共刺激分子4-1BBL基因免疫对HBsAg核酸疫苗诱导小鼠特异性免疫应答的影响

目的 研究共刺激分子4-1BBL基因免疫对HBsAg核酸疫苗诱导小鼠特异性体液和细胞免疫应答的影响.方法 将HBV表面抗原核酸疫苗pcDS2单独或联合共刺激分子4-1BBL质粒肌肉注射免疫C57BL/6小鼠;ELISA法检测小鼠血清抗-HBs IgG及亚型IgG1和IgG2a;迟发型超敏反应(DTH)反应检测体内细胞反应;流式细胞仪检测CD4+ T淋巴细胞分泌IL-4和IFN-γ及CD8+T淋巴细胞分泌IFN-γ水平;流式细胞仪检测小鼠脾细胞HBsAg特异性体外细胞毒性T淋巴细胞杀伤作用(CTL).结果 与单纯免疫核酸疫苗pcDS2组比较,pcDS2和4-1BBL联合免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠经HBsAg脚掌皮下刺激后,联合免疫组小鼠脚掌的厚度显著高于pcDS2组;联合免疫组CD4+T淋巴细胞的IL-4和IFN-γ表达水平及CD8+T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体外CIL杀伤作用高于对照组,其中联合免疫组小鼠的体外CTL杀伤作用最强.结论共刺激分子4-1BBL不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性型细胞免疫反应,尤其增强体内CIL的杀伤活性.     

pcDNA3.1+/MAGE-3 DNA疫苗诱导特异性抗肿瘤免疫应答的实验研究

目的：构建pcDNA3.1+/MAGE-3 DNA疫苗，观察其在小鼠体内诱导特异性抗肿瘤免疫应答的能力。方法： 通过RT-PCR构建重组表达质粒pcDNA3.1+/MAGE-3；以pcDNA3.1+/MAGE-3 DNA疫苗免疫已接种肿瘤细胞的小鼠，每10 d重复免疫1次，共3次，以pcDNA3.1+、PBS为对照。末次免疫后5 d检测血清中MAGE-3抗体滴度、小鼠脾淋巴细胞的细胞毒T细胞（cytotoxic T lymphocytes，CTL）杀伤活性、细胞因子IL-2和IFN-γ的浓度，同时计算抑瘤率。结果： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，用此疫苗免疫已接种B16/MAGE-3细胞的小鼠后，能诱导小鼠脾淋巴细胞MAGE-3特异性的杀伤活性，脾细胞培养上清中细胞因子IL-2和IFN-γ的浓度明显增高，血清中抗MAGE-3抗体在1∶20滴度时阳性，肿瘤生长被显著抑制，与pcDNA3.1+组、PBS组相比，差异显著（P＜0.01）。结论： 成功构建了pcDNA3.1+/MAGE-3 DNA疫苗，该疫苗在小鼠体内既能激活CTL杀伤活性和CD4+ T细胞活性，又能激活体液免疫反应，从而诱导出特异性的抗肿瘤免疫应答。     

结核分枝杆菌Rv2450蛋白诱导小鼠免疫应答的实验研究

目的:以原核表达的结核分枝杆菌Rv2450蛋白在小鼠体内诱导体液和细胞免疫应答。方法:采用皮下包埋的方法,以预先转移到硝酸纤维素膜上的原核表达的Rv2450蛋白免疫小鼠(10只)3次,每次间隔2周。用间接ELISA法检测免疫小鼠血清特异性抗体的滴度。末次免疫完成后4周,处死3只免疫小鼠并分离脾淋巴细胞,体外经PPD(2μg/孔)刺激后,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖指数。用ELISA法检测脾淋巴细胞悬液中IFN-γ、IL-10及IL-12的水平。结果:Rv2450蛋白免疫小鼠血清特异性抗体的滴度为1∶3200,淋巴细胞增殖指数为3.76±0.19。免疫小鼠脾淋巴细胞培养液中IFN-γ、IL-10及IL-12的含量,分别为(1740±19)ng/L、(678±15)ng/L、(469±13)ng/L,均高于各组的生理盐水对照组(P<0.05)。结论:Rv2450有可能作为新型结核疫苗的候选组分。     

口服免疫Ag85A脂质体DNA诱导T细胞亚群应答效应研究

目的:拟采用构建的可在真核细胞内表达Ag85A基因的质粒,以阳离子脂质体为运载体,制成DNA疫苗,经口途径投予小鼠,以观察Ag85A脂质体DNA疫苗诱导免疫应答效应,为口服DNA疫苗的临床应用提供理论和实验依据.方法:ELISA方法检测Ag85A特异性抗体产生水平及血清Th1型细胞因子IFN-γ及Th2型细胞因子IL-4的分泌水平,ELISPOT技术检测口服DNA疫苗后小鼠可分泌IFN-γ和IL-4脾淋巴细胞数量.流式细胞术观察口服DNA疫苗后小鼠脾淋巴细胞CD4+T细胞及CD8+T细胞亚群的变化,从而判断口服DNA疫苗的免疫效果及脂质体是否有免疫增强作用.结果:口服自制Ag85ADNA疫苗可见血清中抗Ag85A特异性抗体的产生;下调了脾CD4+T细胞和CD8+T细胞亚群的数量;分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降,而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高.口服DNA疫苗组诱导Ag85A特异性抗体产生,脂质体具有免疫佐剂作用.结论:应用阳离子脂质体为运载体,重组Ag85A DNA疫苗口服免疫C57BL/6小鼠,诱导了CD4+及CD8+T细胞亚群的下调及IFN-γ的表达减少,而玎IL-4的分泌呈增高趋势;疫苗可诱导Ag85A特异性抗体的分泌,产生了明显的体液应答.     

IL-17作为分子佐剂增强HIV DNA疫苗细胞免疫应答的研究

目的 为了进一步增强HIV DNA疫苗的免疫反应,本研究将IL-17作为HIV DNA疫苗的分子佐剂免疫小鼠,旨在探讨IL-17对HIVDNA疫苗诱导体液和细胞免疫应答的影响.方法 将小鼠IL-17构建到真核表达载体,与HW-1膜蛋白DNA疫苗pGX-Env联合免疫BALB/c小鼠;分别在第0、2周进行免疫,在第4周检测T淋巴细胞增殖指数、抗-Env IgG、细胞因子表达水平和体内细胞毒性T淋巴细胞杀伤作用(CTL)等免疫学指标.结果 IL-17能够增强HIV DNA疫苗的特定免疫反应.与单注射疫苗组相比,IL-17作为佐剂组的T细胞增殖、抗体水平和CD4~+T细胞分泌IFN-γ、IL-4和IL-17的表达均无明显增强,但对CD8~+T细胞分泌IFN-γ的表达和体内CTL的效果影响明显(P<0.05).结论 IL-17作为分子佐剂不足以影响Th细胞分化,然而却能够增强特异性CD8~+T细胞中IFN-γ的表达,尤其是增强体内CTL反应.此结果为增强艾滋病DNA疫苗CD8~+T细胞活性和用于艾滋病的治疗提供了一个新的思路.     

结核分枝杆菌Ag85B-MPT64融合基因疫苗的免疫效果观察

目的:研究并比较结核分枝杆菌(H37Rv)Ag85B、MPT64DNA.以及两者的融合基因(AM)的免疫原性。方法:雌性C57BL/6小鼠25只,随机分为5组,即A组(PBS)、B组(peDNA3.1)、C组(pcDNA/Ag85B)、D组(pcDNA/MPT64)和E组(pcDNA/AM)。分别于胫前肌注射7.5 g/L利多卡因和质粒混合物(1:4,100μL,含质粒70μg/次),间隔2 wk免疫 1次,共3次。末次免疫后4 wk取血分离血清测定总IgG,同时分离脾淋巴细胞,用PPD刺激后分别做脾淋巴细胞增殖实验(MTT比色法)和测定脾淋巴细胞培养上清中IFN-γ的水平。结果:Ag85B、MPT64和AM质粒DNA,均能诱导小鼠产生较高水平的PPD特异性IgG。免疫小鼠脾淋巴细胞体外经PPD刺激后,能产生特异性淋巴细胞增殖和分泌IFN-γ。peDNA/AM组IFN-γ的分泌水平明显高于pcDNA/Ag85B和pcDNA/MPT64免疫组(P<0.05)。结论:结核分枝杆菌Ag85B-MPT64融合基因疫苗,能在小鼠体内诱导特异性细胞和体液免疫。     

重组耻垢分枝杆菌制剂预防幽门螺杆菌感染的作用机理的初步探讨

目的探讨重组耻垢分枝杆菌实验制剂（recombinant Mycobacterium smegmatis，rMs）预防幽门螺杆菌（Helicobacter pylori，Hp）感染的作用机理。方法实验制剂免疫BLAB／c小鼠4周，用Hp攻击后4周，取血清、胃、脾组织，RT-PCR检测小鼠胃黏膜和脾组织的TH1型细胞因子（IFN-γ、IL-2、IL-12）与TH2型细胞因子（IL-4、IL-6、IL-10）；用ELISA检测针对唧的特异性血清IgG、IgG1、IgG2a和IgA水平；用免疫组化方法检测胃黏膜固有层IgA表达；用MTT检测脾淋巴细胞增殖。结果免疫后BALB／c小鼠特异性抗体显示rMs制剂诱导Hp特异性血清IgG、IgA水平都升高，以IgG2a占优势。用Hp抗原刺激后各免疫组小鼠脾淋巴细胞均有明显增殖。免疫组化显示各免疫组小鼠胃黏膜固有层IgA阳性标记腺体数明显高于对照组。RT-PCR证实，跏攻击之前，各免疫组胃和脾组织已出现了IFN-γ、IL-12、IL-2表达而无IL-4、IL-6、IL-10表达。PBS对照组和单纯耻垢分枝杆菌（Ms）组胃黏膜和脾组织各细胞因子均无表达；Hp攻击后，PBS和单纯Ms组胃组织出现以TH1细胞IFN-γ为主的增生，IFN-γ水平显著高于rMs组（P〈0．05）；而3个rMs制剂组脾脏则出现以TH2细胞（IL-4）为主的增生，IL-4水平显著高于对照组（P〈0．05）。提示该制剂的作用机制是诱导TH1，TH2平衡型免疫应答。结论成功地建立了BALB／c小鼠的Hp感染模型，对rMs制荆的免疫保护机理研究结果显示，rMs能产生以TH1细胞和TH2细胞协同作用的保护性免疫应答。     

结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。     

铜绿假单胞菌重组Bb-OprF疫苗诱导小鼠细胞免疫应答的研究

目的研究铜绿假单胞菌重组双歧杆菌(rBb)-OprF疫苗免疫小鼠,PAO1株攻击后产生的细胞免疫应答。方法将rBb-OprF疫苗分别采用皮下注射、肌肉注射、鼻腔黏膜和口服灌胃4种途径免疫Balb/c小鼠,免疫后8周用5×106CFU的PAO1株攻击,攻击1周后杀鼠取脾,MTT法检测特异性脾淋巴细胞增殖,流式细胞术检测脾CD4+T和CD8+T细胞比率,用ELISA法检测脾细胞培养上清IFN-γ、IL-12、TNF-α和IL-10的水平。结果脾T淋巴细胞增殖明显;脾细胞CD4+和CD8+T细胞亚群显著增加;脾细胞培养上清IFN-γ、IL-12、TNF-α和IL-10的水平显著升高,其中IFN-γ最为显著。结论铜绿假单胞菌rBb-OprF疫苗可诱导小鼠产生混合型的Th1和Th2免疫应答。     

Ag85A DNA疫苗加强免疫显著提高卡介苗初免小鼠的抗结核T细胞免疫应答

目的 构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法 以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗;接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果 与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论 本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.     

Polysaccharides of Mycobacterium bovis Ushi 10, Mycobacterium smegmatis, Mycobacterium phlei, and Atypical Mycobacterium P1

Four kinds of polysaccharides, glucan, mannan, arabinomannan, and arabinogalactan, were purified from the defatted cells of four strains of mycobacteria. By the chemical and immunological analyses, we concluded that arabinomannan and arabinogalactan are common serologically active polysaccharides in mycobacteria.     

LightCycler-based differentiation of Mycobacterium abscessus and Mycobacterium chelonae

In this study we introduce a rapid procedure to identify Mycobacterium abscessus (types I and II) and M. chelonae using LightCycler-based analysis of the hsp65 gene. Results from 36 clinical strains were compared with hsp65 gene restriction analysis and biochemical profiles of bacilli. As all three methods yielded identical results for each isolate, this procedure offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnosis of mycobacterial diseases.     

Identification of Mycobacterium fortuitum and Mycobacterium chelonei.

The study of 52 strains of rapidly growing mycobacteria showed that Mycobacterium fortuitum and M. chelonei were clearly distinguished by the aid of seven key tests (nitrate reductase, iron uptake, beta-glucosidase, penicillinase, growth on fructose, resistance to pipemidic acid, and resistance to capreomycin) and by analysis of their respective mycolic acids. However, the subdivision of these species into M. fortuitum var. fortuitum and M. fortuitum var. peregrinum and M. chelonei subsp. chelonei and M. chelonei subsp. abscessus was not satisfactorily accomplished.     

Phenol-Soluble Antigens from Mycobacterium kansasii, Mycobacterium gastri, and Mycobacterium marinum

Many of the demonstrable antigens derived from mycobacteria are common to members of different species. Agglutination tests have yielded the most specific characterizations. An antigen which may be associated with the specific agglutination is soluble in phenol and can be extracted and separated from other antigens by use of this solvent. Phenol-soluble antigens have been extracted from representative cultures of Mycobacterium kansasii, M. gastri, and M. marinum. Most representatives of each of these species yielded an antigen which was characteristic of the species but was distinct from antigen derived from the other two species.     

Differential identification of Mycobacterium fortuitum and Mycobacterium chelonei.

Mycobacterium fortuitum and Mycobacterium chelonei are distinguished unambiguously by the combined use of five test characters: nitrate reductase, beta-glucosidase, acid production from fructose, penicillinase, and trehalase. Typically, M. fortuitum was nitrate reductase positive, beta-glucosidase positive; M. chelonei was nitrate reductase negative, beta-glucosidase negative, penicillinase positive, and trehalase positive and did not produce acid from fructose.     

Proportions of Mycobacterium massiliense and Mycobacterium bolletii strains among Korean Mycobacterium chelonae-Mycobacterium abscessus group isolates

Korean isolates of the Mycobacterium chelonae-Mycobacterium abscessus group, which had been isolated from two different hospitals in South Korea, were identified by PCR restriction analysis (PRA) and comparative sequence analysis of 16S rRNA genes, rpoB, and hsp65 to evaluate the proportion of four closely related species (M. chelonae, M. abscessus, M. massiliense, and M. bolletii). Of the 144 rapidly growing mycobacterial strains tested, 127 strains (88.2%) belonged to the M. chelonae-M. abscessus group. In this group, M. chelonae, M. abscessus, M. massiliense, and M. bolletii accounted for 0.8% (n = 1), 51.2% (n = 65), 46.5% (n = 59), and 1.6% (n = 2), respectively. Two isolates which showed discordant results, M. massiliense by rpoB sequence analysis and M. abscessus by hsp65 sequence analysis, were finally identified as M. massiliense based on the additional analysis of sodA and the 16S-23S internal transcribed spacer. M. abscessus group I isolates previously identified by hsp65 PRA were all found to be M. abscessus, whereas group II isolates were further identified as M. massiliense or M. bolletii by sequencing of rpoB and hsp65. Smooth, rough, or mixed colonies of both M. abscessus and M. massiliense isolates were observed. M. massiliense strains that were highly resistant to clarithromycin had a point mutation at the adenine at position 2058 (A₂₀₅₈) or 2059 (A₂₀₅₉) in the peptidyltransferase region of the 23S rRNA gene.     

Cohort Study of Molecular Identification and Typing of Mycobacterium abscessus,Mycobacterium massiliense,and Mycobacterium bolletii

Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease. Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Health care-associated outbreaks have recently been investigated by the use of molecular identification and typing tools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M. bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult and relies on the sequencing of one or more housekeeping genes. We performed extensive molecular identification and typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at the NIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB, hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and 2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted further sequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonal groups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB, hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliense type strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, a multilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus and closely related species. Molecular typing complements sequence-based identification and provides information on prevalent clones with possible relevant clinical aspects.Rapidly growing mycobacteria (RGM) are ubiquitous organisms increasingly emerging as important human pathogens. Mycobacterium abscessus is commonly associated with wound infections and abscess formation and is the most frequent RGM causing chronic lung disease, often in immunocompromised patients (15, 22, 24). M. abscessus is also notable for its resistance to treatment and the poor clinical outcome of infection with the organism (22, 24). Within the past decade, two new species of mycobacteria closely related to M. abscessus, M. massiliense and M. bolletii, have been described (1, 3). Information on the pathogenic role of M. massiliense and M. bolletii is still scant. Recent reports have described the isolation of M. massiliense from two patients in the United States (29) and one patient in Italy (35) and, lately, the identification of M. massiliense and M. bolletii among South Korean isolates (18). Both M. massiliense and M. bolletii have also been linked to health care-associated outbreaks (8, 19, 37).The species-level identification of RGM can provide the first indication of antibiotic susceptibility and can suggest the appropriate type of patient management. For example, M. abscessus is more resistant to many antibiotics both in vivo and in vitro than M. fortuitum and M. mucogenicum, but it is usually susceptible to amikacin and clarithromycin (6, 15, 24). M. massiliense was originally reported to be distinguishable from M. abscessus and related species by its susceptibility to doxycycline (3); however, resistant isolates have since been described (19, 37), suggesting that antibiotic susceptibility results may not reliably differentiate among these closely related species.Although 16S rRNA gene sequencing has been used for the identification of nontuberculous mycobacteria (NTM), including RGM, it has limited value in distinguishing among some closely related species (9, 14). Therefore, the use of several other gene targets for the identification of mycobacteria has been proposed (2, 5, 11, 23, 25, 31, 32, 39, 41). Discrimination among M. abscessus, M. massiliense, and M. bolletii (which have identical 16S rRNA gene sequences) has proven to be difficult, with sequencing of different gene targets often providing conflicting results. Among these gene targets, partial sequencing of rpoB has increasingly been used (1, 19, 29, 37).Genotypic analysis of NTM has proven useful not only in the investigation of outbreaks and pseudo-outbreaks (38) but also in characterizing the molecular epidemiology of strains, and in assessing clonal distribution and expansion (4, 7, 13, 17). In particular, molecular typing has recently been used for the characterization of health care-related outbreaks of M. massiliense and M. bolletii (19, 37).We sought to perform a thorough molecular investigation, including strain identification and typing, for a series of 42 clinical isolates (CIs) of M. abscessus, M. massiliense, and M. bolleti from patients monitored in our institution between 1999 and 2007. A retrospective patient chart review assessed demographics, underlying conditions, and clinical history.The 42 CIs and 3 type strains were subjected to multilocus sequence analysis, including sequencing of rpoB, hsp65, secA, and the internally transcribed spacer (ITS) region. The relatedness among the isolates was assessed by use of an automated repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). This is the most extensive molecular characterization of non-outbreak-related isolates from patients with M. abscessus, M. massiliense, and M. bolletii infections.     

Characterization of an unusual Mycobacterium: a possible missing link between Mycobacterium marinum and Mycobacterium ulcerans

In an attempt to characterize an unusual mycobacterial isolate from a 44-year-old patient living in France, we applied phenotypic characterizations and various previously described molecular methods for the taxonomic classification of mycobacteria. The results of the investigations were compared to those obtained in a previous study with a set of temporally and geographically diverse Mycobacterium ulcerans (n = 29) and Mycobacterium marinum (n = 29) isolates (K. Chemlal, G. Huys, P.-A. Fonteyne, V. Vincent, A. G. Lopez, L. Rigouts, J. Swings, W. M. Meyers, and F. Portaels, J. Clin. Microbiol. 39:3272-3278, 2001). The isolate, designated ITM 00-1026 (IPP 2000-372), is closely related to M. marinum according to its phenotypic properties, lipid pattern, and partial 16S rRNA sequence. Moreover, fingerprinting by amplified fragment length polymorphism (AFLP) analysis unequivocally classified this strain as a member of the species M. marinum, although it lacked two species-specific AFLP marker bands. However, PCR and restriction fragment length polymorphism analysis based on M. ulcerans-specific insertion sequence IS2404 showed the presence of this element in a low copy number in isolate ITM 00-1026. In conclusion, the designation of this isolate as a transitional species further supports the recent claim by Stinear et al. (T. Stinear, G. Jenkin, P. D. Johnson, and J. K. Davies, J. Bacteriol. 182:6322-6330, 2000) that M. ulcerans represents a relatively recent phylogenetic derivative of M. marinum resulting from the systematic acquisition of foreign DNA fragments.     

Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum

A multicenter study was conducted to assess the inter- and intralaboratory reproducibility of the Etest for susceptibility testing of the rapidly growing mycobacteria. The accuracy also was evaluated by comparing Etest results to those obtained by broth microdilution. Ten isolates (four of the Mycobacterium fortuitum group, three of Mycobacterium abscessus, and three of Mycobacterium chelonae) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole in each of four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) using common lots of media and Etest strips. Interlaboratory agreement among MICs (i.e., mode +/- 1 twofold dilution) varied for the different drug-isolate combinations and overall was best for trimethoprim-sulfamethoxazole (75% for one isolate and 100% for all others), followed by doxycycline and ciprofloxacin. Interlaboratory agreement based on interpretive category also varied and overall was best for doxycycline (100% for all isolates), followed by trimethoprim-sulfamethoxazole and ciprofloxacin. Interlaboratory reproducibility among MICs was most variable for imipenem, and agreement by interpretive category was lowest for imipenem and amikacin. Modal Etest MICs agreed with those by broth microdilution only for doxycycline and the sulfonamides. For all other drugs, the modal MICs by the two methods differed by more than +/- 1 twofold dilution for one or more isolates. In all cases, the Etest MIC was higher and would have caused reports of false resistance. In summary, the Etest in this evaluation did not perform as well as broth microdilution for susceptibility testing of the rapidly growing mycobacteria. It was problematic for most species and drugs, primarily because of a trailing endpoint and/or high MICs compared to broth. Its use will necessitate further investigation, including determination of the optimal medium and incubation conditions and clarification of endpoint interpretation.