Name: adherence of bacteria to plant surfaces measured in the laboratory
Uploaded: 2018-06-19T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Автор благодарит Сьюзан Уитфилд для приготовления цифры и Камилла Мартин и Хиллари Samagaio для помощи с некоторыми из экспериментов.

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Автор заявляет, что она не имеет никаких финансовых интересов.

Важно быть в курсе всех поверхностей, которые бактерии могут придерживаться в ходе эксперимента. Таким образом могут недооценивать бактерий, которые способны привязки к стеклу, если сделали жизнеспособных клеток с использованием стеклянных трубок и пипетки. Если растения выращиваются в агар или почвы и некоторые из агар или почвы остается на растения, бактерии могут связывать приставшего материала, а не к растениям. С другой стороны, мытье поверхности растений, особенно в случае корни, могут удалить природных поверхностных покрытий таких, как слизистые и таким образом изменить результаты испытаний присоединения.
Важно быть уверенным, что бактерии добавляется в смесь инкубации живы во время эксперимента. Таким образом следует регулярно жизнеспособных клеток бактерий, как бесплатные, так и вложенные. Некоторые лечения или бактерий мутации могут снизить темпы роста бактерий или на самом деле причиной смерти часть населения бактериальных. Живые и Мертвые бактерии не могут быть различимы в микроскопе, если используются специальные пятна. Есть полезные пятно комплект для жить/Мертвые бактерии, которая зависит исключение красители из живых бактерий. Однако если присутствует смешанное население бактериальных видов жизнеспособных клеток видов интерес может быть простой способ, чтобы определить ли инкубации привело к бактериальной смерти.
Средний состав будет влиять бактериальных выживания и роста. Корень экссудатом и материалы освобожден от раны и вырезать сайтов предоставит субстрат для поддержки скромный рост бактерий. Фосфат, азота и железа, как правило, ограничения в этих условиях. Двухвалентной катионов кальция и магния могут влиять адгезии. В некоторых случаях источник углерода могут влиять адгезии. также имеет значение pH. В целом pH ризосфере находится между 5.5 и 6.5.
Необходимо быть осторожным при использовании бактерий, отмеченные антибиотикорезистентности. Наиболее часто используемых антибиотиков, рифампицин и налидиксовой кислотой. Сопротивление этих антибиотиков является как правило из-за мутации в генах, хромосомная (РНК-полимераза и гиразы, соответственно) и таким образом не могут легко быть переданы другой штамм во время инкубации. Также этот вид сопротивления не привести к деградации или модификации антибиотика. Маркировка бактерий с маркером плазмида нести ген не рекомендуется, если плазмида не могут быть переданы любой другой бактерии. Антибиотикам не должны быть из-за деградации или химическая модификация антибиотика как антибиотик чувствительных бактерий затем сможет расти на антибиотик пластины, если они расположены недалеко от резистентных бактерий.
Методы, описанные в этом документе являются полезными для небольшой выборки и/или экспериментов, где образцы должны содержаться (например, эксперименты с патогенов человека). Для больших выборок (выше 100 г материала или более чем 50 растений) другие методы или резкого изменения этих методов будет необходимо10,,1932,19,33, 34 , 35 , 36. присутствие большого числа микроорганизмов за исключением видов изучается также может представлять серьезные проблемы. Возможные решения включают в себя использование бактерий, отмеченные флуоресцентный белок или резистентность к антибиотикам, как описано в шагах 6.1.2 и 7.3. Однако когда бактерии интерес редких особей в большой популяции других микроорганизмов эти маркеры не может быть достаточным для однозначной оценки числа бактерий изучается.
Все описанные здесь методы, на основе лабораторных методов. Для исследования парниковых потребуются незначительные изменения. Более крупные изменения, вероятно, будет требоваться для полевых исследований, где простейшие, насекомых и других животных хищничество и климатических вариаций осложнить предоставление определенных условий для экспериментов. В будущем эти методы может быть расширена для включения взаимодействия двух или более микроорганизмов на поверхности растений.

Измерения бактериальной привязки к заводе поверхности стало важным в трех различных ситуациях. Первая ситуация является изучение передачи человеческих патогенов растений поверхностей1,2,3. Цель здесь является для предотвращения бактериального привязки или для удаления или убить привязанных бактерий и тем самым по сокращению передачи заболевания от растительного материала. Вторая ситуация является изучение привязки патогенов растений к заводе поверхности4. Еще раз целью здесь является для предотвращения привязки или удалить или убить привязанных бактерий и тем самым уменьшить болезни. Третья ситуация является изучение привязки симбиотические или растений рост содействия бактерий5,6. Цель здесь заключается в содействии бактериальных обязательную силу и, таким образом, увеличение здоровья растений и культур урожайность.
Методы измерения бактериальных привязки к заводе поверхностей, описанные в этой статье, недорогой и сравнительно легко осуществить. Только требования являются микроскопом и материалы, обычно встречается в бактериологии лаборатории. Для некоторых методов sonicator Ванна является полезным. Методы, описанные предназначены для привязки экспериментов, проводимых с использованием относительно небольшие размеры выборки. Измерения привязки производятся в лаборатории, хотя это может быть возможность изменить некоторые из этих методов для использования в теплицах или в поле.
Эти методы были использованы для измерения бактериальных привязки корни, ростки, вырезать листья, фрукты и нетронутыми помидорами черри в лаборатории,78,9,10,11, 12,13,14,15. Они также используются для измерения корень колонизации растений, растущих в почве или песок в лаборатории16. С многих бактериальных видов, включая Agrobacterium tumefaciens, Sinorhizobium meliloti, кишечная палочка, Salmonella enterica и Pseudomonas fluorescensбыли использованы методы. Полезное описание методов для оценки взаимодействия A. tumefaciens с поверхности можно найти в Мортон и Фукуа (2012)17. Во всех случаях участие выборки были небольшими, обычно меньше чем растения 25-50. Описанные методы подходят для использования с патогенов человека, которые должны храниться содержащихся в ходе экспериментов.

<table><tbody><tr><td>light microscope</td><td>any</td><td>N/A</td><td>phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.</td></tr><tr><td>seeds</td><td>any&amp;nbsp;</td><td>N/A</td><td>make a note of the seed lot number and the cultivar</td></tr><tr><td>bleach</td><td>any</td><td>N/A</td><td></td></tr><tr><td>bath sonicator</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>nutrient agar&amp;nbsp;</td><td>Difco&amp;nbsp;</td><td>001-01-8</td><td></td></tr><tr><td>soytone</td><td>Difco&amp;nbsp;</td><td>24360</td><td></td></tr><tr><td>Sand</td><td>sea sand Fisher Scientific</td><td>S25</td><td></td></tr><tr><td>sand</td><td>&amp;nbsp;Sigma-Aldrich</td><td>S9887</td><td></td></tr><tr><td>conetainers</td><td>Stuewe &amp;amp; Sons, Inc.</td><td>Ray-Leach cone-tainers</td><td>many different sizes are available to suit the type of plant you wish to grow</td></tr><tr><td>parafilm</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>MS salts</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>parrafin</td><td>any</td><td>N/A</td><td></td></tr><tr><td>centrigfuge</td><td>any scientific company</td><td>N/A</td><td>to pellet most bacteri only a small centrifuge with a max force of 10,000 XG is needed</td></tr><tr><td>vortex mixer</td><td>any scientific company</td><td></td><td></td></tr><tr><td>micrometer</td><td>ACCU-SCOPE Accessories</td><td>A3145</td><td></td></tr><tr><td>Sedgwick-rafter counting cell</td><td>Hauser Scientific</td><td>HS3800</td><td></td></tr><tr><td>probe-clip press-seal incubatin chamber</td><td>Sigma-Aldrich</td><td>Z359483</td><td></td></tr><tr><td>rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td></td></tr><tr><td>naladixic acid</td><td>Sigma-Aldrich</td><td>n8878</td><td></td></tr><tr><td>Live/dead stain</td><td>In Vitrogen Molecular Bioprobes</td><td>L7007</td><td></td></tr></tbody></table>

adherence of bacteria to plant surfaces measured in the laboratory

Эта рукопись описывает метод для измерения бактериальных привязки для стерильных завод поверхностей в световой микроскоп, а также путем применения жизнеспособных клеток. Завод материалов включают корни, побеги, листья и разрезать фрукты. Методы, описанные недорогой, легко и подходит для небольших выборок. Привязка измеряется в лаборатории и могут использоваться разнообразные инкубации средств массовой информации и условий. Эффект ингибиторов может быть определена. Можно также оценить ситуации, которые поощряют и подавляют привязки. В некоторых случаях это возможно отличить ли различные условия изменить привязки главным образом из-за их воздействия на заводе или на бактерии.

Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

Простой метод для измерения и характеризующие бактериальной адгезии для растений, особенно корни и побеги, описанные в этой статье.

Присоединение бактерий к заводе поверхности измеряется в лаборатории

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

adherence-of-bacteria-to-plant-surfaces-measured-in-the-laboratory

Research

JoVE Journal

Environment

12.6K Views.  University of North Carolina at Chapel Hill. Простой метод для измерения и характеризующие бактериальной адгезии для растений, особенно корни и побеги, описанные в этой статье.

Video: Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

In Vitro Пробирной бактериальной адгезии на эпителиальных клетках млекопитающих

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Представляем напряжения сдвига в изучении бактериальной адгезии

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Инженерные Приверженец бактерии путем создания единого синтетического Curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioengineering

Обнаружение колонизации ризобактерий на Arabidopsis thaliana

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

В центре внимания автора: Усиление колонизации ризобактерий на корнях растений для повышения эффективности микробных удобрений

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Измерение бактериальной колонизации корнями Arabidopsis thaliana в гидропонном состоянии

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Биомиметические Материалы для характеристики Бактерии-хозяин взаимодействий

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Мониторинг бактериальной колонизации и технического обслуживания на арабидопсис thaliana Корни в плавающей гидропонной системы

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae+ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae+ STEC.

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Here we present a protocol to study the function of fimbriae in bacterial colonization.

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

Присоединение бактерий к заводе поверхности измеряется в лаборатории

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