Separation of bacteria in temperature gradient: micro-thermal focusing field-flow fractionation

The separation of Staphylococcus epidermidis and Rhodococcus erythropolis bacteria was achieved with the use of Micro-Thermal Focusing Field-Flow Fractionation. This is the first performance of separation exploiting the Ludwig-Soret effect (thermal diffusion) of living biological cells, combined with lift forces and resulting in the focusing mechanism of separation. The experiments were carried out under carefully chosen experimental conditions preventing the denaturation of the bacteria.     

Separation of bacteria in temperature gradient: Micro-Thermal Focusing Field-Flow Fractionation

The separation of Staphylococcus epidermidis and Rhodococcus erythropolis bacteria was achieved with the use of Micro-Thermal Focusing Field-Flow Fractionation. This is the first performance of separation exploiting the Ludwig–Soret effect (thermal diffusion) of living biological cells, combined with lift forces and resulting in the focusing mechanism of separation. The experiments were carried out under carefully chosen experimental conditions preventing the denaturation of the bacteria.     

Factors affecting sedimentational separation of bacteria from blood

Rapid diagnosis of blood infections requires fast and efficient separation of bacteria from blood. We have developed spinning hollow disks that separate bacteria from blood cells via the differences in sedimentation velocities of these particles. Factors affecting separation included the spinning speed and duration, and disk size. These factors were varied in dozens of experiments for which the volume of separated plasma, and the concentration of bacteria and red blood cells (RBCs) in separated plasma were measured. Data were correlated by a parameter of characteristic sedimentation length, which is the distance that an idealized RBC would travel during the entire spin. Results show that characteristic sedimentation length of 20 to 25 mm produces an optimal separation and collection of bacteria in plasma. This corresponds to spinning a 12-cm-diameter disk at 3,000 rpm for 13 s. Following the spin, a careful deceleration preserves the separation of cells from plasma and provides a bacterial recovery of about 61 ± 5%.     

Micro-thermal field-flow fractionation of bacteria

The retention of Staphylococcus epidermidis bacteria cells, achieved with the use of micro-thermal field-flow fractionation and described in this paper, represents the first experimental proof that the separation and characterization of the bio-macromolecules and biological particles is possible by exploiting Ludwig-Soret effect of thermal diffusion. The experiments were carried out under gentle experimental conditions preventing the denaturation of the bacteria. Lift forces, appearing at high linear velocities of the carrier liquid, generated the focusing mechanism of the retention which resulted in high-speed and high-performance separation performed in less than 10 min.     

一种联合MALDI-TOF MS直接鉴定阳性血培养瓶方法改进及与Sepsityper Kit试剂盒法、SELTERS法和血清分离胶法的比较

目的评价直接使用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)联合Sepsityper Kit试剂盒法(简称试剂盒法)、SELTERS法和血清分离胶法(简称分离胶法)鉴定阳性血培养瓶血中细菌的符合率,并对SELTERS方法进行改进,以缩短样本处理时间。方法对656例临床血培养阳性标本,应用试剂盒法、SELTERS方法或分离胶法处理后,直接使用质谱仪快速鉴定菌株,同时进行传统培养,比较分析二者之间的差异。结果656例血培养阳性标本共分离出626株单种菌感染和30株多种菌感染标本。MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶法可在1 h内快速鉴定血培养阳性标本。在单种细菌感染中,MALDI-TOF MS联合试剂盒法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是97.1%(367/378)、0.8%(3/378),真菌的种、属符合率分别是32.4%(12/37)、0.0%(0/37);MALDI-TOF MS联合SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是66.8%(141/211)、21.3%(45/211),革兰阴性菌的种、属符合率分别是96.3%(364/378)、2.4%(9/378),真菌的种、属符合率分别是32.4%(12/37)、2.7%(1/37);MALDI-TOF MS联合分离胶法直接鉴定革兰阳性菌的种、属符合率分别是51.2%(108/211)、20.9%(44/211),革兰阴性菌的种、属符合率分别是93.4%(353/378)、1.6%(6/378),真菌的种、属符合率分别是13.5%(5/37)、2.7%(1/37);MALDI-TOF MS联合改良SELTERS法直接鉴定革兰阳性菌的种、属符合率分别是59.1%(13/22)、18.2%(4/22),革兰阴性菌的种、属符合率分别是88.5%(23/26)、3.8%(1/26),真菌的种、属符合率分别是0.0%(0/2)、50.0%(1/2)。而对于多种菌感染的血培养瓶,3种方法鉴定率均较低。结论MALDI-TOF MS联合试剂盒法、SELTERS法或分离胶直接鉴定阳性血标本中的病原菌,其结果可在1 h内获得,并与传统培养结果相比具有较高的符合率。但是这些方法检测更快速、操作更简便,同时改良SELTERS法样本处理时间缩短,成本降低,且符合率与前3种方法没有区别。这4种方法均能满足临床快速诊断和及时有效抗菌治疗的需求,临床可根据自身情况选择。     

Investigation of the first events leading to loss of culturability during Escherichia coli starvation: future nonculturable bacteria form a subpopulation

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.     

Extraction of methane-oxidizing bacteria from soil particles

Abstract: We present a method for extraction of active methane (CH₄)-oxidizing bacteria from soil samples. The method is based on physical dispersion of bacteria from the soil particles followed by separation of bacteria and soil particles by floatation in the density media Nycodenz or Percoll. Separation on Nycodenz produced very pure bacterial suspensions while separation on Percoll produced rather impure suspensions. However, more than 60% of the methane-oxidizing activity was irreversibly inhibited in the procedure using Nycodenz compared to less than 10% irreversible inhibition when Percoll was employed. The bacterial suspensions extracted from soil can be used to study the physiology and ecology of soil bacteria that oxidize methane at atmospheric concentrations. Our data indicated that these bacteria are extremely difficult to dislodge from particles compared to the majority of bacteria in soil. Tentatively, we interpret the strong attachment to long residence time (i.e. slow turnover) of the methane-oxidizing bacteria. A slow turnover/growth rate would explain why soil disturbances, like cultivation, have a long lasting effect on the oxidation of atmospheric methane in soil.     

Separation of bacteria of the Clostridium leptum subgroup from the human colonic microbiota by fluorescence-activated cell sorting or group-specific PCR using 16S rRNA gene oligonucleotides

Molecular methods based on 16S rRNA gene sequence analyses have shown that bacteria of the Clostridium leptum subgroup are predominant in the colonic microbiota of healthy humans; this subgroup includes bacteria that produce butyrate, a source of energy for intestinal epithelial cells. To improve our understanding of the species within this important group, separation methods using fluorescence-activated cell sorting (FACS) and specific PCR were combined with 16S rRNA gene sequence analyses. FACS was developed for bacteria labelled in situ with two rRNA oligonucleotide probes, namely EUB338-FITC for total bacteria and Clep866-CY5/cp or Fprau645-CY5 for bacteria of the C. leptum subgroup. Bacterial cell sorting allowed a selective recovery of members of the C. leptum subgroup from the human microbiota with efficiencies as high as 95%. Group-specific PCR amplification of the C. leptum subgroup was developed, and temporal thermal gradient gel electrophoresis showed host-specific profiles with low complexity, with a sharing of common bands between individuals and bands stable over 2 months for the same individual. A library of 16S rRNA gene cloned sequences (106 sequences) was prepared with DNA obtained from both separation methods, and 15 distinct phylotypes were identified, among which 10 have no cultivable or currently cultivated representative in reference collections.     

毛细管电泳在细菌分离分析中的应用

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

Evolutionary relationship of denitrifying bacteria as deduced from 5S rRNA sequences

The nucleotide sequences of 5S rRNA from seven denitrifying bacteria have been determined. Based on these sequences and those reported in the literature (including two denitrifiers), a phylogenic tree of 104 eubacterial 5S rRNA sequences has been constructed to establish the position of the denitrifying bacteria. These bacteria belong to either one of the three major subgroups of gram-negative bacteria. The grouping based on 5S rRNA sequences is almost compatible with the type of the nitrite reductases, with the one apparent exception of Paracoccus denitrificans ATCC 13543. Moreover, the separation time of most of the denitrifying bacteria from other non-denitrifying bacteria belonging to the same subgroup is recent. These results suggest that the denitrifying systems in these bacteria would have developed polyphyletically, and not so anciently, during eubacterial evolution.     

基于分子印迹技术的细菌传感检测

分子印迹因其材料结构的稳定性及靶标物识别的特异性而被广泛应用于生化分离分析的相关领域。近年来，将具有选择性捕获、分离和富集靶标物等优势的分子印迹技术与生化传感检测技术有机结合，是目前细菌等微生物高效检测领域备受关注的研究热点。本文就分子印迹技术在细菌分析中的印迹方法、分析检测技术和典型应用等方面的最新进展进行综述。首先介绍了细菌分子印迹原理，对表面印迹的材料以及直接压印、间接印迹和电聚合等制备方法进行了总结和归纳；重点对基于荧光、电化学、石英晶体微天平（QCM）等检测模式的细菌印迹传感监测在细菌分析检测及其与微流控芯片技术耦合的应用和进展进行了综述；最后，提出了存在的挑战及发展的趋势。     

Evaluation of the efficiency of extraction for the quantitative estimation of hydrogen bacteria in soil

The efficiency of extraction of hydrogen bacteria from soil for plate counting was evaluated by using pure cultures adsorbed to sterilized soil. The utilization of model materials which interact with bacteria by adhesive, capillary or electrostatic forces and the use of extraction fluids with buffering, detergent or chelating activity demonstrated the major importance of capillary forces for the retention of hydrogen bacteria.Utilization of Tris buffer (pH 7.5) as extraction fluid and separation of extracted bacteria from soil particles by sedimentation for 15 min resulted in the highest recovery. A second extraction step including sonication did not increase the efficiency. The extraction efficiency of 8 different strains of hydrogen bacteria adsorbed to 3 different soils demonstrated a high degree of variation with respect to bacterial strains, but not to soil types. The recovery was inversely related to cell parameters such as size, motility and slime formation.     

基于微流控技术的微生物细胞梯度稀释分离方法

随着微流控分析技术的快速发展,集成化的微流控芯片在满足实验高通量的同时,还在微生物细胞分离领域呈现出独特的优势。本研究基于微流控技术,制备了以聚二甲基硅氧烷(PDMS)、玻片为材料的细菌细胞梯度稀释分离芯片。该芯片的核心是通过一系列复杂的梯度网络来实现对细菌悬液的连续稀释,最终被分离的细菌细胞进入通道末端的存储孔内。结果显示,该方法能分离出的最少细菌细胞数低于10个。此芯片平台操作简单、耗时短、成本低,为微生物单细胞研究提供了新的途径。     

The use of chromogenic bacteria as coloured substitutes for pathogens: A simple strategy during design and development of a new method for sample pretreatment

Aims: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set‐up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red‐coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. Methods and Results: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. Conclusion: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. Significance and Impact of Study: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.     

Induction of stolon settlement in the scyphopolyps ofAurelia aurita (Cnidaria,Scyphozoa, Semaeostomeae) by glycolipids of marine bacteria

The settlement of pedal stolons of scyphopolyps ofAurelia aurita Lamarck could be induced by addition of a species of bacteria from the family Micrococcaceae. After treatment of the bacteria with several organic solvents a crude lipid extract free of bacteria could be obtained which was shown to be effective in inducing stolon settlement. Crude lipids extracted from the late logarithmic growth phase had an optimal effect on stolon attachment, in contrast to previously published experiments showing that all logarithmic phases of bacteria had the same level of effectiveness. After separation of the crude lipid extracts by thin layer chromatography and subsequent bioassay of the reeluated substances, acylgalactosidyldiglyceride and monogalactosidyldiglyceride were identified as the effective substances. Monogalactosidyldiglyceride was only found in bacteria from the medium logarithmic growth phase, whereas the former was found at all stages. The effectiveness of acylgalactosidyldiglyceride was independent of the growth phase of the extracted bacteria.     

Evaluation of methods for extraction of bacteria from soil

Abstract Several methods for dispersion of soil were tested for possible use in procedures for extraction of bacteria. Physical cell damage on cells and efficiency in extraction of indigenous cells from soil, were investigated. Cell damage by the dispersion methods was investigated by measuring the physical cell integrity and viability of pure cultures of Escherichia coli and Bacillus subtilis , as well as soil bacteria extracted from soil, when dispersed in slurries of γ-sterilized soil. Separation of bacteria and soil particles on the basis of buoyant density was conducted with the nonionic density gradient medium Nycodenz. When slurries of γ-sterilized soil with added pure cultured cells were centrifuged (10000 × g ) over cushions of Nycodenz (1.3 g ml⁻¹), practically all the added cells were recovered in a layer on top of the cushion. This proves that a reversible attachment and cosedimentation is not an important phenomenon in this procedure. The efficiency of the different dispersion methods for the extraction of indigenous soil bacteria, was assessed after separation of dislodged and attached soil bacteria. This separation was done either on the basis of sedimentation rate by low speed centrifugation, or buoyant density by Nycodenz density gradient centrifugation. The physical dispersion by ultrasonic treatment and chemical dispersion by the use of a chelating agent together with a detergent, were inferior to physical dispersion either by Waring blender (for large volumes) or a rotating rubber pestle treatment (for smaller volumes). The physical dispersion did not appear to be destructive to the cells tested.     

Carbon nanotube clusters as universal bacterial adsorbents and magnetic separation agents

The magnetic susceptibility and high bacterial affinity of carbon nanotube (CNT) clusters highlight their great potential as a magnetic bio‐separation agent. This article reports the CNT clusters' capability as “universal” bacterial adsorbents and magnetic separation agents by designing and testing a multiwalled carbon nanotube (MWNT) cluster‐based process for bacterial capturing and separation. The reaction system consisted of large clusters of MWNTs for bacterial capture and an external magnet for bio‐separation. The designed system was tested and optimized using Escherichia coli as a model bacterium, and further generalized by testing the process with other representative strains of both gram‐positive and gram‐negative bacteria. For all strains tested, bacterial adsorption to MWNT clusters occurred spontaneously, and the estimated MWNT clusters' adsorption capacities were nearly the same regardless of the types of strains. The bacteria‐bound MWNT clusters also responded almost instantaneously to the magnetic field by a rare‐earth magnet (0.68 Tesla), and completely separated from the bulk aqueous phase and retained in the system. The results clearly demonstrate their excellent potential as highly effective “universal” bacterial adsorbents for the spontaneous adsorption of any types of bacteria to the clusters and as paramagnetic complexes for the rapid and highly effective magnetic separations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria

The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5′-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract.Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.     

Rapid physicochemical detachment, separation and concentration of bacteria from beef surfaces

A simple, rapid, physicochemical treatment for the removal of viable bacteria from the surface of raw and cooked beef is described. The detachment method was linked to a differential centrifugation step which removed large amounts of particulate food matter and concentrated the detached bacteria. The method increased the numbers of bacteria released from beef surfaces and increased the numbers detected by at least one and a half orders of magnitude, when compared to the traditional 'stomaching' technique. This 1-h separation and concentration method produced cleaner suspensions of bacteria and improved the sensitivity of detection by DEFT and direct plate count.