基于16S rRNA和gyrB基因的施万菌种水平鉴定分析

目的 构建基于16S rRNA和gyrB基因对施万菌(Shewanella)进行种水平鉴定的方法,比较2个基因的鉴定能力差异.方法 利用DnaSP 6.0软件对施万菌16S rRNA和gyrB基因的信息位点数及其百分比、核苷酸多态性值、平均G+C含量、非同义突变率与同义突变率的比值(Ka/Ks)、Tajima检验进行基...     

革兰阴性杆菌16S rRNA甲基化酶基因检测及作用研究

目的分析上海中医药大学附属普陀医院临床分离的革兰阴性杆菌中16S rRNA甲基化酶基因的流行情况及革兰阴性杆菌的氨基糖苷类耐药机制。方法收集临床分离的对阿米卡星和/或庆大霉素耐药的革兰阴性杆菌53株。采用聚合酶链反应（PCR）法检测16S rRNA甲基化酶基因：armA、rmtA、rmtB、rmtC、rmtD、npmA;PCR阳性产物测序分析。构建PET32-armA和PET32-rmtB的重组质粒并转化入宿主菌BL21中。纸片扩散法（K-B）检测临床分离16S rRNA甲基化酶基因阳性菌株和重组菌对5种氨基糖苷类药物的敏感性。结果 53株耐药菌中5株鲍曼不动杆菌、2株肺炎克雷伯和1株阴沟肠杆菌检出armA基因,2株大肠埃希菌检出rmtB基因。获得PET32-armA和PET32-rmtB的重组BL21菌株。PET32-armA和PET32-rmtB的重组菌均获得氨基糖苷类抗菌药物耐药性。结论本院临床样本检测到16S rRNA甲基化酶基因armA和rmtB阳性菌株,armA基因存在于鲍曼不动杆菌、肺炎克雷伯菌和阴沟肠杆菌中;rmtB基因位于大肠埃希菌中。将armA和rmtB基因转入非耐药的宿主菌中可以诱导其对氨基糖苷类抗菌药物的耐药。     

16S rRNA mutation-mediated tetracycline resistance in Helicobacter pylori

Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet(r)) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tet(s) strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tet(r) strain 181, the Tet(s) strain 26695, and four Tet(r) 26695 transformants showed that a single triple-base-pair substitution, AGA(926-928)-->TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet(r) H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet(r) strain 181.     

不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

目的 评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点.方法 收集41株临床分离的非结核分枝杆菌,以16S rRNA基因测序方法为标准,同时以hsp65基因测序方法及PCR-RFLP方法鉴定菌株,与16S rRNA基因测序结果进行比较.结果 41株非结核分枝杆菌16SrRNA基因测序结果:9株龟分枝杆菌复合群,7株偶发分枝杆菌,7株胞内分枝杆菌,3株鸟分枝杆菌,3株堪萨斯分枝杆菌复合群,3株耻垢分枝杆菌,3株土分枝杆菌,2株草分枝杆菌,2株无色分枝杆菌,1株瘰疬分枝杆菌,1株M.arupense.与16S rRNA基因测序相比较,hs65 PCR-RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌;1株偶发分枝杆菌及1株无色分枝杆菌与其不符;其余菌株鉴定结果一致,符合率为95.1%(39/41).hsp65基因测序结果显示,1株爱尔兰分枝杆菌与16S rRNA测序结果不符,其余菌株鉴定结果与其一致,符合率为97.6%(40/41),并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌.结论 3种方法均能快速鉴定非结核分枝杆菌.与16S rRNA基因测序相比,hsp65基因测序及hsp65 PCR-RFLP更容易鉴定临床最常见非结核分枝杆菌(如堪萨斯分枝杆菌和脓肿分枝杆菌),可在临床推广使用.     

Emergence of ArmA and RmtB aminoglycoside resistance 16S rRNA methylases in Belgium

OBJECTIVES: 16S rRNA methylase-mediated high-level resistance to aminoglycosides has been reported recently in clinical isolates of Gram-negative bacilli only from a limited number of countries. This study was conducted to investigate the occurrence of this type of resistance in clinical isolates of Enterobacteriaceae from two Belgian hospitals and the characteristics of the strains. METHODS: We screened for high-level gentamicin, tobramycin and amikacin resistance in clinical isolates of Enterobacteriaceae consecutively collected between 2000 and 2005 at two laboratories by PCR for the armA, rmtA and rmtB 16S rRNA methylase genes. The beta-lactamase presence in the strains was also determined by phenotypic and genotypic methods. RESULTS: Overall armA genes were detected in 18 Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae and Citrobacter amalonaticus whereas rmtB was detected in a single E. coli isolate. The rmtA gene was not found. All 16S rRNA methylase-bearing strains produced extended-spectrum beta-lactamases (ESBLs), predominantly type CTX-M-3, as well as various types of beta-lactamases. In the majority of the strains, the armA gene was carried by conjugative plasmids of the IncL/M incompatibility group whereas rmtB was borne by an IncFI plasmid. CONCLUSIONS: This is the first report of the emergence of 16S rRNA methylases in Enterobacteriaceae in Belgium. The rapid spread of multidrug-resistant isolates producing both ESBLs and 16S rRNA methylases raises clinical concern and may become a major therapeutic threat in the future.     

肿瘤靶向治疗新抗原筛选与临床研究进展

肿瘤免疫治疗是近年来研究的热点,而基于靶向肿瘤新抗原的免疫治疗极具广阔前景,但是长期以来,缺乏肿瘤特异性抗原是阻碍肿瘤免疫治疗的瓶颈,因此,发现新的肿瘤特异性抗原一直是基础和临床肿瘤免疫学家梦寐以求的目标。二代测序技术的出现推动了肿瘤新抗原筛选与鉴定的发展。目前主要有全外显子结合转录组测序法、串联微基因序列法、靶基因测序法、共享新抗原肽库法和质谱法等筛选肿瘤新抗原的方法。肿瘤新抗原的发现为临床开展抗原疫苗、抗原特异性T细胞、T细胞受体工程T细胞(T cell receptor genetically engineered T cell,TCR-T)和嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)等靶向肿瘤抗原的免疫治疗提供了优质的靶标。     

Fluoroquinolone resistance in atypical pneumococci and oral streptococci: evidence of horizontal gene transfer of fluoroquinolone resistance determinants from Streptococcus pneumoniae

Atypical strains, presumed to be pneumococcus, with ciprofloxacin MICs of > or =4.0 microg/ml and unique sequence variations within the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in comparison with the Streptococcus pneumoniae R6 strain, were examined. These strains were reidentified using phenotypic methods, including detection of optochin susceptibility, bile solubility, and agglutination by serotype-specific antisera, and genotypic methods, including detection of pneumolysin and autolysin genes by PCR, 16S rRNA sequencing, and multilocus sequence typing (MLST). The analysis based on concatenated sequences of the six MLST loci distinguished the "atypical" strains from pneumococci, and these strains clustered closely with S. mitis. However, all these strains and five of nine strains from the viridans streptococcal group possessed one to three gyrA, gyrB, parC, and parE genes whose QRDR sequences clustered with those of S. pneumoniae, providing evidence of horizontal transfer of the QRDRs of the gyrase and topoisomerase genes from pneumococci into viridans streptococci. These genes also conferred fluoroquinolone resistance to viridans streptococci. In addition, the fluoroquinolone resistance determinants of 32 well-characterized Streptococcus mitis and Streptococcus oralis strains from bacteremic patients were also compared. These strains have unique amino acid substitutions in GyrA and ParC that were distinguishable from those in fluoroquinolone-resistant pneumococci and the "atypical" isolates. Both recombinational events and de novo mutations play an important role in the development of fluoroquinolone resistance.     

苏尼特左旗自毙鼠琥珀葡萄球菌分离株检测研究

  目的   拟通过实验室检测,探究2019年内蒙古自治区锡林郭勒盟苏尼特左旗长爪沙鼠成批死亡的原因。  方法   将所有自毙鼠解剖后,取其脏器接种到赫氏培养基和半胱氨酸心琼脂血培养基上。 对于分离菌株先排除鼠疫菌后,再接种到含有抗生素的半胱氨酸心琼脂血培养基上,同时进行土拉弗朗西斯菌（土拉菌）特异抗原乳胶凝集检测。 其次,对于所有分离菌株提取核酸后,采用16S rRNA基因通用引物进行扩增、测序和同源性比对,并与网上已公布的琥珀葡萄球菌16S rRNA基因序列进行系统进化分析。 另外,用琥珀葡萄球菌悬液感染实验小鼠,在14 d内观察实验小鼠的精神和身体状况。  结果   分离的所有菌株在土拉菌选择性培养基上均不生长,土拉菌特异抗原乳胶凝集检测均为阴性,由此可以排除土拉菌。 经过16S rRNA基因测序和比对,所有分离菌株鉴定为4株琥珀葡萄球菌、1株罗伊乳杆菌、1株阴道乳杆菌和1株粪肠球菌。 系统进化分析显示,本研究获得的3株琥珀葡萄球菌,与9株来自世界其他国家的琥珀葡萄球菌聚在主要谱系分支上。 另外1株琥珀葡萄球菌和来自新疆的琥珀葡萄球菌聚在一个分支上。 实验小鼠感染模型得出,琥珀葡萄球菌的最小致死量为5×108 CFU/mL,亚致死量为108 CFU/mL。   结论   首次发现苏尼特左旗自毙长爪沙鼠体内含有琥珀葡萄球菌,推测琥珀葡萄球菌感染可能是当地长爪沙鼠成批死亡的原因。     

Antimicrobial susceptibility of non-enterococcal intrinsic glycopeptide-resistant Gram-positive organisms

Non-enterococcal Gram-positive bacteria that are intrinsically vancomycin-resistant have been infrequently isolated in association with serious infections. However, well-documented infections have lately been reported with increasing frequency. Because these organisms may be pathogens, we tested the MICs of 19 antimicrobial agents by the agar dilution method for predicting susceptibility. The activity of these antimicrobial agents was assessed against 28 strains (Lactobacillus rhamnosus, 6; Lactobacillus acidophilus, 1; Lactobacillus casei, 1; Lactobacillus fermentum, 2; Lactobacillus brevis, 1; Lactobacillus plantarum, 1; Weissella confusa, 2; Leuconostoc mesenteroides, 7; Leuconostoc lactis, 4; Pediococcus acidilactici, 2; Pediococcus pentosaceus, 1), isolated from clinical specimens in an Argentinian university hospital from 1997 to 2003. The MICs of penicillin for 67% of the Lactobacillus strains and 100% of the Leuconostoc spp. and Pediococcus spp. strains tested were in the 0.25-2 microg/mL range. Erythromycin was the most active antimicrobial overall. Multiresistance was observed in 2 strains (Lactobacillus rhamnosus, 1; Lactobacillus plantarum, 1).     

Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene

OBJECTIVE: Bacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia. DESIGN: The results of the T-RFLP analysis are compared with two standard biochemical identification methods. METHODS: Lactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed. RESULTS: T-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations. CONCLUSION: T-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.     

人苍白杆菌深圳分离株16S rRNA基因部分核苷酸序列分析

目的对深圳市布吉人民医院分离鉴定的1株人苍白杆菌进行16S rRNA基因的部分核苷酸序列分析,以探讨该菌株在分类学上的准确地位。方法对该菌株进行PCR扩增及16SrRNA基因序列测定,并与人苍白杆菌标准株及羊种布氏杆菌比较。结果3株细菌均可扩增到大小约900bp的16S rRNA基因片段,经核苷酸序列分析比较,发现人苍白杆菌深圳分离株与人苍白杆菌标准株和羊种布氏杆菌具有高度同源性,同源程度大于98%。结论该院分离的人苍白杆菌不仅与购自美国的人苍白杆菌ATCC株有高度同源性,而且与北京保存的羊种布氏菌在遗传学上也具有非常密切的亲缘关系。     

4株威克汉姆无绿藻的鉴定及基因特征分析

目的对临床分离的4株无绿藻进行菌种鉴定和基因特征分析。方法对临床分离的4株酵母样微生物进行培养特性和形态学特征分析,商品化Vitek 2细菌鉴定系统(配套YST卡)和MALDI-TOF MS微生物鉴定系统鉴定;PCR扩增其16S rRNA基因和28S rRNA基因,并进行系统发育分析,以确定其分类学地位。结果该4株微生物在沙保氏葡萄糖琼脂培养基上培养3 d可形成灰白色、光滑、湿润的"酵母样真菌"菌落;光镜下观察可见大量圆形、卵圆形或椭圆形的孢子囊,且其内含内孢子,形似桑葚状或草莓状,与酵母菌的菌体形态差异显著;Vitek YST和MALDI-TOF MS系统,均鉴定为威克汉姆无绿藻。该4株微生物同时存在代表原核生物的16S rRNA基因以及代表真核生物的28S rRNA基因,其中,16S rRNA基因序列与威克汉姆无绿藻相似度最高,在99.7%以上;28S rRNA基因经克隆测序发现其存在多拷贝,且同一菌株不同克隆序列之间相似度在91.9%～100%。16S rRNA基因和28S rRNA基因系统发育分析均显示,该4株微生物与威克汉姆无绿藻聚类在同一个分枝。结论该4株微生物可鉴定为威克汉姆无绿藻,且单拷贝的小亚基16S rRNA更适合作为其菌种鉴定的靶基因。     

喜马拉雅旱獭源鹑鸡肠球菌的分离及其耐药性检测

目的 了解青藏高原喜马拉雅旱獭携带院内感染病原菌鹑鸡肠球菌情况,并检测其对临床常用抗菌药物的耐药性。方法 选择性培养基、革兰染色、生化反应和16S rRNA基因序列分析方法对细菌进行分离和鉴定;K-B纸片法检测菌株对15种抗菌药物的耐药情况,并应用PCR方法检测毒力基因和耐药基因。结果 79份喜马拉雅旱獭肠道样本中共分离到3株鹑鸡肠球菌,其中1株对利福平中介,3株均对奎奴普丁中介,对其他被检抗菌药物均敏感;未检测到常见肠球菌毒力基因(asa1、esp、hyl、gelE和cylA)和相关耐药基因。结论 首次在青藏高原旱獭粪便中分离到鹑鸡肠球菌,常见的毒力基因检测阴性,对常见抗菌药物敏感。     

16S rRNA Mutation Associated with Tetracycline Resistance in a Gram-Positive Bacterium

A genetic basis for tetracycline resistance in cutaneous propionibacteria was suggested by comparing the nucleotide sequences of the 16S rRNA genes from 16 susceptible and 21 resistant clinical isolates and 6 laboratory-selected tetracycline-resistant mutants of a susceptible strain. Fifteen clinical isolates resistant to tetracycline were found to have cytosine instead of guanine at a position cognate with Escherichia coli 16S rRNA base 1058 in a region important for peptide chain termination and translational accuracy known as helix 34. Cytosine at base 1058 was not detected in the laboratory mutants or the tetracycline-susceptible strains. The apparent mutation was recreated by site-directed mutagenesis in the cloned E. coli ribosomal operon, rrnB, encoded by pKK3535. E. coli strains carrying the mutant plasmid were more resistant to tetracycline than those carrying the wild-type plasmid both in MIC determinations and when grown in tetracycline-containing liquid medium. These data are consistent with a role for the single 16S rRNA base mutation in clinical tetracycline resistance in cutaneous propionibacteria.     

大肠埃希菌16S rRNA甲基化酶基因的接合传递及其与整合子的相关性

目的 对临床分离的多重耐药（MDR）大肠埃希菌株的16S rRNA甲基化酶基因特征与接合传递效率进行研究,探讨其与整合子的相关性。 方法 136株MDR大肠埃希菌经PCR筛检16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC、rmtD;对阳性菌株作整合酶基因intI1、intI2和intI3检测,并扩增Ⅰ类整合子可变区插入片段,对扩增产物进行测序与鉴定所含耐药基因盒;以阳性菌株为供体菌,耐叠氮化钠大肠埃希菌J53为受体菌进行接合试验,并结合质粒图谱对16S rRNA甲基化酶基因进行初步定位。 结果 在136株多重耐药大肠埃希菌中,共检出16S rRNA甲基化酶阳性菌12株（8.8%）,其中,armA阳性3株（2.2%）,rmtB阳性10株（7.4%）,未检出rmtA、rmtC、rmtD基因。阳性菌株均只含Ⅰ类整合子,对其可变区扩增片段（1 000～2 300 bp）的测序结果显示,该区域含有多种耐药基因盒,但不含16S rRNA甲基化酶基因。接合试验与质粒图谱结果初步表明armA和rmtB编码基因位于约23 000 bp的质粒上,接合试验的耐药质粒传递率高达83.3%(10/12)。 结论 在MDR大肠埃希菌中,armA和rmtB编码基因位于约23 000 bp质粒上,其中,rmtB为优势基因,接合试验和质粒图谱证明该类耐药质粒很容易在同种菌间传播。Ⅰ类整合子与16S rRNA甲基化酶基因虽然存在于同一菌体内和/或同处于一个质粒上,但整合子基因盒对该类基因的捕获率很低或根本不捕获。     

Comparison of pulsed-field gel electrophoresis (PFGE) and two different polymerase chain reactions (PCRs) for species identification of Borrelia burgdorferi sensu lato strains

The purpose of the present study was to compare the findings of three different molecular-biological methods for Borrelia burgdorferi sensu lato species identification: (i) large DNA fragments pattern obtained with Mlul restriction endonuclease and separated with pulsed-field gel electrophoresis (PFGE); (ii) polymerase chain reaction (PCR), the region inside the 16S rRNA gene multiplied with species-specific primers; and (iii) PCR, the interspace region between the 5S and 23S rRNA genes amplified, the PCR product restricted with Msel restriction endonuclease and fragments separated in polyacrylamide gel. Forty-eight Borrelia strains isolated from diverse clinical materials and two tick strains were analyzed. Each of the 50 isolates analyzed by PFGE was found to be a single species: 30 B. afzelii, 14 B. garinii, and 6 B. burgdorferi sensu stricto. PCR amplification of 16S rRNA with species-specific primers revealed a single species in 41/50 samples and in nine samples two species were detected. PCR of the 5S-23S interspace region restricted with MseI restriction endonuclease detected a single species in 48/50 samples and a mixture of two species was found in 2/50 samples. In all cases where a single species was identified using PCR the species was in accordance with the PFGE result, and in all cases where a mixture of two species was identified by PCR one of the species was the same as that detected by PFGE. Using a criterion of complete concordance of the results a significant difference in species identification was found when PFGE and the 16S rRNA PCR were compared (p = 0.0026), but not between 5S-23S interspace PCR and PFGE (p = 0.4949) or between 16S rRNA and 5S-23S interspace PCRs (p = 0.0552). PCR assays were faster and easier to perform than PFGE for Borrelia species identification, however PFGE remains a standard procedure for analyzing isolates and demonstrating heterogeneity within species.     

导致阿米卡星双圈耐药肠杆菌科细菌耐药性研究

目的探讨临床分离的纸片扩散法药物敏感性试验对阿米卡星（AMK）呈双圈耐药的肠杆菌科细菌相关携带基因及药物诱导对耐药基因rnRNA表达量的影响。方法收集纸片扩散法药物敏感性试验对AMK呈双圈耐药的大肠埃希菌（编号Ec085）和肺炎克雷伯菌（编号Kpn110）各1株;纸片诱导法探讨耐药表型的改变;聚合酶链反应（PCR）扩增氨基糖苷类修饰酶基因、整合子及其可变区以及16SrRNA甲基化酶基㈥;逆转录PCR分析armA基因在AMK诱导前后菌株中的表达情况;接合试验探讨耐药基因的可传递性。结果Ec085和Kpn110分别在第10代和第16代诱导后转变为双圈消失;2株菌均扩增出I类整合子基因,可变区分别携带aadA5-dfra17和aadA2-dfrA12基因盒,未扩增出Ⅱ和Ⅲ类整合子基因;Ec085扩增出aac（6）-I基因,而Kpnll0扩增出ant（3”）-I基因;Ec085和Kpn110均扩增出armA基因,基因测序未发现突变,未检出rratB基因;经AMK诱导后菌株armA基因mRNA表达量明显上升;接合试验未获成功。结论大肠埃希菌和肺炎克雷伯茵对AMK呈双圈耐药可能与16SrRNA甲基化酶armA基因的诱导表达相关。     

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.     

Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance

Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL(3+), and M. smegmatis rrnB rpsL(3+). M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL(3+) is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL(3+) carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL(3+). Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G-->C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.     

Mutations in ribosomal protein L16 and in 23S rRNA in Enterococcus strains for which evernimicin MICs differ

Mutations in ribosomal protein L16 and in 23S rRNA were investigated in 22 Enterococcus strains of different species and for which the MICs of evernimicin differ (MICs, 0.023 to 16 micro g/ml). Amino acid changes (Arg56His, Ile52Thr, or Arg51His) in protein L16 were found in seven strains, and a nucleotide G2535A mutation in 23S rRNA was found in 1 strain among 13 for which the MICs are > or =1 micro g/ml.