现.     

应用rpoB基因PCR反向斑点杂交快速鉴定分枝杆菌菌种的研究

目的根据分枝杆菌rpoB基因序列建立一种准确、快速的分枝杆菌菌种鉴定方法。方法以分枝杆菌rpoB基因编码序列为靶基因,用聚合酶链反应(PCR)反向斑点杂交技术检测24种分枝杆菌标准株、8种非分枝杆菌标准株、37株分枝杆菌临床分离株。结果分枝杆菌与非分枝杆菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bpDNA片段,非分枝杆菌除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它菌种均未见扩增。敏感性试验可检测出1 pg结核分枝杆菌DNA。探针特异性试验表明,21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交,其余均为特异性杂交。应用该方法对37株分枝杆菌临床分离株进行鉴定,结果与常规方法鉴定结果相符。结论应用rpoB基因序列和PCR反向斑点杂交技术鉴定分枝杆菌菌种快速、准确,具有较高的应有价值。     

Rapid genetic identification system of mycobacteria]

Rapid colorimetric hybridization method was applied for the identification of mycobacteria and phylogenetic detection and identification system of mycobacteria of polymerase chain reaction method was designed. Quantitative DNA-DNA hybridization in microdilution plate was used to identify 22 mycobacterial species. This method could identify 90% (178 among 194 trials) of clinical isolates within 3 hr. Ten percent of clinical isolates did not belong to any of the established 22 species. Through this work, we found Mycobacterium abscessus is genetically independent from M. chelonae and proposed M. abscessus as a distinct species. M. pregrinum had been classified as M. fortuitum, however, it was also found as a independent species. Thus the name M. peregrinum was officially revived and acquired the taxonomic position. Highly sensitive genetic detection system of mycobacteria was designed by using polymerase chain reaction (PCR) method. Common mycobacterial sequence of 16S ribosomal RNA gene was first amplified by a single pair of PCR primers from staining negative sputum and the amplified DNA was identified by species specific DNA probe because the amplified fragment contained species specific sequence.     

Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria

Most mycobacterial infections are still caused by Mycobacterium tuberculosis complex (MTC) strains; however, infections by non-tuberculous mycobacteria (NTM) are increasing, particularly among immunocompromised patients. Conventional species-specific identification and proper patient management are delayed due to the slow-growing nature of mycobacteria. We have developed a multiplex PCR (mPCR) targeting the oxyR-ahpC intergenic region and rpoB gene for direct detection and differentiation of clinical isolates as MTC or NTM in primary culture. Two amplicons of 473 bp and 235 bp from MTC members and a single amplicon of 136 bp from NTM are expected. The mPCR was developed using several mycobacterial species and was evaluated by testing extracted DNA from liquid cultures, flagged as positive for bacterial growth, of 100 consecutive mycobacterial isolates. The results were validated by DNA sequencing of the species-specific 16S-23S internal transcribed spacer (ITS) region. The mPCR with template DNA from reference Mycobacterium spp. yielded the expected amplicons. When 100 consecutive clinical isolates of Mycobacterium spp. were tested, 92 strains yielded MTC member-specific amplicons, and DNA sequences from 10 randomly selected isolates matched completely with the ITS sequence from M. tuberculosis. Eight isolates were identified as NTM, and DNA sequencing of the ITS region confirmed the NTM status of each of these isolates. The mPCR developed in this study allowed rapid detection and differentiation of primary cultures as MTC or NTM, thus helping in timely institution of specific therapy.