嗜麦芽寡养单胞菌ERIC基因分型方法的应用及意义评估

目的:探讨嗜麦芽寡养单胞菌基因分型情况,并结合临床资料对分型方法及意义进行评估.方法:应用重复序列PCR(ERlC-PCR)析方法进行分型,以细菌耐药鉴定系统及改良K-B法进行药敏试验.结果:23株嗜麦芽寡养单胞菌对亚胺培南、氨曲南、头孢噻肟、阿米卡星高度耐药.但对头孢哌酮/舒巴坦、左氧诺氟沙星、复方新诺明敏感率较高.ERIC将嗜麦芽寡养单胞菌分为5型,其中A型10株、B型7株、C型3株、D型2株、E型1株.结论:ERIC-PCR能有效地对嗜麦芽寡养单胞菌进行分型.临床上嗜麦芽寡养单胞菌耐药程度严重,有多种基因型并存,与耐药程度有相关性.     

63株嗜麦芽寡养单胞菌的临床分布与耐药性分析

目的了解嗜麦芽寡养单胞菌的临床分布及对抗菌药物的耐药性。方法采用VITEK-2 COMPACT全自动微生物鉴定仪及纸片扩散(K-B)法对分离的63株嗜麦芽寡养单胞菌的来源分布、药物敏感性结果进行统计分析。结果 63株嗜麦芽寡养单胞菌在临床标本中分布以痰液为主,占87.3%;感染主要发生在新生儿科为17.4%和胸外科17.4%;其次为呼吸科14.3%和重症监护病房(ICU)14.3%;嗜麦芽寡养单胞菌对亚胺培南耐药率为100.0%;而对左氧氟沙星、米诺环素、磺胺甲口恶唑-甲氧苄啶耐药率较低分别为1.6%、3.2%、3.2%。结论应加强对嗜麦芽寡养单胞菌耐药性监测,以指导临床根据药物敏感性试验结果合理选择抗菌药物。     

嗜麦芽寡养单胞菌的耐药性分析及临床分布

对我院2007年1月～2011年6月收治的患者中临床分离的898株嗜麦芽寡养单胞菌进行标本来源、临床科室分布以及耐药性进行分析。结果 :不同科室患者分离出嗜麦芽寡养单胞菌的菌株数有明显的差异性(P<0.05)。对菌落的耐药性研究发现,嗜麦芽寡养单胞菌对多种抗菌药物均产生较大的耐药性,而对磺胺甲噁唑/甲氧苄啶等耐药性较差。     

54株嗜麦芽寡养单胞菌的临床分布及耐药分析

目的调查该院嗜麦芽寡养单胞菌的临床分布和耐药状况。方法回顾性分析54株嗜麦芽寡养单胞菌的标本来源、科室分布及耐药情况。结果 54株嗜麦芽寡养单胞菌主要来源于下呼吸道分泌物,占77.8;其临床分布以重症监护病房为主,占55.6。药敏结果显示,嗜麦芽寡养单胞菌对亚胺培南100耐药,对氨曲南和哌拉西林高度耐药(耐药率75),但对复方新诺明、左氧氟沙星、头孢哌酮/舒巴坦、阿米卡星、哌拉西林/他唑巴坦仍保持一定的敏感性,耐药率30。结论嗜麦芽寡养单胞菌的耐药性严重,加强耐药性监测、合理应用抗菌药物十分重要。     

285株嗜麦芽寡养单胞菌临床分布特点及耐药性分析

目的 研究该院嗜麦芽寡养单胞菌临床分离株的分布特点及耐药性,为临床合理用药及感染防控提供依据.方法 回顾性分析2009～2011年嗜麦芽寡养单胞菌培养、鉴定及药敏试验结果.结果 共分离获得285株嗜麦芽寡养单胞菌,87.72%分离自痰液标本,90.18%来自ICU、脑外科、呼吸科及老年病科;对米诺环素、左氧氟沙星、复方新诺明的耐药率分别为1.05%、93.33%和5.61%.结论 嗜麦芽寡养单胞菌极易导致呼吸道感染,米诺环素和复方新诺明对嗜麦芽寡养单胞菌有较高抗菌活性;应依据药敏试验结果优化选择抗菌药物,提高感染治愈率.     

210株嗜麦芽寡养单胞菌的临床分布与耐药性分析

目的 了解医院嗜麦芽寡养单胞菌临床分布情况及其对常用抗菌药物的耐药率,为临床合理使用抗菌药物提供依据.方法 回顾分析医院2006年1月至2009年12月检出的210株嗜麦芽寡养单胞菌采用纸片扩散法得出的药敏结果资料.结果 210株嗜麦芽寡养单胞菌在临床标本中分布以痰液为主,占80.0%.感染主要发生在重症监护病房、呼吸...     

54株分枝杆菌国际参考株16S rRNA序列分析

目的分析54株分枝杆菌国际参考株16S rRNA序列,为临床分离株的鉴定提供参考。方法用16S rRNA序列分析法对54株分枝杆菌国际参考株进行分析,构建系统发育树,计算相似性百分比。结果除11株（共9种4组）分枝杆菌,即产鼻疽分枝杆菌与塞内加尔分枝杆菌;溃疡分枝杆菌与海分枝杆菌;堪萨斯与胃分枝杆菌;3株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌16S rRNA基因序列完全相同无法相互鉴别,其它41株（共41种）分枝杆菌菌种间16S rRNA均不相同可以得到很好的鉴别。结论 16S rRNA基因序列分析是一种很好的鉴定分枝杆菌的方法,国际参考株16S rRNA基因序列的研究弥补了基因数据库的不足。     

54株分枝杆菌国际参考株16S rRNA序列分析

目的分析54株分枝杆菌国际参考株16S rRNA序列,为临床分离株的鉴定提供参考。方法用16S rRNA序列分析法对54株分枝杆菌国际参考株进行分析,构建系统发育树,计算相似性百分比。结果除11株(共9种4组)分枝杆菌,即产鼻疽分枝杆菌与塞内加尔分枝杆菌;溃疡分枝杆菌与海分枝杆菌;堪萨斯与胃分枝杆菌;3株结核分枝杆菌与田鼠分枝杆菌、非洲分枝杆菌16S rRNA基因序列完全相同无法相互鉴别,其它41株(共41种)分枝杆菌菌种间16S rRNA均不相同可以得到很好的鉴别。结论 16S rRNA基因序列分析是一种很好的鉴定分枝杆菌的方法,国际参考株16S rRNA基因序列的研究弥补了基因数据库的不足。     

139株嗜麦芽寡养单胞菌的临床分布及耐药性分析

目的了解嗜麦芽寡养单胞菌的标本来源、临床分布及耐药状况。方法细菌培养和鉴定严格按照《全国临床检验操作规程》进行;检测139株嗜麦芽寡养单胞菌对临床常用18种抗菌药物的敏感性。结果嗜麦芽寡养单胞菌主要来源于痰标本,占41.01%(57/139);科室分布主要分离自重症监护室(ICU)(33.09%,46/139)和呼吸内科(27.34%,38/139)。该菌对米诺环素、复方新诺明、左氧氟沙星的耐药率较低,分别为15.11%、16.55%、19.42%,对其他抗菌药物的耐药率较高。结论嗜麦芽寡养单胞菌具有高度和多重耐药性,临床抗感染治疗应根据药敏试验结果选用敏感药物。     

嗜麦芽寡养单胞菌的医院感染特征及耐药性分析

目的 了解嗜麦芽寡养单胞菌的医院感染特征及耐药状况,为临床合理用药提供依据.方法 分析184株嗜麦芽寡养单胞菌的标本来源、感染科室分布及耐药状况.结果 嗜麦芽寡养单胞菌痰液检出率最高;临床分布以呼吸科病区和重症监护病区最多;耐药率较低的抗菌药物为复方磺胺、哌拉西林/他唑巴坦、左旋氧氟沙星、莫西沙星、多黏菌素;而亚胺培南、美罗培南耐药率最高;其中62株合并了其他病原菌的感染.结论 嗜麦芽寡养单胞菌具有高度和多重耐药性,临床应重视抗菌药物的合理使用,积极预防院内感染,以提高疗效和减缓多重耐药株的产生.     

Beta-lactam resistance and beta-lactamase expression in clinical Stenotrophomonas maltophilia isolates having defined phylogenetic relationships

AIMS: To test the hypothesis that Stenotrophomonas maltophilia isolates from certain phylogenetic groups have predictable beta-lactamase expression and beta-lactam resistance profiles. METHODS: Isolates were grouped using sequences of the 16S rRNA gene and smeT-smeD intergenic region. beta-Lactamase activities in cell extracts were quantified spectrophotometrically and beta-lactam MICs were determined using agar dilution methodology and Etest as appropriate. RESULTS: A collection of 50 clinical S. maltophilia isolates from Europe and North, South and Central America were phylogenetically grouped. Group 'A' (22 out of 50) includes remarkably genetically homogeneous isolates; group 'B' (17 out of 50) includes isolates that are genetically heterogeneous and quite distinct from those of group A. Members of these two groups are, however, indistinguishable in terms of their beta-lactam resistance and beta-lactamase expression phenotypes. In contrast, isolates from group 'C', which are less common (8 out of 50), are considerably more susceptible to beta-lactams owing to reduced inducibility of beta-lactamase expression following beta-lactam challenge. CONCLUSIONS: The majority of S. maltophilia clinical isolates behave similarly in terms of beta-lactamase expression and beta-lactam resistance properties, despite considerable phylogenetic variability.     

The sigH gene sequence can subspeciate staphylococci

In an evolutionarily conserved gene organization (syntenic region), the sigH gene shares exceptionally low homology among staphylococcal species. We analyzed the "positionally cloned" sigH sequences of 39 staphylococcal species. The topology of the SigH phylogenetic tree was consistent with that of 16S rRNA. Certain clinical isolates were successfully differentiated at the species level with the sigH sequence data set. We propose that the sigH gene is a promising molecular target in genotypic identification because it is highly discriminative in differentiating closely related staphylococcal species.     

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。     

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检验进行基因多态性分析。 用MEGA 6.06软件的邻接距离矩阵法对90株测试菌株和54株模式菌株构建16S rRNA 和gyrB 基因的进化树。 用MEGA 6.06软件的Kimura’s 2-parameter模型,确定90株测试菌株的菌种后,计算16S rRNA 和gyrB 基因的遗传距离和序列相似性。 比较两者对施万菌种水平鉴定能力差异。   结果   16S rRNA和gyrB基因序列相似性平均值分别为95.0%、80.8%。 在16S rRNA基因进化树中,S. marinintestina和S. sairae、S. livingstonensis和S. vesiculosa的进化分支几乎完全相同,gyrB基因则能在种水平将所有菌株区分开。 16S rRNA基因的种内和种间相似性范围小。   结论   与16S rRNA基因相比,gyrB基因能够更准确的用于施万菌的种水平鉴定。     

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.     

玫瑰单胞菌一株的鉴定及分析

目的 确定临床脓液标本中1株革兰阴性球杆菌K8756的分类学位置.方法 抽取患者深部脓肿物,置于Amies培养基室温保存、运输.脓液标本分区划线接种血平板、巧克力平板,置于35 ℃含5%CO2培养箱.采用API、Vitek2细菌鉴定系统,结合传统形态学检查、手工生化实验对分离株进行鉴定.从纯培养物提取脂肪酸、甲基化,采用气相色谱仪分析脂肪酸成分.PCR扩增16SrRNA基因并测序,对所测得的核酸序列进行同源性比对及系统发育分析.结果 血平板、巧克力平板分离到1株缓慢生长的革兰阴性球杆菌K8756.API 20NE生化编码为1245045(72 h),提示为人苍白杆菌;Vitek2 GN鉴定卡重复实验3次,提示为皮氏罗尔斯顿菌或支气管炎鲍特菌.但基于16SrRNA基因序列的系统发育分析表明,菌株K8756属于玫瑰单胞菌的成员,与该属的合格发表种黏液玫瑰单胞菌MDA5527T核酸匹配度高达100%(菌株K8756的GenBank登录号为GU207841).其菌落形态、表型特征及主要细胞脂肪酸成分亦与黏液玫瑰单胞菌相似.结论 菌株K8756(=GIMCC 1.0030)在分类学上属于黏液玫瑰单胞菌.16S rRNA基因序列分析是鉴定临床疑难菌株及新发现菌株的重要手段.

Abstract：

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria.     

Staphylococcus pseudolugdunensis sp. nov., a pyrrolidonyl arylamidase/ornithine decarboxylase-positive bacterium isolated from blood cultures

Twelve coagulase-negative staphylococcal isolates recovered from blood cultures with positive pyrrolidonyl arylamidase and ornithine decarboxylase reactions were identified as Staphylococcus lugdunensis by the clinical microbiology laboratory. However, none of these 12 isolates were recognized by a S. lugdunensis translation elongation factor Tu (tuf) gene-specific probe. Under the API STAPH V4.0 identification system (bioMérieux, Durham, NC), 8 of these 12 isolates could not be identified with low discrimination scores, and 4 were identified as Kocuria varians/rosea with identification probabilities that ranged from 95.5% to 99.6%. All 12 isolates possessed identical partial 16S rRNA gene sequences, and the full 16S rRNA gene sequences of the prototype strain B006 were closely related to a tentatively assigned “Staphylococcus pettenkoferi”. All 12 isolates had identical partial tuf gene sequences corresponding to 666 to 858 nucleotide positions, and the 1188-base pair full tuf sequences of B006 strain were mostly related to 2 Staphylococcus epidermidis isolates with a 93.02% similarity. Two isolates, which were recovered from the same patient over a 16-day interval, were considered to be a pathogen causing an intravenous line-associated infection; the remaining 10 isolates were considered to be skin contaminants. Biochemical tests currently used in the clinical microbiology laboratory to identify S. lugdunensis appear to lack specificity in identifying these isolates. On the basis of the close biochemical reactions with S. lugdunensis and phylogenetic evidence, these isolates are proposed the designation Staphylococcus pseudolugdunensis sp. nov.     

富西亚分枝杆菌从血液与静脉插管中的分离与鉴定

目的对从同一患者血液和静脉插管中分离的两株抗酸阳性细菌B0793、V1948进行菌种鉴定。方法广谱PCR扩增细菌的16S核糖体核糖核酸(16SrRNA)基因与核糖核酸(RNA)聚合酶β亚单位(rpoB)基因,测序其核苷酸序列并与相关菌种进行同源性比对及系统发育分析,以传统的表型实验对基因鉴定的结果进行验证,并参考CLSI M24-A的方法和解释标准进行分离株抗菌药物的药物敏感性试验。结果该2株细菌均为血平板上生长缓慢的革兰染色着色较淡的抗酸阳性杆菌,但2株细菌的菌落形态差别较大,B793在血平板培养4d形成直径1cm、米黄色、圆形、突起、不溶血、易乳化的光滑型菌落,V948则为直径约2cm、米黄色、豆腐渣样、边缘不规则的粗糙型菌落。16SrRNA基因和rpoB基因序列与富西亚分枝杆菌(mycobacterium phocaicum)的同源性均在99%以上,其半定量触酶、68℃触酶试验、5%NaCl耐受试验与脲酶试验阴性,亦符合富亚分枝杆菌的特征。结论该2株抗酸阳性细菌在分类学上属于富西亚分枝杆菌,其光滑型/粗糙型菌落变异为国际上首次发现。     

Comparison between two PCR-based bacterial identification methods through artificial neural network data analysis

The 16S ribosomal ribonucleic acid (rRNA) and 16S-23S rRNA spacer region genes are commonly used as taxonomic and phylogenetic tools. In this study, two pairs of fluorescent-labeled primers for 16S rRNA genes and one pair of primers for 16S-23S rRNA spacer region genes were selected to amplify target sequences of 317 isolates from positive blood cultures. The polymerase chain reaction (PCR) products of both were then subjected to restriction fragment length polymorphism (RFLP) analysis by capillary electrophoresis after incomplete digestion by Hae III. For products of 16S rRNA genes, single-strand conformation polymorphism (SSCP) analysis was also performed directly. When the data were processed by artificial neural network (ANN), the accuracy of prediction based on 16S-23S rRNA spacer region gene RFLP data was much higher than that of prediction based on 16S rRNA gene SSCP analysis data (98.0% vs. 79.6%). This study proved that the utilization of ANN as a pattern recognition method was a valuable strategy to simplify bacterial identification when relatively complex data were encountered.