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Mar 2021

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Assessing Swarming of Aerobic Bacteria from Human Fecal Matter
人类粪便中需氧细菌群集的评估   

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Abstract

Swarming – swift movement across a surface via flagella propulsion – is a unique property of many bacteria. The role of swarming, particularly among bacterial populations of the human gut microbiome, is not yet fully understood; although, it is becoming an area of increased scientific and clinical inquiry. To further characterize bacterial swarming in human health, an effective assay for swarming that utilizes complex material, such as fecal matter, is necessary. Until now, the vast majority of swarming assays have only been able to accommodate bacteria grown in culture, most often Pseudomonas. These assays tend to use a standard lysogenic broth (LB) agar medium; however, the reagents involved have not been tailored to the inoculation of complex material. In this paper, we offer a specialized protocol for eliciting the swarming of bacteria from frozen human fecal samples. We describe the simple, yet reproducible steps required to perform the assay, identifying an ideal volume of 7.5 μl for inoculation of material, as well as an ideal agar concentration of 0.4%. This protocol typically allows researchers to identify swarming within 24 h after incubation in a standard incubator.

Keywords: Bacterial swarming (细菌群集), Human gut microbiome (人类肠道微生物组), Frozen samples (冷冻样本), Complex material (复合材料), Agar (琼脂)

Background

Swarming is a unique bacterial property that ensures mass movement across a surface in a collective fashion (Verstraeten et al., 2008; Kearns, 2010). The exact host consequences of bacteria that swarm are not yet fully known; however, recent research has aimed to elucidate such a role in pathogenesis (Barak et al., 2009). It has been theorized that swarming enables bacteria to adhere to and disperse from localized spots of infection (Kearns, 2010), as well as to escape engulfment by macrophages (Amendola, 1998). For instance, the swarming of Proteus mirabilis has been implicated in catheter-associated urinary tract infections (Jones et al., 2004). Moreover, bacteria of many species become resistant to a range of antibiotics when they swarm, in a manner that appears unrelated to normal antibiotic efflux mechanisms (Butler et al., 2010).


More recently, our group has shown for the first time that swarming bacterial activity from human fecal matter is a specific indicator of intestinal inflammation and/or polyp formation (De et al., 2021). This represents a novel advancement since patient feces may now be used to inform clinicians in the assessment of “vague abdominal” symptoms among patients at risk for inflammatory bowel disease, ulcerative colitis, and polyps, among other conditions. This knowledge could help to guide clinical decision-making, such as determining which patients need a colonoscopy and further evaluation versus those who do not, ultimately reducing overall healthcare costs. As a result, the importance of optimizing an assay that detects swarming bacteria from feces is critical to the validation of our findings. Thus far, assays that focus on detecting swarming bacteria have been restricted to the study of cultured single strains of bacteria, namely Pseudomonas (Tremblay et al., 2008; Morris et al., 2011; Ha et al., 2014; Morales-soto et al., 2015). Ha et al., (2014), Morris et al., (2011), and Tremblay et al., (2008) describe thorough protocols for assaying bacterial swarming in Pseudomonas aeruginosa (Tremblay and Deziel, 2008; Morris et al., 2011; Ha et al., 2014); however, little, if any, attention has been focused on designing a protocol for eliciting bacterial swarming from human fecal samples, which are often the most convenient materials from which researchers can learn more about the microbial communities of the human gut. In this protocol, which we used in our group’s recent publication in De et al. (2021), we describe an assay to observe swarming in the shortest amount of time and with the least consumption of laboratory reagents. To the best of our knowledge, this is the first comprehensive description and optimization of an assay protocol that detects swarming bacteria from fecal matter.

Materials and Reagents

  1. Petri dish, stackable lid, 100 mm × 15 mm, sterile, polystyrene (Fisherbrand, Fisher Scientific, catalog number: FB0875713)

  2. Fecal specimen collection cup, 650 ml (Fisherbrand, Commode Specimen Collection System, catalog number: 23-038032)

  3. Microcentrifuge tube, 1.5 ml (Fisherbrand, Fisher Scientific, catalog number: 05-408-130)

  4. Adjustable 0.5-10 μl pipette (Eppendorf Research, catalog number: EPPR4402)

  5. 10 μl pipette Tips, racked, sterile (Thomas Scientific, catalog number: 1148U48)

  6. Agar, bacteriological grade (Research Products International, CAS number: 9002-18-0)

  7. Sodium chloride (Fischer Chemicals, Fisher Scientific, CAS number: 7647-14-5)

  8. Tryptone, microbiologically tested (Sigma-Aldrich, CAS number: 91079-40-2)

  9. Yeast extract (Acumedia, Neogen Corporation, catalog number: 7184B)

  10. Distilled water (Thermo Fisher Scientific, catalog number: 15230001)

  11. Agar LB plates (see Recipes)

Note: All materials and reagents are stored at room temperature.

Equipment

  1. Epson Expression 1600 Scanner (Epson, Expression 1600, catalog number: E1600-PRO)

  2. Incubator (Thermo Scientific, NAPCO Series 8000DH Incubator, catalog number: 3541)

  3. -80°C freezer (Thermo Fisher Scientific, Revco ExF, catalog number: EXF40086D)

  4. Autoclave (Amsco Scientific, Eagle, catalog number: SV120)

  5. Hotplate (Thermolyne, Cimarec, catalog number: HP131225)

  6. Biological hood (Thermo Fisher Scientific, 1300 Series Class II, Biological Safety Cabinet, catalog number: 1353)

Software

  1. ImageJ Version 1.59e (National Institutes of Health, https://imagej.nih.gov/ij/download.html)

Procedure

  1. Prepare the agar LB plates with a final agar concentration of 0.4% (see Recipes).


  2. Remove fecal samples from the freezer

    1. Fresh human fecal samples were obtained in 2015 (under protocols IRB# 2009-446 and 2015-4465) from healthy donors without intestinal distress and donors known to suffer from intestinal inflammation. Specimens were collected in sterile fecal specimen cups without preservatives. Specimens were then kept at 4°C for up to 15 min before transport to the laboratory at ambient temperature for processing, which consisted of aliquoting the specimen into a 100-μl microcentrifuge and storage at -80°C since 2015.

    2. Remove the fecal samples from the freezer and allow to thaw at room temperature on the bench.

    3. Under a biological hood, use a pipette to inoculate 7.5 μl healthy sample on one side of a plate (as a control) and 7.5 μl disease sample on the other half of the plate.

      Note: If the samples are not pre-homogenized, homogenization is preferable using a homogenizer for uniformity.

    4. Take care to avoid puncturing the agar with the pipette tip, as this may impede the ability to identify future swarming.

    5. Leave the cover off the Petri dish and allow to dry under a biological hood for 10 min.


  3. Incubation

    1. (Optional) Once dry, take an image of the plate on the scanner, which will serve as a “before image” for later reference. See Figure 1A below.

    2. Place the plate upright (not inverted) in an incubator, set to 37°C and a humidity of 80%.

    3. Remove the plate from the incubator after 24 h and assess visually for the presence of swarming.

    4. (Optional) Scan an image of the plate and label as “after image” for record-keeping. See Figure 1B below.

    5. (Optional) Input the before and after images into the NIH’s ImageJ program to quantitatively measure the folds of expansion. Use the “Free Select” tool to highlight an area in the ‘before image’, then highlight the area of swarming in the ‘after image’. This method allows measurement of the pixels of growth and enables quantitation even when swarming occurs in irregular directions.



      Figure 1. Bacterial swarming on LB agar plates before and after incubation. A. 7.5 μl fecal samples were inoculated onto 20 ml 0.4% LB agar plates, which were then scanned. B. Plates were placed in an incubator, set to 37°C and a humidity of 80%, and were removed and scanned after 24 h.

Data analysis

An agar concentration of 0.4% was found to elicit the most swarming (compared with agar concentrations of 0.5%, 0.6%, and 0.7%), as was a sample volume of 7.5 μl (compared with volumes of 2.5 μl or 5.0 μl). At the same time, the thawing method for the samples (on ice or at room temperature) was found to have no significant impact on the presence of swarming. These conclusions were drawn from the statistical analysis of 10 independent experiments (performed in duplicate) for 6 separate patient samples. One-way ANOVA was performed in GraphPad Prism (v8) to determine significant differences between agar and inoculant volume conditions.

Recipes

  1. Agar LB plates

    1 g Tryptone

    0.5 g Yeast extract

    0.5 g NaCl

    0.4 g Agar

    100 ml dH2O

    1. Combine the water and dry ingredients while stirring on a hotplate, until the water begins to boil, then autoclave to sterilize (121°C, 15 PSI, 65 min)

    2. After autoclaving, allow the 0.4% LB agar solution to cool until warm to the touch, stirring continuously to prevent clumping or hardening

    3. Pour 20 ml 0.4% LB agar solution into sterile Petri dishes

    4. Allow LB agar plates to cool and harden on the bench at RT for at least one h

Acknowledgments

Research was conducted under a Student Research Fellowship Award from the Crohn’s and Colitis Foundation of America (CCFA) #640806 awarded to A.B, as well as #431602 awarded to S.M. The data acquisition was also partially supported by the Broad Medical Research Program (BMRP) grant #362520 at CCFA to S.M. The authors would like to thank Arpan De and Hao Li for their advice, suggestions for variables to explore, and help with formatting and editing. This protocol was modified from work completed by our group, published in March of 2021 in Gastroenterology (De et al., 2021) https://doi.org/10.1053/j.gastro.2021.03.017.

Competing interests

The authors have no competing interests or financial disclosures to report.

Ethics

All described protocols have been approved by the Albert Einstein College of Medicine Institutional Review Board (IRB# 2009-446 and 2015-4465). Informed consent was given by the human subjects from whom fecal samples were acquired.

References

  1. Amendola, A., Geisenberger, O., Anderson, J. B., Givskov, M., Schleifer, K. H. and Eberl, L. (1998) Serratia liquefaciens swarm cells exhibit enhanced resistance to predation by Tetrahymena sp. FEMS Microbiol Lett 164(1): 69-75.
  2. Barak, J. D., Gorski, L., Liang, A. S. and Narm, K. E. (2009). Previously uncharacterized Salmonella enterica genes required for swarming play a role in seedling colonization. Microbiology 155(Pt 11): 3701-3709.
  3. Butler, M. T., Wang, Q. and Harshey, R. M. (2010) Cell density and mobility protect swarming bacteria against antibiotics. Proc Natl Acad Sci 107(8):3776-3781.
  4. De, A., Chen, W., Li, H., Wright, J. R., Lamendella, R., Lukin, D. J., Szymczak, W. A., Sun, K., Kelly, L., Ghosh, S., Kearns, D. B., He, Z., Jobin, C., Luo, X., Byju, A., Chatterjee, S., San Yeoh, B., Vijay-Kumar, M., Tang, J. X., Prajapati, M. et al. (2021). Bacterial Swarmers Enriched during Intestinal Stress Ameliorate Damage. Gastroenterology S0016-5085(21)00524-2.
  5. Ha, D. G., Kuchma, S. L. and O'Toole, G. A. (2014). Plate-based assay for swarming motility in Pseudomonas aeruginosa. Methods Mol Biol 1149: 67-72.
  6. Jones, B. V., Young, R., Mahenthiralingam, E. and Stickler, D. J. (2004) Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection. Infect Immun 72(7): 3941-3950.
  7. Kearns, D. B. (2010). A field guide to bacterial swarming motility. Nat Rev Microbiol 8(9): 634-644.
  8. Morales-Soto, N., Anyan, M. E., Mattingly, A. E., Madukoma, C. S., Harvey, C. W., Alber, M., Deziel, E., Kearns, D. B. and Shrout, J. D. (2015). Preparation, imaging, and quantification of bacterial surface motility assays. J Vis Exp 98.
  9. Morris, J. D., Hewitt, J. L., Wolfe, L. G., Kamatkar, N. G., Chapman, S. M., Diener, J. M., Courtney, A. J., Leevy, W. M. and Shrout, J. D. (2011). Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production. Appl Environ Microbiol 77(23): 8310-8317.
  10. Tremblay, J. and Deziel, E. (2008). Improving the reproducibility of Pseudomonas aeruginosa swarming motility assays. J Basic Microbiol 48(6): 509-515.
  11. Verstraeten, N., Braeken, K., Debkumari, B., Fauvart, M., Fransaer, J., Vermant, J. and Michiels, J. (2008). Living on a surface: swarming and biofilm formation. Trends Microbiol 16(10): 496-506.

简介

[摘要]蜜蜂分群 -跨冲浪动作敏捷ACE通过鞭毛推进-是许多细菌的独特性质。蜂群的作用,特别是在人类肠道微生物组的细菌种群中的作用,尚未得到充分了解;虽然,它正在成为越来越多的科学和临床研究领域。Ť ○在人类健康进一步表征细菌蜂拥,一个有效的测定法蜂拥利用复杂的材料,例如粪便物,是必要的。到现在为止,绝大多数群分析仅能适应培养物中生长的细菌,最常见的是假单胞菌。这些测定法倾向于使用标准 升ysogenic肉汤(LB)琼脂培养基; 但是,该试剂涉及尚未定制到复杂物料的接种。在本文中,我们提供了引发特殊协议的从冷冻的人类粪便样品细菌的蜂拥。我们描述了简单的,但代表roducible需要至p步骤erform测定,确定一个理想的体积7.5微升用于inoculat材料的离子,以及理想的琼脂的0.4%的浓度。该协议通常允许研究人员在标准培养箱中孵育后24小时内识别群居。

[背景]集群是一种独特的细菌特性,可确保物质以集体方式在整个表面上运动(Verstraeten等人,2008; Kearns,2010)。尚不完全了解成群细菌的确切宿主后果。然而,最近的研究旨在阐明这种在病原体逸出中的作用(Barak等,2009)。据推测,蜂拥使细菌坚持和分散感染的局部位置(卡恩斯,2010) ,以及以逃避吞噬的巨噬细胞(阿门多拉,1998年)。例如,在中蜂拥奇异变形杆菌已在导管相关尿路感染牵连(琼斯等人。,2004)。此外,许多物种的细菌在成群时对一系列抗生素具有抗性,其方式似乎与正常的抗生素外流机制无关(Butler et al。,2010)。

更最近,我们小组已经示出了用于第一次蜂拥细菌一个从人类粪便ctivity是肠炎症和/或息肉形成的特定指示符(德等人。,20 21 )。Ť他代表了一种新的进步,因为病人的粪便现在可以用来在炎症性肠疾病,溃疡性结肠炎,息肉,其他条件中的风险告知临床医生在患者的“模糊腹部”症状的评估。这方面的知识可以帮助以指导临床决策,如确定哪些患者需要结肠镜检查,并进一步评估对那些谁不这样做,最终降低总体医疗费用。因此,优化检测粪便中成群细菌的检测方法的重要性对于验证我们的发现至关重要。到目前为止,专注于检测群体细菌的检测方法仅限于培养单一细菌菌株,即假单胞菌(Tremlay等人,2008; Morris等人,2011; Ha等人,2014; Morales - soto等人,2015年)。Ha等人(2014),Morris等人(2011)和Tremblay等人(2014)。,(2008)描述了用于测定铜绿假单胞菌中细菌群体的彻底方案(Tremblay和Deziel ,2008; Morris等,2011; Ha等,2014)。^ h H但是,很少,如果有的话,注意力一直集中在设计方案从人类粪便样品,这往往是最方便的材料引发细菌蜂拥小号从中研究人员可以更多地了解人类肠道的微生物群落。在这个协议中,我们在我们的小组最近在出版物中使用消化科(https://doi.org/10.1053/j.gastro.2021.03.017),我们描述了一个实验,观察的时间,并与最短量蜂拥最少消耗实验室试剂。为了最好的我们所知,这是检测来自排泄物的细菌蜂拥化验协议的第一个全面的描述和优化。

关键字:细菌群集, 人类肠道微生物组, 冷冻样本, 复合材料, 琼脂



材料和试剂


陪替氏d杂交,可堆叠盖子,100毫米× 15毫米,无菌,聚苯乙烯(FISHERBRAND ,Fisher Scientific公司,目录号:FB0875713)
粪便标本收集杯,650 ml (Fisherbrand ,座厕标本收集系统,目录号:23-038032)
1.5 ml微量离心管(Fisherbrand ,Fisher Scientif ic,目录号05-408-130)
可调0.5- 10微升p ipette仪(Eppendorf研究,目录号:EPPR4402)
10个微升p ipette提示,- [R ACKED,小号terile(托马斯科学,目录号:1148U48)
琼脂,b acteriological摹RADE(研究产品国际,CAS号:9002-18-0)
钠Ç hloride(费舍尔化工,Fisher Scientific公司,CAS编号:7647-14-5)
胰蛋白,,经过微生物测试(Sigma-Aldrich,CAS编号:91079-40-2)
酵母提取物(Acumedia ,Neogen Corporation,目录号:7184B)
蒸馏水(Thermo Fisher Scientific,目录号:15230001)
琼脂LB p鲈(见食谱)
注意:所有材料和试剂均在室温下保存。


设备


Epson Expression 1600扫描仪(Epson,Expression 1600,目录号:E1600-PRO)
孵化器(Thermo Scientific,NAPCO Series 8000DH孵化器,目录号:3541)
-80°C冰箱(Thermo Fisher Scientific,Revco ExF ,目录号:EXF40086D)
高压灭菌器(Amsco Scientific ,Eagle,目录号:SV120)
加热板(Thermolyne ,Cima rec ,目录号:HP131225)
生物^ h OOD(赛飞世尔科技,1300系列II类,生物安全柜,目录号:1353)


软件


ImageJ的版本1.59e(研究所小号健康,https://imagej.nih.gov/ij/download.html)


程序


准备的一个GAR LB p与拉泰什一个的0.4%最终浓度的琼脂(参见食谱)。


从删除粪便样品的冰箱
在2015年(根据IRB#2009-446和2015-4465协议)从没有肠道困扰的健康捐献者和已知患有肠道炎症的捐献者中获得了新鲜的人类粪便样品。标本收集在不含防腐剂的无菌粪便标本杯中。然后样品保持在4℃下进行最多15分钟,运输前,实验室在环境温度下处理,其中由等分的所述样品置于一个100-微升在-80微量和存储℃下自2015。
删除了从粪便样品的冰箱,并允许在室温解冻彩画TURE在板凳上。
下一个生物罩,使用一个移液管来接种7.5微升在板的一侧(作为对照)和7.5健康样品微升疾病在板的另一半样品。
注意:如果样品未预先均质化,则最好使用均质器进行均质化以确保均匀性。


小心避免刺破的琼脂的吸管尖端,因为这可能会妨碍对确定今后的群聚能力。
离开所述的罩F中的P ETRI培养皿中并允许下干燥一个生物罩10分钟。


孵化
(ø ptional)一旦医生Ý ,取在板的n个图像的扫描仪,其将作为“前图像”以供以后参考。参见下面的图1A。
放置在在板直立(未反相)的培养箱中,设定为37℃和80%的湿度。
除去的从板的24小时后培养箱并目视评估蜂拥的存在。
(ø ptional)扫描的所述板和标签为“后的图像的图像”用于记录保存。参见下面的图1B。
(ø ptional)输入的之前和之后的图像到所述NIH的ImageJ的程序定量测定膨胀的褶皱。使用“自由选择”工具来凸显一个在“图像前区” ,牛逼母鸡突出“的形象后,蜂拥的区域” 。此方法允许生长的像素的测量并且使孔定量吨即使当在蜂拥不规则的方向发生通货膨胀。




图1.孵育前后,LB a gar平板上的细菌群。一。7.5微升粪便样品小号被接种到20毫升0.4%LB琼脂平板上,其然后被扫描。乙。板置于一个保温箱,设定为37℃和80%的湿度,并取出并扫描在24小时后。




数据分析


的琼脂浓度0.4%,发现引起最群聚(比较用琼脂精矿的0.5%,0.6%,和0.7%ations),作为是样品体积7.5微升(比较用2.5体积微升或5.0微升) 。同时,发现样品的解冻方法(在冰上或在室温下)对成群的存在没有显着影响。这些结论来自对6个独立患者样本的10个独立实验(一式两份)的统计分析。在GraphPad Prism(v8)中执行单向ANOVA,以确定琼脂和接种物体积条件之间的显着差异s 。


菜谱


琼脂LB p乳胶
1克胰蛋白Try


0.5克酵母ë XTRACT


0.5克氯化钠


0.4克琼脂


100毫升dH 2 O


结合的水和干成分而上搅拌一热板上,直到所述水开始沸腾,然后高压灭菌消毒(121 ℃,15 PSI,65分钟)
高压灭菌后,允许将0.4%LB一个GAR溶液冷却至温热到的触摸,搅拌连续吨ö防止结块或硬化
倾20毫升0.4%LB一个GAR小号olution到无菌P ETRI菜ES
允许LB一个GAR板以冷却并硬化于所述在RT长椅为至少一个H


致谢


研究从克罗恩病和美国结肠炎基金会(CCFA)#下一个学生研究奖学金奖进行了640806授予AB ,以及#431602颁发给SM 。数据采集也部分受支持的广泛的医学研究发展计划(BMRP)摹咆哮#362520在CCFA到SM作者竟被d要感谢Arpan德和豪礼为他们出谋划策,为变量的建议探索,并与格式帮助和编辑。该协议从工作由我们集团,完成修改发表在2021年三月消化科(德等人。,2021)https://doi.org/10.1053/j.gastro.2021.03.017。


利益争夺


作者们没有竞争利益小号或财务披露报告。


伦理


所有描述的方案均已获得阿尔伯特·爱因斯坦医学院学院机构审查委员会的批准(IRB#2009-446和2015-4465)。获得粪便样本的人类受试者给予了知情同意。


参考


Amendola,A.,Geisenberger,O.,Anderson,JB,Givskov ,M.,Schleifer,KH和Eberl ,L.(1998)沙雷氏菌液群细胞对四膜虫(Tetrahymena sp。)FEMS Microbiol Lett 164(1):69-75。
Barak,JD,Gorski,L.,Liang,AS和Narm ,KE(2009)。群聚所需的以前未鉴定的肠沙门氏菌基因在幼苗定殖中起作用。微生物学155(Pt 11):3701-3709。
Butler,MT,Wang,Q.和Harshey ,RM(2010)细胞密度和流动性可以保护成群的细菌免受抗生素的侵害。PROC国家科科学院科学107(8):3776-3781。
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引用:Byju, A. S., Patel, D., Chen, W. and Mani, S. (2021). Assessing Swarming of Aerobic Bacteria from Human Fecal Matter. Bio-protocol 11(9): e4008. DOI: 10.21769/BioProtoc.4008.
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