Name: deferred growth inhibition assay to quantify the effect of bacteria-derived antimicrobials on competition
Uploaded: 2016-09-03T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition at JoVE.com

JCM由BBSRC-IPA奖与联合利华公司（BB / L023040 / 1）资助。 HAG由高等教育博士助学金的沙特阿拉伯教育部资助。

下面的方法适合用7,8抑制剂生产菌株，以测试应变竞争对手。 注:该协议涉及气溶胶的产生与细菌培养。这只能在包含的空间与执行本地认可的安全方面的考虑来执行。一个2级安全柜内到琼脂平板培养物喷洒将最小化的琼脂平板的污染和周围环境。这也将防止喷射细菌雾化吸入的研究者，这是一个主要问题，如果研究者使用包含级别2的生物体。适当的个人防护设备必须佩戴。围绕培养皿的区域最好与一次性吸湿组织覆盖。使用UV光喷雾消毒后建议。 1.准备琼脂平板<ol><li>准备脑心脏浸液（BHI）琼脂按照给马努厂家的说明。 </li><li>高压灭菌的BHI琼脂在121℃，15根据高压釜制造商的建议psi的20分钟，或者，进行消毒。 </li><li>倾灭菌BHI琼脂15毫升分装到无菌培养皿中，并允许它设置。 注:确保要使用的每一个琼脂平板内测定从琼脂的相同库存始发，并且有在每个板中的相同量的琼脂。这限制了速效养分和板材允许可比性的抑制剂扩散的可变性。 </li></ol> 2.准备无菌肉汤<ol><li>根据制造商的说明准备BHI肉汤。 </li><li> BHI添加10毫升分装到28毫升玻璃奶瓶普遍。 </li><li> ，15磅20分钟，或根据高压釜制造商的建议高压釜在121℃的BHI液体培养基，灭菌。 注:确保在试验中使用的所有肉汤同一批肉汤AUT的起源同时以限制的营养成分，它可能导致结果变异变异oclaved。 </li></ol> 3.准备无菌蒸发器瓶（S） <ol><li>拆开vapourizer瓶。 </li><li>喷雾5％的表面活性清洗剂通过vapourizer，消除内部的污染物，泡vapourizer瓶子，盖子和vapourizer在5％的表面活性清洗剂过夜。 注:手套和适当的个人防护装备应处理的表面活性剂清洗时使用。 </li><li>在干燥无菌空气流罩蒸发器，盖子和瓶子。 </li><li>把瓶子装满70％的乙醇，重新连接vapourizer并通过vapourizer喷些乙醇。 </li><li>放在蒸发器盖子，里面直到使用乙醇储存。 </li><li>使用，无菌冲洗瓶子用无菌BHI肉汤，并通过vapourizer喷汤前夕。 注意:这可以防止乙醇AFF阿拉斯加入到瓶子中6.3节接种。 </li></ol> 4.准备细菌过夜培养（S） <ol><li>条纹的竞争者和抑制剂株上的无菌BHI琼脂平板上，并在37°C孵育需氧大约18小时（过夜）。 注意:此板产生时保存在4℃下，可以被用于在同一周内多次重复细菌的库存。 </li><li>接种10毫升无菌BHI肉汤的与所需的竞争者细菌菌株的28毫升普遍瓶子内的单个菌落。 </li><li>通过在轨道摇床以250rpm摇动孵育在37℃的普遍瓶需氧过夜。 </li></ol> 5.抑制剂生产隔离现货文化<ol><li>在第4节准备过夜培养（S）。 </li><li>确保琼脂平板完全干燥后，盖上盖子不结露，在接种前（ 例如 ，通过培育解放军德在37℃下60分钟）;这防止了接种物从接种部位穿过该板传播。 </li><li>吸移管25微升过夜培养物到琼脂平板的中央。避免完全放开吸管柱塞，防止气泡横跨琼脂喷文化。 </li><li>允许培养在室温下干燥，以限制文化的传播。在37℃下孵育琼脂平板需氧过夜。 </li></ol> 6.准备测试竞争者应变的喷雾接种（S） <ol><li>如第4稀释的过夜培养10倍的BHI肉汤中所述，得到约4×10 6 CFU毫升-1制备过夜培养，并悬浮液的OD 600 0.3±0.05的。 注:10倍稀释不会放弃4×106 CFU毫升-1所有的细菌种类，通 ​​过传播板之间1×10系列稀释计算所需的稀释倍数<s达&gt; -1 -1×10 -6。 </li><li>倒入稀释文化带入无菌塑料香水瓶喷雾器。 注:总体积必须更对于被喷琼脂板的数量比是必要的。大约每板250μl的是适合于9厘米直径的陪替氏培养皿。 </li></ol> 7.递延生长抑制试验<ol><li>通过各蒸发器的瓶子的喷雾的培养，以确保培养物在喷雾机构喷涂到琼脂之前加载。 </li><li>从约15厘米的琼脂上面的距离，喷约250稀释的培养的微升（典型50毫升汽化器3喷雾剂）在整个琼脂表面。在37℃下孵育琼脂平板需氧过夜。重复具有相同数量的喷雾剂的其它板。 </li></ol> 8.递延解读生长抑制试验结果<ol><li>测量喷雾competi的生长抑制区器菌株（ 图1A - C，"X"为"X"）以毫米为单位，减去抑制剂生产者的中央点的直径（ 图1A - C，"Y"为"Y"）。 注: 图1E表示这个数据是如何被呈现</li><li>记录清晰度得分抑制区。 注:这表示观察到抑制程度。实例示于图1A - D，可用于标准化的菌株中的代表性结果部分的建议。以下尺度可用于清晰度评分:0清晰度评分来描述竞争者菌株的生长的完全抑制的区域。 1分来形容的增长几乎完全抑制的区域，与目前在空地的区域只有个别殖民地。 2分来形容降低经济增长的可见区域，这个区域内的增长是骗子流利或接近汇合但&gt; 50％密度减少。 3分来形容只有最小的增长减少，生长汇合并低于50％降低，区域仍然是可见和可衡量的。 4分来形容生长没有明显的抑制作用。将5得分来描述的竞争者菌株的生长在测试抑制剂生产菌株的接近而增加。参见图1中的例子。这些分数是主观的实验者。 </li></ol> 9.重复<ol><li>至少在生物一式三份重复试验，以确定获得的结果的可重复性。 注意:如果重复在不同的天进行的，进行测量，并在4℃下从温育中取出后立即得分板，然后或者商店板或将其废弃之前采取板的高品质的照片。存储或板的照片，使不同天之间的结果易于比较，T他是整个天/实验证实可重复性的得分尤为重要。注意，在板的存储具有改变的结果的可能性。 注:对于清晰的照片代替板直立没有他们在漫射光的面积的哑光黑色的背景盖，使用高分辨率相机（≥8MP）能够专注于物体靠近镜头（微距模式）。或者，用反式照明部分凝胶成像仪设置为白色光可以生产出高品质，高重现性的图像。 </li></ol>

<ol>
	<li>Bolnick, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+intraspecific+trait+variation+matters+in+community+ecology.">Why intraspecific trait variation matters in community ecology.</a> Trends Ecol Evol. 26 (4), 183-192 (2011).</li><li>Christensen, G. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+between+Staphylococcus+epidermidis+and+Propionibacterium+acnes+and+its+genomic+basis.">Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.</a> BMC Genomics. 17 (152), (2016).</li><li>Faust, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+co-occurrence+relationships+in+the+human+microbiome.">Microbial co-occurrence relationships in the human microbiome.</a> PloS Comput Biol. 8 (7), (2012).</li><li>Frank, D. N., Feazel, L. M., Bessesen, M. T., Price, C. S., Janoff, E. N., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+nasal+microbiota+and+Staphylococcus+aureus+carriage.">The Human nasal microbiota and Staphylococcus aureus carriage.</a> PLoS One. 5 (5), e10598 (2010).</li><li>Fredheim, E. G., Flaegstad, T., Askarian, F., Klingenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonisation+and+interaction+between+S.+epidermidis+and+S.+aureus+in+the+nose+and+throat+of+healthy+adolescents.">Colonisation and interaction between S. epidermidis and S. aureus in the nose and throat of healthy adolescents.</a> Eur J Clin Microbiol Infect Dis. 34, 123-129 (2015).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat Rev Microbiol. 8, 15-25 (2010).</li><li>Libberton, B., Coates, R. E., Brockhurst, M. A., Horsburgh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+intraspecific+trait+variation+among+nasal+bacteria+shapes+the+distribution+of+Staphylococcus+aureus.">Evidence that intraspecific trait variation among nasal bacteria shapes the distribution of Staphylococcus aureus.</a> Infect Immun. 82 (9), 3811-3815 (2014).</li><li>Libberton, B., Horsburgh, M. J., Brockhurst, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+spatial+structure,+frequency+dependence+and+resistance+evolution+on+the+dynamics+of+toxin-mediated+microbial+invasions.">The effects of spatial structure, frequency dependence and resistance evolution on the dynamics of toxin-mediated microbial invasions.</a> Evol appl. 8, 738-750 (2015).</li><li>Liu, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+and+the+ecology+of+the+nasal+microbiome.">Staphylococcus aureus and the ecology of the nasal microbiome.</a> Sci Adv. 1, e1400216 (2015).</li><li>Nascimento, J. S., Ceotto, H., Nascimento, S. B., Giambiagi-deMarval, M., Santos, K. R., Bastos, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+are+alternative+agents+for+control+of+multiresistant+staphylococcal+strains.">Bacteriocins are alternative agents for control of multiresistant staphylococcal strains.</a> Lett Appl Microbiol. 42, 215-221 (2006).</li><li>Park, B., Iwase, T., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+application+of+S.+epidermidis+prevents+colonization+by+methicillin-resistant+Staphylococcus+aureus+in+mice.">Intranasal application of S. epidermidis prevents colonization by methicillin-resistant Staphylococcus aureus in mice.</a> PLoS One. 6 (10), e25880 (2011).</li><li>Uehara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+interference+among+nasal+inhabitants:+eradication+of+Staphylococcus+aureus+from+nasal+cavities+by+artificial+implantation+of+Corynebacterium+sp.">Bacterial interference among nasal inhabitants: eradication of Staphylococcus aureus from nasal cavities by artificial implantation of Corynebacterium sp.</a> J Hosp Infect. 44, 127-133 (2000).</li><li>Wos-Oxley, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+poke+into+the+diversity+and+associations+within+human+anterior+nare+microbial+communities.">A poke into the diversity and associations within human anterior nare microbial communities.</a> ISME J. 4, 839-851 (2010).</li></ol>

作者什么都没有透露。

从其他人的区别就这个细菌物种竞争协议的一个关键步骤，是抑制剂的孵育过夜除了竞争对手分离的前隔离开来。抑制剂应变需要这种增长时期产生的抑制化合物。作为竞争者菌株在相同介质的顶部重叠，此方法允许研究人员观察到的全部潜力 抑制剂菌株，干扰竞争者菌株的生长。其结果是，该测定反映了与自然发生的情况;一个孤立完全建立在一个利基，另一个必须能够抵消任何抑制变量能够成功入侵的利基。 有两个部分，以该协议最有可能执行的，如果不正确会导致错误的结果。首先，蒸发器瓶杀菌不完全可以引进污染细菌进入检测。 INCU用于冲洗汽化器瓶在步骤3.6在无菌容器中的肉汤bation可以用来确认瓶完全灭菌。在消毒液，如卫可附加洗涤，可先于表面活性清洁剂洗涤，在步骤3.2中所述，如果需要额外的杀菌使用。 可能导致错误的结果的第二步骤是竞争者菌株（步骤6.1）的稀释液。如果稀释并非最佳，抑制的明确测量可能不容易确定。该测定是使用不同的鼻菌株作为抑制剂生产在大范围的革兰氏阳性和革兰氏阴性物种与链球菌菌株优化金黄色葡萄球菌 SH1000作为竞争对手的应变7,8。它后来被用各种凝固酶阴性和凝固酶阳性葡萄球菌作为竞争对手和抑制剂生产菌株进行测试。当测试除上述作为竞争者其他属应变，对于接种稀释的一些优化可能是必要的。 如果清晰度分数重复之间显著变化，可能有必要通过将其设置为特定的光学密度进行标准化竞争者菌株接种物。然而，这将增加该测定需要来设置，并有可能降低菌株的量是可能每个实验进行处理的时间。在视觉上调整文化，用麦氏标准可以提供快速的替代适当的OD。 这种技术的一个限制是，它只能适用于培养细菌。这就是为什么许多研究使用的16S rRNA测序预测竞争排斥相互作用3,4,13。然而，遗传和宏基因组技术只能社区成员区分的物种水平，和/或不考虑特定种内性状的变化。能够筛选一个particul的关联AR基因，该基因编码具有细菌物种5的存在或不存在的性状，但是这需要可能与一个物种的排斥相关联的基因的预先知识。 这种技术的安全性的考虑是，由于气雾产生的细菌，它只能在实验室使用具有2级安全柜，或具有类似的安全预防措施。有替代技术测试为占种内性状变异，不产生细菌菌株气溶胶之间的竞争。一种这样的方法是同时拮抗试验2。在该方法中，一个分离株的接种物涂布在琼脂平板和第二分离接种物点样或刺伤顶部一旦第一已干燥。这将产生其中菌株直接竞争（同时对抗）的环境中，既竞争产生的抑制化合物和第一清除营养素。该advantag递延生长抑制试验在同时对抗法的e是该抑制剂分离应该总是有竞争对手分离的入侵之前被完全建立竞争优势。这是更加反射性的环境中，会发生什么，因为在大多数龛，加入前另一个应变，而不是在同一时间8被加入了两个分离一种隔离群将被建立。 另一种替代，以避免气溶胶的产生菌的是添加竞争者隔离在顶层琼脂，这可能是倾而不是喷洒在抑制剂分离物。然而，上层琼脂中加入的体积将需要约3毫升至确保即使板的覆盖范围。在该卷中，竞争者分离物将不为相同的介质作为抑制剂分离物上生长，所以将不从减少营养物受损。竞争对手也不会与任何UNT抑制化合物直接接触IL它们扩散到顶层琼脂。这样一个试验不能被描述为递延抑制或同时拮抗作用。 类似的方法以前已经用来评估之间临床S.杆菌肽抗性水平金黄色葡萄球菌菌株10。由10描述这里描述的协议和之间的主要区别，是后者只分类竞争者菌株为敏感（完成inhibition-清晰度评分0）或抗性（部分无inhibition-清晰度分数1-5）。延迟生长抑制试验可以因此还可以用于筛选对特定抗微生物剂，或者甚至是生产者的抗性水平来搜索对耐多药病原体活性新颖抗微生物剂。 有人建议，该宿主微生物可以被操纵以消除细菌群落9的不希望的成员。棒状杆菌或金黄色葡萄球菌中的应用ylococcus表皮已经被证明消除S.葡萄球菌定植在人口11,12的一个显著比例鼻孔。因此，研究为特定菌株能竞争排除主机菌群的不良成员，通过技术如这里所描述的人，可能会导致未来的治疗。

Bacteria living in communities compete at the interspecific and intraspecific level for space and nutrients 6. Competition between microbes can often lead to exclusion of particular species or strains within a community. Exclusion can be due to competition for a particular receptor or nutrient, or the production of an antimicrobial compound by a member of the community 6.
Studies investigating competitive exclusion frequently examine the presence and absence of species using 16S rRNA sequencing 3,4,13. However, intraspecific trait variation, for example the presence or absence of antimicrobial peptide production, can hugely impact the ability of one species to inhibit another 1. Since 16S rRNA sequencing cannot distinguish between strains of the same species, intraspecific trait variation cannot be assessed. The technique described here can be used to assess the potential of individual strains to exclude other bacteria. This method has previously been used to show a positive correlation between the presence of Staphylococcus aureus inhibitory bacteria and the absence of S. aureus in a nasal community 7.
The deferred growth inhibition (competition) assay can identify production of antimicrobial compounds or nutrient competition by the inhibitor strain. It can also detect positive interactions, whereby the presence of the inhibitor strain actually improves the growth of the competitor strain. This assay can be used to create quantitative data (zone of clearing size) and qualitative data (degree of inhibition), both of which can be overlaid onto data of the presence or absence of a species within a community to find correlations between phenotype (trait) and competitive exclusion 7.

<table><tbody><tr><td>Brain heart infusion broth</td><td>Lab M</td><td>lab049</td><td>rich general purpose media</td></tr><tr><td>agar-agar</td><td>Merck Milipore</td><td>101614</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt</td><td>82.1473.001</td><td></td></tr><tr><td>Plastic perfume vaporizer</td><td>not known</td><td>-</td><td>10 cm high with 3.5 cm diameter clear plastic spray bottle with lid, purchased from the travel section of a supermarket</td></tr><tr><td>Universal bottles</td><td></td><td>no longer in production, alternative SLS BOT5006</td><td>28 ml glass cylindrical bottle (~280 mm diameter, 850 mm height), flat bottomed with plastic screw cap</td></tr><tr><td>Decon 90</td><td>Decon&amp;nbsp;</td><td></td><td>surface active cleaning agent&amp;nbsp;</td></tr></tbody></table>

deferred growth inhibition assay to quantify the effect of bacteria-derived antimicrobials on competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

该试验的结果应当是观察到的作为在靠近中央斑点抑制剂生产菌株（ 图1）叠加的竞争者应变的差。这些差异可以解释为充分抑制（ 图1A），部分抑制（ 图1B和1C），无抑制（ 图1D）或正相关。在广泛使用的乳酸菌菌株S.表皮 Tü3298和S.表皮 Rp62A和S.表皮 Rp62A和S.金黄色葡萄球菌 Newman是在图1B和图1D分别使用。我们建议，这些菌株可用于测试此协议。 根据测试种类，充分抑制和部分抑制最有可能抗菌肽，抗生素或其他扩散性增长inhibito的结果由抑制剂生产菌株生产的RS。部分抑制也被链接到降低的养分可用性由抑制剂应变6由于竞争者应变摄取或营养物的螯合剂。 针对同一竞争者应变测试了不同的抑制剂生产菌株之间抑制区域的大小的比较表明在任一以下的差异:通过抑制剂生产菌株产生的抗微生物的量;抑制剂通过媒体扩散的能力;抑制剂的类型产生和正在生产的竞争者应变的抑制剂的抗性程度。针对同一抑制剂生产菌株测试不同竞争者菌株之间抑制区域的大小的比较是表示竞争者菌株所生产的抑制剂的抗性水平的。 如果已经发生的细菌的不一致应用不应使用所述板。例如，如果测试竞争者培养是太稀抑制区域将出现大的并用比通常所观察到的（ 图2A）不太明确界定边缘;这会影响任何比较区测量的可靠性。如果该抑制剂生产菌株点没有充分孵育前干燥过夜，抑制剂菌株是可能传播，而不是形成一个明确定义的点（ 图2B），这通常会影响区的测量，但也可以影响透明度得分。如果参赛者应变不均匀喷洒这可能会导致细菌的不均匀扩散（ 图2C）。如果参赛者应变过于集中可能的是所述竞争者菌株可以在抑制剂菌株（ 图2C）的顶部生长，在极端情况下，这可能会导致竞争者/抑制剂应变角色的一些反转。 <img alt="图1" src="/files/ftp_upload/54437/54437fig1.jpg" /> 图 1: 可从延迟生长抑制试验中获得的的定性和定量结果范围结果生长抑制的范围可以从（A）的完全抑制（0分），（B）的部分抑制（评分为1） （ 三 ）部分抑制（2分）至（D）没有抑制（4分）。标签指示抑制剂菌株（Y）的增长的边缘和抑制的最外边缘区（X）的。抑制范围的区域的从0毫米到大于10mm的大小，如图（E）所示，其示出对A拍摄到的板的测量结果，B，C和D的结果示于最近葡萄球菌分离株（A ，C）或普通实验室分离株金黄色葡萄球菌S.表皮 Tü3298（抑制剂）和S.表皮 Rp62A（竞争者）（B）和S.表皮 Rp62A（抑制剂）和S.金黄色葡萄球菌 Newman（竞争对手）（D）。 <a href="https://www.jove.com/files/ftp_upload/54437/54437fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54437/54437fig2.jpg" /> 图 2: 由于技术不佳的结果不一致的例子 ，结果可能会有所不同，如果比预期的（A。 </stroNG&gt;）竞争对手菌株太稀，（B）抑制剂菌株没有完全干的孵化/板前不干燥或（C）如果参赛者菌株并不均匀喷洒并过于集中， <a href="https://www.jove.com/files/ftp_upload/54437/54437fig2large.jpg" target="_blank">请点击这里查看大图这一数字。</a>

Watch this Scientific Journal Video about Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition at JoVE.com

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

递延生长抑制试验来量化竞争性细菌来源的抗菌剂的影响

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

deferred-growth-inhibition-assay-to-quantify-effect-bacteria-derived

Research

JoVE Journal

Biology

14.5K 次观看.  University of Liverpool. The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

视频: Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

科研

免疫与感染

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

微生物相互作用研究的高通量共培养测定

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

生物学

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app.jove.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.

递延生长抑制试验来量化竞争性细菌来源的抗菌剂的影响

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