Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

轮状病毒感染与健康儿童肠道菌群结构的比较

目的比较轮状病毒(RV)感染与健康儿童肠道菌群结构的差异,为临床治疗提供有价值的参考依据。方法采集20例RV感染及6例健康儿童粪便样品,提取粪便样品中细菌的混合DNA,先通过肠道菌重复性基因内一致性序列(ERIC)-聚合酶链反应(PCR)结合分子杂交技术分析两组儿童之间肠道微生物组成的相似性;再扩增粪便样品中菌群的16 S rDNA基因V3区,利用PCR-TGGE技术分析肠道菌群的组成情况,研究两组儿童肠道微生物菌群组成的差异。结果肠道菌群的组成有很强的宿主专一性。RV感染与健康儿童相比,肠道菌群中糖皮质激素(GC)水平较低的细菌明显减少。微生态制剂治疗组温度梯度凝胶电泳(TGGE)条带数有一个逐渐增加的过程。结论轮状病毒感染时,可致患儿肠道菌群紊乱,但用抗生素治疗将更致肠道微生态失调,不利于患儿肠道菌群恢复,建议临床医师在轮状病毒感染时,慎用抗生素。

微生物强化对椒坯发酵群落多样性及性质的影响

描述了应用磷脂脂肪酸(phosphoric acid fatty acids,PLFAs)及聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术研究微生物强化发酵椒坯群落多样性变化的规律。研究表明,高盐发酵使椒坯的细菌生物量显著降低;对于强化的样品,因所使用的菌株及强化方式不同,其群落多样性及优势菌差异显著,假丝酵母菌Candida versatilis强化提高了椒坯总生物量,鲁氏酵母Zygosaacharomyce rouxii则相反;共培养强化提高了椒坯以Tetragenoccus和Zygosaacharomyces为主的优势菌的生物量,同时发现嗜盐四链球菌Tetragenoccus halophilus对Vagococcus具有明显的抑制作用。理化研究表明,不同强化方式使样品的总酸(TA)、还原糖(RS)和氨基态氮(FN)等参数略有差异。不同强化方式对挥发性组分贡献不同,其中,共培养强化使椒坯挥发性组分总含量提高了28.46%。该研究揭示了强化发酵对椒坯微生物群落和品质的影响规律,对微生物强化技术在传统发酵的应用有一定的指导作用。

Biostimulation-enhanced Biodegradation of Nitrobenzene in Contaminated Groundwater

In this study, biostimulation technology was used for bioremediation of nitrobenzene-contaminated groundwater by adding a mixture of lactose and phosphate, peptone, and beef extract. During the process of biostimulation, the remediation effectiveness, microbial dehydrogenase activities and microbial densities were investigated; the varieties of microbial community structure and composition were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE） technique and the relative abundances of nitrobenzene-degrading gene（nbzA） were determined by fluorescence quantitative real-time PCR（RT-PCR）. Findings show that the removal rate of nitrobenzene in groundwater could reach about 60% by biostimulation with lactose and phosphate, 70% with peptone and 68% with beef extract. The microbial dehydrogenase activities and microbial densities were all improved obviously via biostimulation. The results of PCR-DGGE show that the microbial diversities were improved, and more than ten kinds of dominant microorganisms were detected after biostimulation. RT-PCR results show that the relative abundances of nbzA gene of microbes in groundwater were increased significantly, which indicated that biostimulation actually enhanced the growth of nitrobenzene-degrading bacteria. Therefore, biostimulation is a cost-effective and feasible bioremediation technique for nitrobenzene-contaminated groundwater.

Influence of Temperature on Symbiotic Bacterium Composition in Successive Generations of Egg Parasitoid, Anagrus nilaparvatae

Anagrus nilaparvatae is the dominant egg parasitoid of rice planthoppers and plays an important role in biological control. Symbiotic bacteria can significantly influence the development, survival, reproduction and population differentiation of their hosts. To study the influence of temperature on symbiotic bacterial composition in the successive generations of A. nilaparvatae, A. nilaparvatae were raised under different constant temperatures of 22 °C, 25 °C, 28 °C, 31 °C and 34 °C. Polymerase chain reaction-denaturing gradient gel electrophoresis was used to investigate the diversity of symbiotic bacteria. Our results revealed that the endophytic bacteria of A. nilaparvatae were Pantoea sp., Pseudomonas sp. and some uncultured bacteria. The bacterial community composition in A. nilaparvatae significantly varied among different temperatures and generations, which might be partially caused by temperature, feeding behavior and the physical changes of hosts. However, the analysis of wsp gene showed that the Wolbachia in A. nilaparvatae belonged to group A, sub-group Mors and sub-group Dro. Sub-group Mors was absolutely dominant, and this Wolbachia composition remained stable in different temperatures and generations, except for the 3rd generation under 34 °C during which sub-group Dro became the dominant Wolbachia. The above results suggest that the continuous high temperature of 34 °C can influence the Wolbachia community composition in A. nilaparvatae.

嗜酸性细菌浸出废旧线路板过程中微生物群落的聚合酶链式反应变性梯度凝胶电泳分析

以富集的嗜酸性细菌为接种物，进行废旧线路板的生物浸出实验，同时收集浸出不同时间的微生物样品，利用聚合酶链式反应一变性梯度凝胶电泳技术分析了浸出过程中微生物群落结构的变化．结果表明，嗜酸性细菌能在48h内浸出废旧线路板中96．36％的铜．从变性梯度凝胶上挑选的明亮条带分析结果显示，Z1-Z7七个条带序列与氧化亚铁硫杆菌叫cidithiobacillusferrooxidans）的序列同源性均在99％以上，即均为Acidithiobacillusferrooxidans．浸出0，5，24和60h的样品中ZI~Z7的组成比例变化不大，其中Z3相对丰度一直最高，分别为72．70％．82．90％，79．00％和85．80％．浸出过程中微生物群落结构演变较平缓，始终为Acidithiobacillusferrooxidans菌群，菌株Z3是整个浸出过程中的优势菌种．

Applicability of anoxic-oxic process in treating petrochemical wastewater

To explore the applicability of anoxic-oxic (A/O) activated sludge process for petrochemical wastewater treatment,the relationship between bacterial community structure and pollutants loading/removal efficiencies was investigated by gas chromatograph-mass spectrometry (GC-MS),polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and other conventional techniques.It showed that when the concentrations of the influent chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) were 420～560mg/L and 64～100mg/L,respectively,the corresponding average effluent concentra-tions were 160mg/L and 55mg/L,which were 1.6 and 2.2times higher than those of the national standards in China,respectively,demonstrating the inefficient performances of A/O process.Analysis of GC-MS indicated that refractory pollutants were mainly removed by sludge adsorption,but not by biodegradation.PCR-DGGE profile analysis suggested that the biological system was species-rich,but there was apparent succession of the bacterial community structure in different locations of the A/O system.Variations of bacterial community structure and pollutant loadings had obvious influences on pollutants removal efficiencies.Thus,A/O process was inapplicable for the treatment of complicated petrochemical wastewater,and strategies such as the reinforcement of pre-treatment and two-stage A/O process were suggested.

Microbial and hydrodynamic properties of aerobic granules in a sequencing batch reactor treating landfill leachate

A sequencing batch reactor （SBR） seeded with activated sludge was established for landfill leachate treatment. Small bio-aggregates began to appear after 40-d operation, and gradually changed to mature aerobic granules, with a mean size of 0.36-0.60 ram. Their sludge volume index at 5 min （SVI5 rain）, mixed liquor volatile suspended solids （MLVSS）, and wet density were around 35 ml/g, 3.4 g/L, and 1.062 g/cm3, respectively. The settling velocities of the granules in distilled water ranged from 0.3 to 1.3 cm/s, which were faster than those in landfill leachate with a salt content of 1.4% （w/v）, and also slightly faster than those predicted by Stokes＇ law for porous but impermeable particles. Microbial community evolution during the granulation process and stages under different nitrogen loading rates （NLRs） were monitored and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE）, cloning, and sequencing of 16S ribosomal RNA （rRNA） fragments. Results revealed that some primary and dominant communities in inoculating activated sludge died out gradually; while a few common bacteria, inhabiting soils, municipal wastewater, or activated sludge systems, dominated in the SBR system throughout. In addition, some other dominant species, associated with the aerobic granulation process, were thought to play a significant role in the formation and growth of aerobic granular sludge. During the stable operation time under low NLR, a few species were present in abundance, and may have been responsible for the high organic removal efficiency at this time.

Seasonal dynamic changes in bacterial compositions in the Inner Mongolia desert steppe

Polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)technique was employed to examine the seasonal dynamic changes in bacterial community composition in the Inner Mongolia desert steppe using specific primers F954 and R1369.Bright and reproducible bands were sequenced,and the phylogenic tree was constructed.The results show that the bacterial community composition changed between different seasons.The specific bands were different between the sampling sites with light and heavy levels of degraded grassland.Three main types of bacteria constituting the microbial community in the Inner Mongolia desert steppe belonged to the α,γ and δ-sub phyla of Proteobacteria,Bacteroidetes and Acidobacteria.The unculturable bacteria accounted for 69%of the whole bacterial community of the Inner Mongolia desert steppe.