Dynamics and estimates of growth and loss rates of bacterioplankton in a temperate freshwater system

The growth rate and losses of bacterioplankton in the epilimnion of an oligo-mesotrophic reservoir were simultaneously estimated using three different methods for each process. Bacterial production was determined by means of the tritiated thymidine incorporation method, the dialysis bag method and the dilution method, while bacterial mortality was assessed with the dilution method, the disappearance of thymidine-labeled natural cells and ingestion of fluorescent bacterial tracers by heterotrophic flagellates. The different methods used to estimate bacterial growth rates yielded similar results. On the other hand, the mortality rates obtained with the dilution method were significantly lower than those obtained with the use of thymidine-labeled natural cells. The bacterial ingestion rate by flagellates accounted on average for 39% of total bacterial mortality estimated by the dilution method, but this value fell to 5% when the total mortality was measured by the thymidine-labeling method. Bacterial abundance and production varied in opposite phase to flagellate abundance and the various bacterial mortality rates. All this points to the critical importance of methodological aspects in the elaboration of quantitative models of matter and energy flows over the time through microbial trophic networks in aquatic systems, and highlights the role of bacterioplankton as a source of carbon for higher trophic levels in the studied system.     

用气溶胶法和摇床法建立铜绿假单胞菌生物膜体外模型

目的模拟体内环境,体外建立细菌生物膜模型,为进一步深入研究细菌生物膜生物学特点提供基础。方法将粘附载体置于气溶胶法和摇床法模拟体内细菌生物膜形成的微环境中,将铜绿假单胞菌株培养3d后,取出标本分别进行通过FITC—ConA染色及SYT09／PI染色,然后分别进行荧光显微镜检测及激光共聚焦检测,观察细菌生物膜的形成情况;进行电子显微镜扫描观察形成的细菌生物膜的形态特点。结果在气溶胶的微环境下,FITC—ConA染色后在荧光显微镜观察到明亮成片状的细菌生物膜;SYT09／PI染色后在激光共聚焦检测,观察到片状,层叠如积云状,棉絮样的细菌生物膜;在电子显微镜扫描观察到大量细菌成团聚集,团状丛生突出表面,具有立体结构的细菌生物膜。在摇床法的微环境下,用3种检测方法都观察到成流线状的细菌生物膜。结论运用气溶胶法、摇床法可成功建立分别模拟体内呼吸系统及循环、泌尿系统的微环境下生物膜形成模型。     

Rapid lab-on-a-chip profiling of human gut bacteria

The human gut microbiota has a substantial impact on human health. Different factors such as disease, diet and drug use can have significant impacts on the gut microbiota. Therefore, it is of interest to have simple, rapid methods for analysis of the composition of the gut microbiota for clinical diagnostic purposes. Since only a minor fraction of the gastrointestinal bacterial community is presently possible to cultivate, molecular approaches are currently the best suited to investigate its composition. However, most of these molecular approaches require technical expertise and expensive equipment to run and they are not routinely available. Ideally, the analyses should be point-of-care options that can be run on a chip. In this study, an existing lab-on-chip (LOC) system for sizing/quantifying DNA was combined with length heterogeneity PCR (LH-PCR), a PCR-based profiling method targeting bacterial 16S rRNA gene sequences, to develop a fast, straightforward, reproducible, and economical method for profiling bacterial communities. The LOC LH-PCR method was first evaluated using a standardized gut cocktail containing genomic DNA from eight different bacterial species representing different genera of relevance for human health. The method was also tested on DNA that was directly extracted from human faecal samples and it was consistently capable of detecting alterations in the bacterial samples before and after antibiotic treatment. Although the resolution of the method needs improvement, this study represents the first step towards development of a diagnostic LOC for profiling gut bacterial communities.     

Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures

A new radiometric method for the automatic detection of bacterial growth in blood cultures has been compared with conventional methods. A total of 2,967 cultures from 1,280 patients suspected of having bacteremia were studied. A 2-ml amount of blood was inoculated into culture media in which the glucose was labeled with carbon-14. The release of (14)CO(2) by bacterial metabolism was checked hourly for 18 to 24 hr, daily for the next 2 days, and, on the 12th day, with an automated instrument. A 10-ml amount of blood was studied by conventional bacteriological techniques. In 125 cultures from 50 patients, there was bacterial growth in at least one of the routine media. Of these, the radiometric method detected 102 cultures from 40 patients. In 111 cultures from 48 patients, there was radiometric detection of bacterial growth. In all of these cultures, there was detection of bacterial growth in subcultures from the radioactive medium. Of these, the routine laboratory detected 98 cultures from 40 patients. Neither method detected all patients with bacteremia. Among the 57 patients positive by one or both methods, routine techniques detected bacteria in 87% and the radiometric method detected bacteria in 85%. Seventy per cent of the cultures were detected first by the radiometric method, 65% on the day of inoculation. Our results suggest that the radiometric method is faster than conventional techniques and comparable in accuracy. Its great advantage is that it is simple, automatic, and can be extended to automatic detection of bacterial sensitivity to antibiotics.     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

Effects of two different application methods of Burkholderia ambifaria MCI 7 on plant growth and rhizospheric bacterial diversity

In order to acquire a better understanding of the effects of the different delivery modes of bacterial inoculants on plant growth and on the community structure of rhizosphere bacterial populations, Burkholderia ambifaria MCI 7 (formerly B. cepacia MCI 7) was inoculated into the rhizosphere of maize plants by either seed adhesion or incorporation into soil. Plant growth was evaluated at different inoculum concentrations. The community structure of rhizosphere bacterial populations was evaluated by analysing the restriction patterns of the DNA coding for 16S rRNA amplified by polymerase chain reaction (PCR) (ARDRA) of 745 bacterial isolates. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from control and treated plants according to the concept of the r/K strategy. Moreover, the analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that the method of application can be an essential element in determining the effects of the inoculant on plant growth. In fact, when applied as a maize seed treatment, B. ambifaria MCI 7 promoted plant growth significantly; on the contrary, when incorporated into soil, the same strain reduced plant growth markedly. As far as the bacterial community structure is concerned, B. ambifaria MCI 7 affected the indigenous microflora of treated plants according to the application method: seed treatment brought about an abrupt decrease in bacterial diversity, whereas incorporation into soil increased bacterial diversity. Moreover, changes in bacterial diversity were limited to r-strategist bacteria. In conclusion, B. ambifaria MCI 7 can act as both a plant growth-promoting rhizobacterium and a deleterious rhizobacterium depending on the inoculation method.     

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.     

Bacterial mRNA purification by magnetic capture-hybridization method

A magnetic capture-hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A(260 nm) / A(280 nm) value, denatured gel electrophoresis and RT-PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture-hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.     

Rapid, high-throughput extraction of bacterial genomic DNA from selective-enrichment culture media

AIMS: To create a fast, sensitive and inexpensive high-throughput method for the extraction of bacterial genomic DNA from selective-enrichment culture media. METHODS AND RESULTS: Lysis of bacteria was achieved using guanidinium isothiocyanate, and DNA was extracted using 96-well glass microfibre filtration plates. Extraction-PCR detected the presence of 1 cfu Yersinia ruckeri and 16 cfu Lactococcus garvieae 200 microl(-1) sample of selective-enrichment medium. CONCLUSION: An efficient method for high-throughput extraction of bacterial genomic DNA from selective-enrichment culture media was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: This method enables detection of covert bacterial infections in fish. The simultaneous extraction of large numbers of samples allows for its use in bacterial monitoring programmes and quarantine.     

Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods

Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.     

Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.     

需氧菌阴道炎／细菌性阴道病联合测定技术诊断需氧菌性阴道炎的临床价值

目的探讨需氧菌阴道炎／细菌性阴道病联合测定技术诊断需氧菌性阴道炎（AV）的临床价值。方法分别采用镜检法和需氧菌阴道炎／细菌性阴道病联合测定技术对200例疑似AV患者进行检测。结果在200例疑似AV患者中,镜检法检出阳性患者174例,需氧菌阴道炎／细菌性阴道病联合测定法检出阳性患者169例。以镜检法为诊断标准,需氧菌阴道炎／细菌性阴道病联合测定技术的敏感性为94．83％、特异性为84．62％。两种方法的符合率为93．50％[（165＋22）／200],两者呈中度一致性（Kappa=0．735,P〉0．05）。结论需氧菌阴道炎／细菌性阴道病联合测定技术诊断AV具有很高的敏感度和特异度,与目前临床使用的常规镜检法符合率高,且该方法操作简便、快速,值得临床推广使用。     

菜豆种质资源抗普通细菌性疫病鉴定

采用针刺叶片接种法、温室地面水层保湿方式,对146份普通菜豆种质资源进行抗普通细菌性疫病鉴定和评价,结果表明,该接种方法简便实用,保湿方式效果良好,鉴定结果准确;从146份普通菜豆种质中筛选出抗病种质(R)2份、中抗(MR)种质50份、感病(S)种质81份、高感种质(HS)13份。本研究表明针刺叶片接种、温室地面水层自然蒸发保持湿度可以作为大规模菜豆抗普通细菌性疫病鉴定的适宜方法。     

A simple method for quantitative determination of bacterial adherence to human and animal epithelial cells

A simple quantitative method for determining bacterial adherence to epithelial cells was devised. The method involved incubation of fluorescein-labeled bacteria with oral epithelial cells. Non-adherent bacteria were subsequently removed by Percoll density centrifugation. The degree of bacterial adherence was then determined fluorospectrophotometrically by measuring the fluorescein extracted from the adherent bacteria. The degree of adherence corresponded well with the microscopic counts of adherent bacteria. The method proved to be suitable for assaying adherence of both gram-positive and gram-negative bacteria to human as well as animal epithelial cells.     

DNA疫苗的细菌内毒素检测

目的 :考察DNA疫苗细菌内毒素的检查方法的可行性及DNA疫苗的干扰作用。方法 :干扰试验和对比试验。结果 :供试品阴性对照系列样品溶液无干扰作用 ,与家兔法结果一致。结论 :将疫苗稀释 2倍可用灵敏度为 0 5EU ml的鲎试剂作细菌内毒素检查。     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10¹ to 10² bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.     

A simple method for the measurement of bacterial particle conductivities

A method was developed for the measurement of the bacterial particle conductivity, based on the measurement of the conductivity of a bacterial cell suspension sigma(s) and the suspending medium sigma(m). A line plotted through sigma(s) - sigma(m) versus sigma(m) crosses the x-axis at sigma(m) = sigma(p), independent of the bacterial cell concentration. The method does not require anything more complex than a centrifuge and a conductivity meter. Knowledge of the bacterial particle conductivity is of importance in, for example, the dielectrophoretic separation, manipulation and trapping of bacterial cells, as well as the study of their physiological state.     

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing – tubular bioreactor system

A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10⁻³ h⁻¹ and increased approximately four-fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.     

粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。