Validation of PCR for the detection of Pseudomonas aeruginosa from corneal samples

AIM: To determine a species-specific real-time polymerase chain reaction (PCR) assay to detect Pseudomonas aeruginosa (PA),a secondary DNA target for PA that may provide a universal target for other bacterial pathogens,and validate both assays for diagnostic testing.METHODS: PCR detection was established against the ecfX PA gene and the 16S rRNA gene using known PA keratitis isolates.The outcome parameters for both assays were 'limit of detection' (LOD),amplification efficiency (AE),and PAGE amplified product analysis.Both assays were validated against 20 true-positive clinical samples positive for PA DNA and 20 true-negative samples containing no PA DNA.Descriptive statistics and PAGE analysis were used as outcome parameters.RESULTS: AE of the ecfX assay was 96.6%,and LOD was 33.6 copies of target DNA per microliter.AE of the 16S rRNA assay was 103.4%,and LOD was 8.12 copies per microliter.The sensitivity,specificity,positive predictive value,negative predictive value,and efficiency for the ecfX and 16S rRNA assays were [75%,95%,94%,79%,and 85%],and [70%,100%,100%,77%,and 85%],respectively.Both PCR assays were validated,followed by confirmation of DNA patterns from PAGE analysis.CONCLUSION: The PCR methodology described here may be a useful adjunct to standard methods in the diagnosis of PA keratitis.

根据16SrRNA序列对假单胞菌属分类学的研究进展

假单胞菌属是植物病原细菌中一个重要的属 ,而 1 6SrRNA序列分析是当前细菌分类中最精确的一种技术 ,应用这一技术对假单胞菌属进行分类有助于解决目前假单胞菌属分类的混乱 ,弄清假单胞菌属的系统进化关系。

Polyphyly in 16S rRNA-based LVTree Versus Monophyly in Whole-genome-based CVTree

We report an important but long-overlooked manifestation of low-resolution power of 16S rRNA sequence analysis at the species level, namely, in 16S rRNA-based phylogenetic trees polyphyletic placements of closely-related species are abundant compared to those in genomebased phylogeny. This phenomenon makes the demarcation of genera within many families ambiguous in the 16S rRNA-based taxonomy. In this study, we reconstructed phylogenetic relationship for more than ten thousand prokaryote genomes using the CVTree method, which is based on wholegenome information. And many such genera, which are polyphyletic in 16S rRNA-based trees, are well resolved as monophyletic clusters by CVTree. We believe that with genome sequencing of prokaryotes becoming a commonplace, genome-based phylogeny is doomed to play a definitive role in the construction of a natural and objective taxonomy.

Neomicrocoleus gen.nov.(Microcoleaceae,Oscillatoriales),a novel cyanobacterial genus from benthic mats in a water channel

A new filamentous cyanobacterial strain(CHAB 4127)was successfully isolated from the algal mat during the field investigation of the open channel from Luanhe River to Tianjin,China.The polyphasic approach combining morphological,ultrastructural,ecological,and molecular features was used to characterize this studied strain.The strain is morphologically similar to the Microcoleus-like cyanobacterial taxa under light microscopy,and the radial arrangement of thylakoids is also consistent with that of Microcoleus-like groups.The phylogenetic position of CHAB 4127 based on 16S rRNA gene sequences,is shown to be clearly clustered into an independent clade with the newly established genus Microcoleusiopsis.The maximum similarity of 16S r RNA gene of the studied strain with other existing related cyanobacterial taxa is 93.97%,and the ITS secondary structures is also obviously different from other members of Microcoleaceae.Based on all the above evidence,we proposed the establishment of this novel cyanobacterial genus as Neomicrocoleus,with its type species,Neomicrocoleus tianjinensis.

一株草鱼源荧光假单胞菌的分离鉴定及其耐药特性分析

为探究临沂市某草鱼养殖场发生病害的原因,从患病的草鱼(Ctenopharyngodon idellus)肝脏和肾脏混合组织中分离得到一株优势致病菌G-13,通过细菌形态学观察、革兰氏染色、生理生化特征、16S rRNA基因测序,以及系统发育分析等完成菌株鉴定,并测定其耐药特性。形态特征显示G-13是一种短杆状的革兰氏阴性菌,在LB固体平板上形成乳白色、圆形、整体呈隆起状、边缘较为整齐、表面光滑湿润的菌落;16S rRNA系统发育进化分析发现G-13与荧光假单胞菌(Pseudomonas fluorescens)聚在一支,序列同源性高达100%;生理生化试验显示葡萄糖、精氨酸双水解酶、鸟氨酸脱羧酶、赖氨酸脱羧酶、氧化酶、无盐和3%NaCl胰胨水等7项指标为阳性,与假单胞菌属特征相近,确定分离株G-13为P.fluorescens。药敏试验结果显示菌株G-13对丁胺卡那、多西环素、四环素、新霉素、环丙沙星、氧氟沙星等13种抗生素敏感,对氨苄西林、头孢拉定、克林霉素、呋喃唑酮等7种抗生素耐药。

水蛭来源荧光假单胞菌的分离鉴定及系统发育树分析

为调查山东某水蛭养殖场病情暴发原因,从该场患病水蛭中分离得到优势菌株SZ-1,对其进行生理生化特性及分子生物学分析。形态学、生理生化分析显示,菌株SZ-1为革兰氏阴性菌,杆状,两端钝圆,有鞭毛;Biolog GenIII分析表明,该菌株生长代谢利用情况与假单胞菌属吻合,与荧光假单胞菌B特征相似。基于16S rRNA基因序列分析同源性,该菌株16S rRNA基因序列与荧光假单胞菌(Pseudomonas fluorescens)相聚类,聚合置信度可达99%。综合生物学及分子特征,判定SZ-1为荧光假单胞菌。

A new species of the genus Theloderma Tschudi, 1838 (Amphibia： Anura： Rhacophoridae) from Tay Nguyen Plateau, central Vietnam

A new species of small tree frog from a primary montane tropical forest of central Vietnam, Tay Nguyen Plateau, is described based on morphological, molecular, and acoustic evidence. The Golden Bug-Eyed Frog, Theloderma auratum sp. nov., is distinguishable from its congeners and other small rhacophorid species based on a combination of the following morphological attributes： （1） bony ridges on head absent; （2） smooth skin completely lacking calcified warts or asperities; （3） pointed elongated tapering snout; （4） vocal opening in males absent; （5） vomerine teeth absent; （6） males of small body size （SVL 21.8-26.4 mm）; （7） head longer than wide; ED/SVL ratio 13%-15%; ESL/SVL ratio 16%-20%; （8） small tympanum （TD/EL ratio 50%-60%） with few tiny tubercles; （9） supratympanic fold absent; （10） ventral surfaces completely smooth; （11 ） webbing between fingers absent; （12） outer and inner metacarpal tubercles present, supernumerary metacarpal tubercle single, medial, oval in shape; （13） toes half-webbed： I 2-21/4 II 11/^-23/~ III 2-31/4 IV 3-11/2 V; （14） inner metatarsal tubercle present, oval; outer metatarsal tubercle absent; （15） iris bicolored; （16） dorsal surfaces golden-yellow with sparse golden-orange speckling or reticulations and few small dark-brown spots; （17） lateral sides of head and body with wide dark reddish-brown to black lateral stripes, clearlyseparated from lighter dorsal coloration by straight contrasting edge; （18） ventral surfaces of body, throat, and chest greyish-blue with indistinct brown confluent blotches; （19） upper eyelids with few （3-5） very small flat reddish superciliary tubercles; （20） limbs dorsally reddish-brown, ventrally brown with small bluish-white speckles. The new species is also distinct from all congeners in 12S rRNA to 16S rRNA mitochondrial DNA fragment sequences （uncorrected genetic distance P〉8.9%）. Advertisement call and tadpole morphology of the new species are described. Our molecular data showed Theloderma auratum sp. nov. to be a sister species of Th. palliatum from Langbian Plateau in southern Vietnam.

Description of two new species of Pseudoaliinostoc(Nostocales,Cyanobacteria)from China based on the polyphasic approach

Two cyanobacterial strains CHAB5870 and CHAB5871 morphologically identifi ed as Nostoc-like species were isolated from different habitats in China,and they were phylogenetically and taxonomically characterized based on a polyphasic approach combining morphological,ecological,and molecular data.In the 16S rRNA gene phylogeny inferred using maximum likelihood,maximum-parismony,and bayesian inference methods,these two strains clustered within the Pseudoaliinostoc clade.The 16S rRNA gene sequences of these two strains displayed≥95.5%and≤98%similarity to Pseudoaliinostoc species,which indicated them to represent new species of the genus Pseudoaliinostoc.Furthermore,the unique pattern of D1-D1′and Box-B helix of the 16S–23S rRNA internal transcribed spacer(ITS)secondary structure also revealed that two strains represented novel species.These results supported the establishment of two new Pseudoaliinostoc species with the name P.jiangxiense sp.nov.and P.yunnanense sp.nov.

Composition and Molecular Identification of Bacterial Community in Seawater Desalination Plants

Biofouling is an important problem for reverse osmosis (RO) membrane manufacturers. Bacteria are mainly involved in generating fouling and obturating RO membranes. Insights into biofilm bacteria composition could help prevent biofouling, reduce the cost of using RO-fouling membranes and guarantee safe water. Culture-dependent and independent techniques were then performed in order to identify bacteria associated with RO membranes. Bacteria cultures described the presence of six pure colonies, four of which were identified through API testing. Based on 16s rRNA gene analysis, a predominant bacterium was identified and annotated as Sphingomonas sp. The 16s rRNA gene clone library, on the other hand, showed that the bacterium, Pseudomonas marincola, accounted for nearly 30% of the clone library, while the rest of bacteria were chimeras (62%) and non-representative species (3%). In conclusion, culture-dependent and independent approaches showed that two dominant bacteria were commonly observed in RO desalination membranes.

鸡源铜绿假单胞菌氨基糖苷类耐药特性研究

为了解铜绿假单胞菌(PA)的氨基糖苷类耐药机制,用纸片扩散法检测分离自鸡场的50株PA对6种氨基糖苷类药物的敏感性,用PCR法检测7种氨基糖苷类修饰酶(AMEs)基因和5种16 S rRNA甲基化酶基因。结果显示:50株PA中45株对氨基糖苷类药物耐药,36株表现出多种氨基糖苷类药物耐药。50株菌中共有45株检测到耐氨基糖苷类药物基因,总检出率为90.0%。AMEs基因aac(3)-Ⅱ、ant(3′′)-Ⅰ、aph(3′′)-Ⅰb、aph(6′)-Ⅰd、aac(6′)-Ⅰb、ant(2′′)-Ⅰ的阳性率分别为88.0%、50.0%、44.0%、24.0%、20.0%、4.0%,16 S rRNA甲基化酶armA基因的阳性率为2.0%,其余5种基因均未检出。提示PA对氨基糖苷类耐药与产AMEs和16 S rRNA甲基化酶有关。

A new species of Crocodile Newt, genus Tylototriton(Amphibia, Caudata, Salamandridae) from the mountains of Kachin State, northern Myanmar

We describe a new species of the genus Tylototriton from Ingyin Taung Mt.,Mohnyin Township,Kachin State,Myanmar,based on morphological and molecular evidence.The new species is assigned to the subgenus Tylototriton s.str.and is clearly distinct from all known congeners by the following characters:medium body size;thin,long tail,lacking lateral grooves;rough skin;truncate snout;wide,protruding supratemporal bony ridges on head,beginning at anterior corner of orbit;weak,almost indistinct sagittal ridge;long,thin limbs,broadly overlapping when adpressed along body;distinct,wide,non-segmented vertebral ridge;13 or 14 rib nodules;brown to darkbrown background coloration with dull orange-brown to yellowish-brown markings on labial regions,parotoids,rib nodules,whole limbs,vent,and ventral tail ridge.We also briefly discuss biogeography and species diversity of the genus Tylototriton in Myanmar.

基于16 S rRNA序列研究华重楼植株可培养内生细菌的多样性

目的分析华重楼Paris polyphylla健株和茎腐病感病植株内生可培养内生细菌的多样性。方法用牛肉膏蛋白胨培养基分离华重楼茎腐病及健康植株根茎、茎、叶等不同部位的细菌,采用16 S rRNA通用引物27F/1492R进行PCR扩增结合DNA测序技术,对分离自华重楼健株及茎腐病植株不同部位的细菌进行初步鉴定。结果从华重楼的健康植株和感病植株中分离到11属23种细菌。从健株分离到4属11种内生细菌,其中根茎、茎、叶分别分离到9、10和5种内生细菌。从病株分离得到10属14种内生细菌,其中根茎、茎、叶分别分离到11、8和3种内生细菌。华重楼健株的根茎部内生细菌量最高,达2.999×105 cfu/g,叶部内生细菌量较低,为7.32×104 cfu/g,华重楼健株根茎、茎、叶中的芽孢杆菌量最高,比例分别为73.3%、67.1%和81.8%。华重楼病株的茎部内生细菌量最高,达2.817×105 cfu/g,叶部内生细菌量较低,为2.76×104 cfu/g,华重楼病株的根茎、茎、叶部的假单胞菌量最高,比例分别为35.6%、50.3%和60.5%。华重楼健株不同部位的多样性指数和均匀度指数均高于病株。结论华重楼健康植株以芽孢杆菌为优势细菌类群,华重楼茎腐病植株以假单胞菌为优势细菌类群。华重楼健株相比病株具有更为丰富的内生细菌群落多样性。

Prokaryote phylogeny meets taxonomy: An exhaustive comparison of composition vector trees with systematic bacteriology

We perform an exhaustive, taxon by taxon, comparison of the branchings in the composition vector trees (CVTrees) inferred from 432 prokaryotic genomes available on 31 December 2006, with the bacte-riologists' taxonomy-primarily the latest online Outline of the Bergey's Manual of Systematic Bacteri-ology. The CVTree phylogeny agrees very well with the Bergey's taxonomy in majority of fine branchings and overall structures. At the same time most of the differences between the trees and the Manual have been known to biologists to some extent and may hint at taxonomic revisions. Instead of demonstrating the overwhelming agreement this paper puts emphasis on the biological implications of the differences.

On monospecific genera in prokaryotic taxonomy

A monospecific genus contains a single species ever since it was proposed.Though formally more than half of the known prokaryotic genera are monospecific,we pick up those which actually raise taxonomic problems by violating monophyly of the taxon within which it resides.Taking monophyly as a guiding principle,our arguments are based on simultaneous support from 16S rRNA sequence analysis and whole-genome phylogeny of prokaryotes,as provided by the LVTree Viewer and CVTree Web Server,respectively.The main purpose of this study consists in calling attention to this specific way of global taxonomic analysis.Therefore,we refrain from making formal emendations for the time being.