Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.     

Development of 16S rRNA-based probes for the identification of Gram-positive anaerobic cocci isolated from human clinical specimens

Fluorescent probes targeted at 16S rRNA were designed for Peptostreptococcus anaerobius and Peptostreptococcus stomatis (Pana134), Parvimonas micra (Pamic1435), Finegoldia magna (Fmag1250), Peptoniphilus asaccharolyticus (Pnasa1254), Peptoniphilus ivorii (Pnivo731), Peptoniphilus harei (Pnhar1466), Anaerococcus vaginalis (Avag1280) and Anaerococcus lactolyticus (Alac1438), based on the 16S rRNA sequences of reference strains and 88 randomly chosen clinical isolates. These strains were also used for validation of the probes. Application of the probes to an additional group of 100 clinical isolates revealed that 87% of Gram-positive anaerobic cocci (GPAC) could be identified with this set of probes. The 16S rRNAs of 13 clinical isolates that could not be identified were sequenced. Most of these isolates were GPAC that were not targeted by the probes. No clinical isolates of Pn. asaccharolyticus were encountered. Near full-length sequences were obtained from 71 of 101 (n = 88 + 13) sequenced clinical isolates. Of these, 25 showed <98% similarity with the homologues of the closest established species. The Fmag1250, Pamic1435, Pnhar1466, Pana134, Pnasa1254 and Pnivo731 probes allowed reliable identification and hybridised with all corresponding isolates. The Avag1280 and Alac1438 probes failed to hybridise with two isolates and one isolate, respectively, because of intra-species variation. However, overall, the set of probes yielded fast and reliable identification for the majority of clinical isolates.     

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background : Currently, increasing attention is being paid to the important role of in-testinal microbiome in diabetes. However, few studies have evaluated the characteris-tics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. Methods : In this study, a mini- pig diabetes model (DM) was established by 9- month high- fat diet (HFD) combined with low- dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini- pigs. Results : The results showed that microbial structure in diabetic mini- pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1 , which were positively correlated with diabetes, and decreases in levels of the bac-teria Rikenellaceae , Clostridiales_vadinBB60_group , and Bacteroidales_RF16_group , which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt- predicted pathways related to the glycolysis and Entner- Doudoroff super-pathway, enterobactin biosynthesis, and the L - tryptophan biosynthesis were signifi-cantly elevated in the DM group. Conclusion : These results reveal the composition and predictive functions of the in-testinal microbiome in the mini- pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini- pig diabetes model in gut microbiome research.     

Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.     

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.     

16SrRNA基因在快速诊断新生儿败血症的病原菌研究

目的探讨血液16SrRNA基因检测在新生儿败血症诊断中的应用价值。方法分析细菌16SrRNA基因保守区,设计一对通用引物扩增已知实验菌株,检测其特异性,用倍比稀释法检测其敏感性,同时进行血培养。结果已知实验菌株均获得920bp扩增产物,对照组中的人类基因组DNA、HBV—DNA和白色假丝酵母菌无相应产物。敏感性测试能达到lpg大肠杆菌DNA。PCR阳性率为31．7％（20／63）,血培养阳性率为14．3％（9／63）,两者比较有显著性差异（P〈0.05）。结论PCR检测血液细菌16SrRNA基因,具有特异性强,敏感度高等特点,能在临床推广应用。     

细菌直接PCR和双引物策略鉴定16S rRNA基因技术的建立

目的探索一种基于16S rRNA基因的快速鉴定细菌方法,为临床诊断治疗及耐药菌的分子遗传分析提供科学依据。方法对卫生部室间质评菌株和临床病人标本分离培养纯菌落,用双蒸水稀释菌落,然后直接以菌液为模板优化反应体系PCR扩增16S rRNA基因片段,再测序扩增片段。将测序结果在细菌Ribosomal数据库中进行同源性比对,根据序列同源性鉴定病原细菌。结果本实验鉴定的卫生部质评菌株结果与卫生部报告结果一致。本实验能够一次性鉴定出临床病人标本分离的菌株。结论本研究优化了一种免纯化细菌核酸直接PCR鉴定细菌16S rRNA基因的方法。本研究建立的基于病原细菌16S rRNA基因鉴定方法可用于细菌的快速诊断。     

Early prosthetic joint infection due to Ureaplasma urealyticum: Benefit of 16S rRNA gene sequence analysis for diagnosis

Detection of the most frequently bacteria involved in prosthetic joint infection (PJI) is usually performed by conventional cultures. We report a case of early PJI due to Ureaplasma urealyticum, diagnosed by 16S rRNA gene sequence analysis, which highlights the interest of molecular methods if fastidious bacteria are involved in PJI.     

Uncultured bacterial diversity in tropical maize (Zea mays L.) rhizosphere

Structure of maize (Zea mays L.) rhizosphere bacteria was evaluated to explore the feasibility of identifying novel rhizosphere bacteria using culture-independent method based on direct amplification and analysis of 16S rRNA gene (rRNA) sequences and especially to obtain a better understanding of bacterial community structure and diversity from maize. A total of 274 sequences were analyzed and assigned 48.00% Proteobacteria, 10.30% Actinobacteria, 9.90% Bacteroidetes, 6.60% Verrucomicrobia, 4.80% Acidobacteria, 1.80% Firmicutes, 1.50% Chloroflexi, 1.50% TM7, 1.10% Deinococcus-Thermus, 0.70% Planctomycetes, 0.70% Gemmatimonadetes and 0.40% Cyanobacteria. Economically important phyla Actinobacteria was second most dominant group after Proteobacteria, in our clone library. It would be interesting to hypothesize that root exudates from maize rhizosphere favors growth of Actinobacteria like microbes to eliminate pathogenic bacteria and decompose plant matter, for enhanced plant and soil health. An additional 12.8% of clone library (35 operational taxonomical units (OTUs) from 43 clones) with less than 94% similarity to any GenBank sequence could not be assigned to any known phylum and may represent unidentified bacterial lineages and suggests that a large amount of the rhizobacterial diversity remains to be characterized by culturing.     

同时产ArmA型16S rRNA甲基化酶及KPC-2型β-内酰胺酶泛耐药阴沟肠杆菌的研究

目的 探讨上海交通大学医学院附属瑞金医院分离的5株泛耐药阴沟肠杆菌产碳青霉烯酶及16S rRNA甲基化酶的情况及两者之间的相关性.方法 用E test法检测5株泛耐药阴沟肠杆菌对10种抗菌药物的MIC值.PCR扩增16S rRNA甲基化酶基因armA、mtA、rmtB、rmtC、rmtD、npmA,β-内酰胺酶基因TEM、SHV、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、CTX-M-25、PER-1、VEB-1.鸟枪克隆法克隆碳青霉烯酶基因,并对克隆片段进行测序.质粒接合试验验证碳青霉烯酶基因及16S rRNA甲基化酶基因是否具有转移性.脉冲场凝胶电泳(PFGE)法对5株菌进行分子分型.等电聚焦电泳法检测β-内酰胺酶等电点.Southern杂交法对耐药决定因子进行定位.结果 5株阴沟肠杆菌对10种抗菌药物的MIC值均＞32 mg/L.克隆的碳青霉稀酶基因为blaKPC-2,酶编码基因上游为一转座酶基因,两侧为复制靶位,下游为ISKpn6的插入序列,该序列包括一个重复序列和tnpA转座子,酶编码基因位于54.2 kb的一个非接合性大质粒上.等电聚焦电泳显示5株菌均产4种β-内酰胺酶(TEM-1,pI5.4;KPC-2,pI6.7;SHV-12,pI8.2;CTX-M-14,pI8.4).16S rRNA甲基化酶基因位于接合性质粒上,而blaKPC-2基因则位于非接合性质粒上,5株菌PFGE型别一致.结论 5株泛耐药阴沟肠杆菌对碳青霉烯类耐药由产碳青霉烯酶KPC-2所介导.blaKPC-2与armA型16S rRNA甲基化酶基因由两条不同的质粒编码,不存在相关性.临床医院应加强监控,以防止交叉感染.     

The interpersonal and intrapersonal diversity of human-associated microbiota in key body sites

The human body harbors 10 to 100 trillion microbes, mainly bacteria in our gut, which greatly outnumber our own human cells. This bacterial assemblage, referred to as the human microbiota, plays a fundamental role in our well-being. Deviations from healthy microbial compositions (dysbiosis) have been linked with important human diseases, including inflammation-linked disorders, such as allergies, obesity, and inflammatory bowel disease. Characterizing the temporal variations and community membership of the healthy human microbiome is critical to accurately identify the significant deviations from normality that could be associated with disease states. However, the diversity of the human microbiome varies between body sites, between patients, and over time. Environmental differences have also been shown to play a role in shaping the human microbiome in different cultures, requiring that the healthy human microbiome be characterized across life spans, ethnicities, nationalities, cultures, and geographic locales. In this article we summarize our knowledge on the microbial composition of the 5 best-characterized body sites (gut, skin, oral, airways, and vagina), focusing on interpersonal and intrapersonal variations and our current understanding of the sources of this variation.     

Effect of comprehensive validation of the template isolation procedure on the reliability of bacteraemia detection by a 16S rRNA gene PCR

The influence of the DNA extraction method on the sensitivity and specificity of bacteraemia detection by a 16S rRNA gene PCR assay was investigated. The detection limit of the assay was 5 fg with purified DNA from Escherichia coli or Staphylococcus aureus, corresponding to one bacterial cell. However, with spiked blood samples, the detection limits were 10(4) and 10(6) CFU/mL, respectively. The sensitivity of the S. aureus assay was improved to the level of the E. coli test with the addition of proteinase K to the commercial DNA extraction kit protocol. Ten (16.6%) of 60 amplification reactions were positive with templates isolated from sterile blood, while PCR reagent controls were negative, thereby indicating contamination during the DNA extraction process. Blood samples were spiked with serial dilutions of E. coli and S. aureus cells, and six PCR results were obtained from three extractions for each blood sample. A classification threshold system was devised, based on the number of positive reactions for each sample. Samples were deemed positive if at least four positive reactions were recorded, making it possible to avoid false-positive results caused by contamination. These results indicate that a comprehensive validation procedure covering all aspects of the assay, including DNA extraction, can improve considerably the validity of PCR assays for bacteraemia, and is a prerequisite for the meaningful detection of bacteraemia by PCR in the clinical setting.     

椰毒假单胞菌部分16SrRNA序列测定及其系统发育地位

采用聚合酶链反应（ＰＣＲ）扩增了椰毒假单胞菌代表菌株Ｔ７７０７的部分１６ＳｒＲＮＡ基因片段，将扩增片段克隆到Ｍ１３ｍｐ１９噬菌体，扩增后提取其单链ＤＮＡ，用ＤＮＡ测序仪进行测序。所得结果与已发表的其它已知假单胞菌的相应序列进行比较，计算出各菌之间的差异，并据此进行聚类。结果表明椰毒假单胞菌在系统发育上与Ｂｕｒｋｈｏｌｄｅｒｉａ属的成员关系密切，而与其它假单胞菌种的关系很远。这表明，椰毒假单胞菌应为Ｂｕｒｋｈｏｌｄｅｒｉａ属的一个种。     

一株不典型山夫登堡沙门氏菌的鉴定及其16SrRNA序列分析

目的对一株从广州市食品从业人员肛拭子中分离的蔗糖发酵型沙门氏菌进行鉴定。方法应用培养特性试验、生化分析、血清型鉴定,及16S rRNA序列分析进行鉴定。结果该菌培养特性及生化特性符合沙门氏菌定义,但与普通沙门氏菌在蔗糖利用、硫化氢产生上存在差异。血清型鉴定为山夫登堡沙门氏菌（Salmonella enterica Subsp·enterica serovar Senftenberg）,16S rRNA序列分析为肠道沙门氏菌亚种（Salmonella enterica subsp）。结论该菌为一株蔗糖发酵型、硫化氢阴性的不典型山夫登堡沙门氏菌。     

用16S rRNA基因序列分析从西藏的微小牛蜱中检出的埃立克体

用埃立克体属特异性套式PCR和DNA序列测定从西藏微小牛蜱检出边缘无形体和埃立克体;用半套式PCR扩得该埃立克体的16S rRNA基因,并测定了它的序列;将该埃立克体的16S rRNA基因序列与其它埃立克体的16S rRNA基因序列进行对比分析,结果证明它的16S rRNA基因与埃立克体属的犬埃立克体群16S rRNA基因相似水平最高(97%～98%);用16S rRNA基因序列做系统发育分析,结果显示该采自西藏的微小牛蜱的埃立克体可能是与查菲埃立克体密切相关的一种新埃立克体.     

The microbiome of a striped dolphin (Stenella coeruleoalba) stranded in Portugal

Infectious diseases with epizootic consequences have not been fully studied in marine mammals. Presently, the unprecedented depth of sequencing, made available by high-throughput approaches, allows detailed comparisons of the microbiome in health and disease. This is the first report of the striped dolphin microbiome in different body sites. Samples from one striped female edematous dolphin were acquired from a variety of body niches, including the blowhole, oral cavity, oral mucosa, tongue, stomach, intestines and genital mucosa. Detailed 16S rRNA analysis of over half a million sequences identified 235 OTUs. Beta diversity analyses indicated that microbial communities vary in structure and cluster by sample origin. Pathogenic, Gram-negative, facultative and obligate anaerobic taxa were significantly detected, including Cetobacterium, Fusobacterium and Ureaplasma. Phocoenobacter and Arcobacter dominated the oral-type samples, while Cardiobacteriaceae and Vibrio were associated with the blowhole and Photobacterium were abundant in the gut. We report for the first time the association of Epulopiscium with a marine mammal gut.The striped dolphin microbiota shows variation in structure and diversity according to the organ type. The high dominance of Gram-negative anaerobic pathogens evidences a cetacean microbiome affected by human-related bacteria.     

Emergence of rmtC and rmtF 16S rRNA Methyltransferase in Clinical Isolates of Pseudomonas aeruginosa

Occurrence of aminoglycoside (AG) resistance in clinical isolates of Pseudomonas aeruginosa is investigated in this study. Antimicrobial susceptibility test and minimum inhibitory concentration (MIC) for amikacin and gentamicin were performed followed by polymerase chain reaction amplifications of AG modifying enzyme genes (aac(6′)-I, aac(6′)-II, aac(3)-II/VI, ant(2″)-I, aph(3′)-VI) and 16S methylases (rmtA-D, rmtF and armA). MIC₅₀ and MIC₉₀ were 64, 128 and > 256, >256 for amikacin and gentamicin, respectively. Four types of genes (aac(6′)-I, aac(3)-II/VI, ant(2″)-I and aph(3′)-VI) were found in 53 (57.6%) isolates. ant(2″)-I was the most predominant gene (28 isolates) followed by aac(6′)-I (23 isolates). Nineteen (20.6%) isolates were positive for 16S RMTases (rmtB, rmtC, rmtF and armA) and two isolates co-harboured rmtB + rmtC + rmtF.     

Identification of randomly selected colonies of lactobacilli from normal vaginal fluid by pyrosequencing of the 16S rDNA variable V1 and V3 regions

The present study aimed to characterize lactobacilli in vaginal fluid from 23 adult healthy women by using high-throughput DNA sequencing for identification of a large number of randomly selected colonies appearing on Rogosa and blood agar. The typing method was based on broad-range PCR of 16S rRNA gene variable regions V1 and V3, pyrosequencing, and classification of the fragments by alignment with NCBI-catalogued sequences and type strain sequences. Four major groups of sequences were found among the 402 isolates clearly corresponding to Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii when compared to the sequences obtained for type strains. Our results indicate that pyrosequencing of 16S rRNA gene fragments as used here is a fast and reliable method well suited for identification to the species level, even within the Lactobacillus acidophilus complex.