Name: adherence of bacteria to plant surfaces measured in the laboratory
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作者感谢苏珊·威菲尔, 准备的数字和卡蜜尔马丁和希拉里 Samagaio 帮助一些实验。

<ol>
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撰文人宣称她没有竞争的经济利益。

重要的是要知道所有的表面, 细菌可以坚持在实验中。因此, 如果能用玻璃管和吸管来完成可行的细胞计数, 就可以低估能够与玻璃结合的细菌。如果植物生长在琼脂或土壤中, 一些琼脂或土壤留在植物上, 那么细菌就会粘附在附着物上, 而不是植物。另一方面, 洗涤植物表面, 特别是在根的情况下, 可能去除天然表面涂层如粘液, 从而改变粘附试验的结果。
重要的是要确定, 添加到孵化混合物中的细菌在实验中仍然活着。因而可生存细胞计数自由并且附有的细菌应该通常被做。一些治疗或细菌突变可能会降低细菌的生长速度, 或者实际上导致部分细菌数量的死亡。除非使用特殊污渍, 否则活体和死细菌在显微镜下可能无法区分。有一个有用的染色试剂盒的活/死细菌, 这取决于排除染料的活菌。然而, 如果有混合的细菌种类的人口存在, 那么可行的细胞计数的兴趣物种可能是最简单的方法来确定孵化是否导致细菌死亡。
培养基组成会影响细菌的生存和生长。根渗出物和从伤口和切割部位释放的物质将提供基质以支持适度的细菌生长。在这些条件下, 磷酸盐、氮和铁往往会受到限制。价阳离子如钙和镁可能影响黏附力。在某些情况下, 碳源会影响粘附。pH 值也很重要。一般来说, 根际的 pH 值介于5.5 和6.5 之间。
必须小心使用有抗生素耐药性的细菌。最常用的抗生素是利福平和萘啶酮酸。对这些抗生素的抗药性通常是由于染色体基因 (RNA 聚合酶和回旋酶) 的突变, 因此在孵化过程中不易转移到另一株。这种耐药性也不会导致抗生素的降解或改性。除非质粒不能转移到任何其他细菌, 否则不推荐用质粒传播的基因标记标记细菌。抗生素耐药性不应归因于抗生素的降解或化学改性, 因为抗生素敏感的细菌如果靠近耐药性细菌, 就能在抗生素板上生长。
本文所描述的方法对于小样本大小和/或实验中需要包含样本 (例如, 涉及人类病原体的实验) 非常有用。对于大样本大小 (超过100克的材料或超过50种植物), 其他方法或对这些方法的剧烈修改将需要10,19,32,19,33,34,35,36. 除了正在研究的物种以外, 存在大量的微生物也会造成重大问题。可能的解决办法包括使用标记有荧光蛋白或耐药性的细菌, 如步骤6.1.2 和7.3 所述。然而, 当有兴趣的细菌是稀有的个体在大量的其他微生物中, 这些标记可能不足以允许对所研究的细菌数量进行明确的评估。
这里描述的所有方法都是基于实验室的方法。温室研究需要稍加修改。对于那些原生动物、昆虫和其他牲畜捕食和气候变化使实验条件的提供复杂化的实地研究, 可能需要进行更多的重大修改。在未来, 这些方法可以扩展到包括两个或更多的微生物在植物表面的相互作用。

在三种不同的情况下, 对植物表面的细菌结合的测量变得非常重要。第一种情况是检查人类病原体在植物表面的传播1,2,3。这里的目标是防止细菌结合, 或清除或杀死绑定细菌, 从而减少疾病传播的植物材料。第二种情况是检查植物病原体对植物表面的约束力4。再次, 这里的目标是防止捆绑或删除或杀死绑定细菌, 从而减少疾病。第三种情况是对共生或植物生长促进细菌的结合的检查5,6。这里的目标是促进细菌的结合, 从而提高植物的健康和作物产量。
本文所描述的对植物表面细菌结合的测量技术是廉价的, 相对容易进行。唯一的要求是显微镜和材料一般发现在细菌学实验室。对于一些技术, 浴 sonicator 是有用的。所描述的技术是为使用相对较小的样本尺寸进行的结合实验设计的。对装订的测量是在实验室中进行的, 尽管可能可以修改其中的一些技术用于温室或田间使用。
这些技术被用来测量细菌结合到根, 芽, 切叶, 切水果, 和完整的樱桃西红柿在实验室7,8,9,10,11, 12,13,14,15。它们也被用来测量实验室16中生长在土壤或沙子中的植物的根系定植。该技术已应用于多种细菌种类, 包括农杆菌、根瘤菌苜蓿、大肠杆菌、沙门氏菌 enterica和铜绿假单胞菌荧光。在莫顿和福库 (2012)17中, 可以找到一个有用的描述方法来评估一个.在所有情况下, 所涉及的样本大小是小的, 一般少于 25-50 植物。所描述的技术适用于人类病原体, 需要在实验中加以保存。

<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

本手稿描述了一种方法, 以测量细菌结合无菌植物表面在光显微镜和通过使用可行的细胞计数。所用的植物材料包括根、芽、叶和切果。所描述的方法便宜, 容易, 适合小样本大小。在实验室中测量装订, 并可使用各种孵化介质和条件。可以确定抑制剂的作用。还可以评估促进和抑制绑定的情况。在某些情况下, 有可能区分各种条件是否会因其对植物或细菌的影响而改变约束力。

殖民根表面。为了确定纤维素的细菌生产是否在根系定植中起着重要作用, 研究了防止纤维素合成的细菌突变对16的影响。使用了步骤1.3 和7.1 中描述的技术。番茄种子在无菌水中进行表面杀菌和发芽。当根约2厘米长, 他们被浸泡在悬浮 105细菌每毫升和种植在巴氏杀菌土中的容器。植物生长了14天在25°c 在 12 h light/12 h 黑暗的周期。在被表明的时间以后植物从容器被去除了。根被洗涤和微气泡在浴 sonicator 去除束缚的细菌。细菌数量是用可行的细胞计数来确定的。图 2显示了两种不同的纤维素-负突变对细菌对番茄根部的殖民能力的影响。虽然一些测量的标准偏差高达0.9 日志10 (这类测量的一个共同问题), 但纤维素-负突变体的束缚的减少显然是明显的, 我们可以得出结论, 细菌生产的纤维素在番茄根系的定植中有助于细菌的生长。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56599/56599fig2.jpg"> 图 2: 由野生类型和纤维素减去突变体的根癌.日志10以野生a 型为基础的 C58 和纤维素-减去突变体 C58:1 和 C58:A60, 从番茄根中回收的每 cm 根长度细菌总数。显示的数字是从最少的四个单独实验的手段。条形表示方法的标准偏差。根接种了浸泡在 105细菌每毫升一分钟的悬浮。这些植物是在容器中生长的, 而松散附着的细菌则是通过在玻璃瓶中洗涤缓冲液中的根部来去除的。紧密贴附的细菌被清除使用浴 sonicator 和由此产生的悬浮镀, 以确定可行的细胞计数16。此数字已从 Matthysse 和威廉·达拉戈·麦默恩修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
研究了多糖在大肠杆菌和其他细菌对苜蓿芽中的结合作用。由于大肠杆菌O157:H7, 一些腹泻病的暴发被追踪到受污染的苜蓿芽。用步骤1.1、5.1 和7.2 中描述的方法测量了无法使各种多糖的野生型细菌和突变体的结合。苜蓿芽在不育的水中以25摄氏度在黑暗中被消毒并发芽一天。四芽与附着的种子大衣被放置在无菌的塑料菜肴含有5毫升的水。在 Luria 汤中生长的细菌被添加到最后浓度约 5 x 103每毫升。接种芽在25摄氏度在黑暗中孵化了3天。用马达驱动的聚四氟乙烯玻璃均质机在洗涤缓冲器中进行剧烈倒置和匀质, 在5毫升的无菌水中洗净豆芽两次。以前的实验中使用的细菌标记的质粒携带 GFP 基因显示没有内化细菌, 虽然表面细菌很容易观察到。结果见表 1。检查了两株大肠杆菌O157:H7。在这两种菌株中, poly-β-1,6-glucuronic 酸 (PGA) 的产生对致病性大肠杆菌对植物表面的束缚作用最大。结肠酸在装订中也起着重要作用。虽然纤维素-负突变体中的结合量的减少是显著的, 但它并不像其他两种多糖那么大。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="4">多糖生产基因突变对大肠杆菌 O157:H7 与芽苗和开放种衣结合的影响</td>
		</tr>
<tr>
<td rowspan="2">菌株</td>
			<td rowspan="2">突变或基因型 (相关表型)</td>
			<td colspan="2">记录10每芽或种衣的细菌数量</td>
		</tr>
<tr>
<td>苜蓿芽b
</td>
			<td>打开种子大衣</td>
		</tr>
<tr>
<td>86-24</td>
			<td>无 (野生类型)</td>
			<td>4.7 @ 0。6</td>
			<td>5.6 @ 0。2</td>
		</tr>
<tr>
<td>8624N</td>
			<td>
yhjN(纤维素-减去)</td>
			<td>2.9 @ 0.7c
</td>
			<td>3.5 @ 0.6c
</td>
		</tr>
<tr>
<td>8624C</td>
			<td>
wcaD(结肠酸-减)</td>
			<td>1.8 @ 0.7c
</td>
			<td>2.4 @ 0.5c
</td>
		</tr>
<tr>
<td>8624P</td>
			<td>
pgaC(PGA-减去)</td>
			<td>< 1.0c
</td>
			<td>1.0 @ 1.0c
</td>
		</tr>
<tr>
<td>DEC4A</td>
			<td>无 (野生类型)</td>
			<td>5.6 @ 0。2</td>
			<td>6.1 @ 0。3</td>
		</tr>
<tr>
<td>DEC4AN</td>
			<td>
yhjN(纤维素-减去)</td>
			<td>4.8 ~ 0.8d
</td>
			<td>4.1 ~ 0.8d
</td>
		</tr>
<tr>
<td>DEC4AC</td>
			<td>
wcaD(结肠酸-减)</td>
			<td>3.9 @ 0.5c
</td>
			<td>4.8 ~ 0.8d
</td>
		</tr>
<tr>
<td>DEC4AP</td>
			<td>
pgaC(PGA-减去)</td>
			<td>< 1.0c
</td>
			<td>1.2 @ 0.7c
</td>
		</tr>
<tr>
<td colspan="4">一个平均的标准偏差至少三测量的数量 (日志10) 的细菌绑定3天后。</td>
		</tr>
<tr>
<td colspan="4">b 芽在测量前洗净。</td>
		</tr>
<tr>
<td colspan="4">c 显着不同于野生类型: P < 0.01。</td>
		</tr>
<tr>
<td colspan="4">d 显着不同于野生类型: P < 0.05。</td>
		</tr>
<tr>
<td colspan="4">此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。</td>
		</tr>
</tbody></table>表 1: 多糖生产基因突变对其结合性的影响大肠杆菌 O157:H7 芽。为了确定各种多糖和脂多糖对苜蓿芽苗的致病性大肠杆菌O157:H7 菌株的作用, 对一组突变体对芽苗和开皮种衣的约束力进行了研究, 采用了方法步骤6中所述。结果表明, poly-ß-1, 6n-乙酰氨基葡萄糖 (PGA) 似乎是必不可少的绑定到芽, 并认为纤维素和 colanic 酸是必需的最大结合大肠杆菌O157。此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。
为了确定 pga 的生产是否足以引起细菌对植物表面的结合, 采用克隆操纵编码的质粒 (pMM11) 将 pga 生产所需的基因引入到两种不同的细菌中, 这将不会通常可以绑定到西红柿根部10。农杆菌A1045 是野生型菌株 C58 的突变体, 不能使 cyclic-β-1,2 葡聚糖, 也不能与植物表面29结合。在苜蓿上形成根结节的根瘤菌苜蓿1021 未与非豆科植物 (包括西红柿12) 结合。步骤1.1、5.1、6.3 和7.1 中描述的技术用于确定是否能够使 PGA 通常增加细菌对根表面的结合。番茄种子在无菌水中进行表面杀菌和发芽。根被切成1厘米长的段, 放在不育的水中, 细菌接种。由于这两种细菌以不同的速率生长, 所以在不同的时间对结合进行了测定, 以允许大致相等的细菌生长量。质粒 pMM11 的存在导致了两种物种的束缚细菌数量的相似显著增加 (表 2)10。在光镜中也看到了捆绑的显著增加, 但这两种物种的结合是非常不同的 (图 3)。 A1045 作为单个细菌被绑定到根表面。苜蓿在大团簇中, 只有少数细菌直接附着在根部, 大多数细菌附着在其他细菌上。这个例子表明, 简单的分析细菌的数量, 而不包括显微观察, 可以给一个误导的印象, 实验结果。虽然两种方法 (可行细胞计数和显微观察) 表明, pMM11 增加了对番茄根系的细菌结合, 但由 PGA 生产所造成的结合类型不同的两个细菌10。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="3">质粒 pMM11 对番茄根系细菌结合的影响</td>
		</tr>
<tr>
<td>菌株</td>
			<td>质 粒</td>
			<td>每毫米根长度的细菌数量</td>
		</tr>
<tr>
<td>A1045 农杆菌
</td>
			<td>没有</td>
			<td>0.25 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td></td>
			<td>pBBR1mcs (矢量)</td>
			<td>0.25 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td></td>
			<td>pMM11 (PGA 合成)</td>
			<td>10 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td>S. 苜蓿 1021b
</td>
			<td>没有</td>
			<td>未检测到</td>
		</tr>
<tr>
<td></td>
			<td>pBBR1mcs (矢量)</td>
			<td>未检测到</td>
		</tr>
<tr>
<td></td>
			<td>pMM11 (PGA 合成)</td>
			<td>50 x 103 @ 5 x 103
</td>
		</tr>
<tr>
<td colspan="3">2小时后测定细菌结合量</td>
		</tr>
<tr>
<td colspan="3">b. 18 小时后细菌结合量的测定</td>
		</tr>
<tr>
<td colspan="3">此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。</td>
		</tr>
</tbody></table>表 2: 质粒携带基因对 PGA 合成的影响 A1045 和苜蓿 1021 到西红柿根段。为了研究 poly-ß-1, 6n-乙酰氨基葡萄糖 (PGA) 的能力, 以促进细菌对植物根系的结合, 一个质粒的影响, 使 PGA (pMM11) 的能力, 对两株植物相关细菌的结合, 以西红柿根被检查。在缺乏质粒或质粒存在的情况下, 两种细菌对番茄根部均无明显的约束力, 而不存在 PGA 合成 (pBBR1mcs) 的基因编码。加入 PGA 合成基因的质粒增加了两种细菌的结合。因为. ................. .........苜蓿后2小时的苜蓿结合量。所使用的技术是步骤7中描述的方法。此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56599/56599fig3.jpg"> 图 3: 质粒 pMM11 携带 PGA 生物合成基因对 A1045 和苜蓿1021 至番茄根毛的结合作用。对西红柿根毛的约束力) a.农杆菌 A1045, B) A1045 pMM11, C) s. 苜蓿1021 和 D) s. 苜蓿1021 pMM11。虽然两种细菌的结合度的增加大致相似, 但在光镜中看到的结合的细节却是截然不同的。所使用的技术是步骤6中描述的方法。此数字已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
有时可以使用绑定到非生物表面, 以帮助区分细菌和植物在特定的相互作用的贡献。单极多糖 (UPP) 已被证明能够调解细菌结合到各种生物和非生物表面30。钙被观察, 以抑制约束的一个. 农杆菌的植物表面介导的 UPP28。为了确定细菌结合对植物表面的钙离子的抑制是由于对细菌或在植物表面的作用, 细菌的捆绑对尼龙螺纹被审查了。使用了步骤8.2 中描述的技术。在步骤1中描述的番茄种子表面被杀菌, 在水中发芽。细菌生长在最小的培养基与蔗糖, 并添加到番茄根或尼龙线的最终浓度约 105/毫升, 如步骤5.1 所述。显微镜观察了添加 CaCl2对番茄根系和尼龙丝细菌结合的影响。图 4显示了类似的抑制钙结合使用任何表面表明, 钙的影响主要是在细菌10。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56599/56599fig4.jpg"> 图 4: 钙对农杆菌结合对番茄根毛和尼龙丝的影响.在1:10 稀释 MS 盐和1:20 稀释 AB 最小培养基的情况下, 用西红柿根 (a 和 B) 或尼龙线 (C 和 D) 在24小时内培育了农杆菌; 0.4% 蔗糖 (A 和 C) 或1:10 稀释的 MS 盐和1:20 稀释的 AB 最小培养基, 0.4% 蔗糖含有60毫米 CaCl2 (B 和 D)31。添加的 CaCl2抑制细菌结合的根和尼龙线表明, 抑制主要是由于对细菌的影响, 而不是在植物表面。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

本文介绍了一种简单的方法来测量和表征细菌对植物的黏附力, 特别是根和芽。

细菌附着在实验室测量的植物表面

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

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JoVE Journal

Environment

12.5K 次观看.  University of North Carolina at Chapel Hill. 本文介绍了一种简单的方法来测量和表征细菌对植物的黏附力, 特别是根和芽。

视频: 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.

到哺乳动物的上皮细胞在体外细菌粘附的含量

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

科研

免疫与感染

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.

在细菌粘附的研究剪应力

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.

通过创建一个的合成Curli的操纵子工程粘附的细菌

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

生物工程

拟南芥根际细菌定植检测

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.

作者聚焦:增强根际细菌在植物根部的定植以提高微生物肥料效率

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.

在水培条件下测量 拟南芥 根上的细菌定植

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

环境科学

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.

仿生材料表征细菌宿主相互作用

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.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

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.

细菌附着在实验室测量的植物表面

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