评估穆林伤口的生物膜分散
Summary
在这里，我们描述了 前活体 和 体内 方法，以评估细菌分散从伤口感染在小鼠。该协议可用于测试局部抗菌和抗生物膜疗法的疗效，或评估不同细菌菌株或物种的分散能力。
Abstract
生物膜相关感染与各种慢性疾病有关，如非愈合性糖尿病足溃疡、慢性鼻窦炎、复发性中耳炎介质等。这些感染中的微生物细胞受细胞外聚合物（EPS）的保护，它可以防止抗生素和宿主免疫细胞清除感染。为了克服这一障碍，研究人员已经开始开发分散剂作为潜在的治疗方法。这些制剂针对生物膜EPS中的各种成分，削弱结构，并启动细菌的分散，从理论上讲，这可以提高抗生素的效力和免疫清除。为了确定分散剂对伤口感染的疗效，我们制定了测量前 活体 和 体内生物膜分散的方案。我们使用鼠标手术切除模型，该模型已被很好地描述，以创建生物膜相关的慢性伤口感染。为了监测 体内的分散，我们用表达红十字路西法酶的细菌菌株感染伤口。一旦成熟感染已经确定，我们灌溉伤口的溶液含有酶，降解生物膜EPS的成分。然后，我们监测伤口和过滤器官中发光信号的位置和强度，以提供有关达到的分散水平的信息。对于生物膜分散的 活体 分析，受感染的伤口组织被淹没在生物膜降解酶溶液中，之后对组织中剩余的细菌负荷与溶液中的细菌负荷进行评估。这两种协议都有优点和缺点，可以优化，以帮助准确确定分散治疗的功效。
Introduction
全世界抗生素耐药性上升，导致缺乏抗生素治疗各种细菌感染的选择。除了抗生素耐药性外，细菌还可以通过采用与生物膜相关的生活方式2获得抗生素耐受性。生物膜是由多糖、细胞外DNA、脂质和蛋白质3的基质保护的微生物群落，统称为细胞外聚合物（EPS）。随着抗生素耐药性危机的继续，迫切需要延长抗生素使用或提高抗生素疗效的新战略。反生物膜制剂是一个有前途的解决方案4。
在已提出的不同反生物膜策略中，针对生物膜EPS不同成分的分散剂的利用处于治疗开发的前沿。糖苷水解（GH）是此类分散剂之一。GH 是一大类酶，它催化多糖内部不同键的，为 EPS 提供结构完整性。我们的小组，以及其他人，已经表明，GH可以有效地降解生物膜，诱导分散和提高抗生素疗效的一些不同的细菌物种，无论是体外和体内6，7，8，9，10，11。
随着人们对生物膜分散性的兴趣日益浓厚，开发评估分散疗效的有效方法非常重要。在这里，我们提出了一个详细的协议，用于治疗生物膜相关伤口感染与分散剂在小鼠，并评估分散效力， 在体内 和 前活体。总体目标是提供有效的方法，可用于前科模型，以有效和高效地测量生物膜的分散。
在这些研究中使用了一种穆林手术切除感染模型来建立与生物膜相关的感染。我们使用这个模型已超过15年，并广泛发表我们的观察7，9，12，13，14，15，16，17，18，19，20，21。一般来说，这是一种非致命性感染模式，细菌保持局部化到病床上，并且与生物膜相关（在EPS包围的聚合物中看到的细菌），建立一种持续长达3周的慢性感染。然而，如果小鼠免疫功能低下（例如1型糖尿病），它们可能更容易在这种模式下出现致命的全身感染。
在这份报告中，我们提供了评估细菌从伤口中传播的协议，无论是体内还是前体内。这两种协议都可用于检查分散剂的功效，并有自己的长处和短处。例如，评估体内的分散性可以提供重要的实时信息，说明细菌在分散后扩散到身体其他部位，以及宿主的反应。另一方面，评估分散前活体可能更可取地筛选多种制剂、剂量或配方，因为组织可以分为多个部分，可以单独测试，从而减少所需的小鼠数量。在评估多个制剂时，我们通常测量分散首先体外，如前所述的6，9，22。然后，我们测试最有效的前活体和储备在体内测试为有限的数量非常有前途的代理。
Protocol
该动物协议由得克萨斯理工大学健康科学中心机构动物护理和使用委员会（协议编号07044）审查和批准。这项研究是严格按照《国家卫生研究院实验室动物护理和使用指南》中的建议进行的。 1. 为小鼠感染准备细菌 注：在这里，我们描述感染小鼠只与 伪多莫纳斯阿鲁吉诺萨。然而，其他细菌物种可能被用来引起感染。细菌菌株和材料详见 材?…
Representative Results
在这个实验中，8-10周大的瑞士雌性韦伯斯特小鼠感染了携带发光质粒pQF50-lux的PAO1的104 个CFU。如上所述，感染允许建立48小时之前，管理3×30分钟的治疗PBS（车辆控制）或10%GH（治疗），以消化生物膜EPS。小鼠在治疗后（0 h）和治疗后10小时和20小时进行成像预处理。 图2A 和 补充图1 显示伤口内已建立的感染，产生明亮的生物发光信号。GH 治疗 （0 h） 后?…
Discussion
在这里，我们描述了可用于研究生物膜分散剂功效的协议。这些协议可以很容易地适应使用不同类型的分散剂，细菌物种或前活体样本，包括临床脱尿样本。该协议还提供了一个临床相关的模型，以收集和研究分散的细菌细胞。分散细菌细胞的表型已证明与浮石或生物膜细胞5、24、25、26的表型…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家卫生研究院（R21 AI137462-01A1）、泰德·纳什长寿基金会、贾斯珀·L和杰克·丹顿·威尔逊基金会以及国防部（国防部MDRP W0318_19_NM_PP）的资助。
Materials
| 1.5 mL microcentrifuge tube | Fisher | 14823434 | Use to complete serial dilutions of samples |
| 25G 58 in needle | Fisher | 14823434 | Attaches to 1 mL syringe |
| Ampicillin Sodium Salt | Fisher | BP1760-5 | Make a 50 mg/ mL stock solution and add 100 µL to 10 mL of LB broth for both overnight and subculture |
| Amylase | MP Biomedicals | 2100447 | Make a 5% w/v solution, vortex- other dispersal agents can be used |
| Buprenorphine SR-LAB 5 mL (1 mg/mL) | ZooPharm | RX216118 | Use as pain mainagement- may use other options |
| Cellulase | MP Biomedicals | 2150583 | Add 5% w/v to the 5% w/v amylase solution, vortex, activate at 37 °C for 30 min- other dispersal agents can be used |
| Depilatory cream | Walmart | 287746 | Use a small amount to massage into the hair follicles on the back of the animal and allot 10 min to remove hair |
| Dressing Forceps, Serrated Tips | Fisher | 12-460-536 | Can use other forms of forceps |
| Erlenmeyer flasks baffled 125 mL | Fisher | 101406 | Use to grow overnights and sub-cultures of bacteria |
| FastPrep-24 Benchtop Homogenizer | MP Biomedicals | 6VFV9 | Use 5 m/s for 60 s two times to homogenize tissue |
| Fatal Plus | Vortech Pharmaceuticals | 0298-9373-68 | Inject 0.2 mL intraperitaneal for each mouse |
| Homogenizing tubes (Bead Tube 2 mL 2.4 mm Metal) | Fisher | 15340151 | Used to homogenize samples for plating |
| Isoflurane | Diamond Back Drugs | ||
| Ketamine hydrochloride/xylazine hydrochloride solution C-IIIN | Sigma Aldrich | K4138 | Use as anasethia- other options can also be utilized to gain a surgical field of anasethia |
| LB broth, Miller | Fisher | BP1426-2 | Add 25 g/L and autoclave |
| Lidocaine 2% Injectable | Diamond Back Drugs | 2468 | Inject 0.05 mL through the side of the marked wound bed area so it is deposited in the center of the mark. Allot 10 min prior to cutting |
| Meropenem | Sigma Aldrich | PHR1772-500MG | Make 5 mg/mL to add to the GH solution to apply topically and a 15 mg/mL solution to inject intraperitaneal 4 h prior and 6 h post-treatment |
| Non-sterile cotton gauze sponges | Fisher | 13-761-52 | Use to remove the depilatory cream |
| PAO1 pQF50-lux bacterial strain | Ref [13] | N/A | PAO1 pgF50-lux was used as the P. aeruginosa strain of interest in this paper's representative results |
| Petri dishes | Fisher | PHR1772-500MG | |
| Phosphate Buffer Saline 10x | Fisher | BP3991 | Dilute 10x to 1x prior to use |
| Polyurethane dressing | Mckesson | 66024007 | Cut the rounded edge off and cut the remaining square into 4 equal sections |
| Pseudomonas isolation agar | VWR | 90004-394 | Add 20 mL/L of glycerol and 45 g/mL to water, autoclave, and pour 20 mL into petri dishes |
| Refresh P.M. | Walmart | Use on eyes to reduce dryness during procedure. | |
| Sterile Alcohol Prep Pads | Fisher | 22-363-750 | Use to clean the skin immediately prior to wounding to disinfect the area |
| Straight Delicate Scissors | Fisher | 89515 | Can also use curved scissors |
| Swiss Webster mice | Charles River | 551NCISWWEB | Other mice strains can be used |
| Syring Slip Tip 1 mL | Fisher | 14823434 | Used to administer drugs and enzyme treatment |
Referenzen
- Rossolini, G. M., Arena, F., Pecile, P., Pollini, S. Update on the antibiotic resistance crisis. Current Opinion in Pharmacology. 18, 56-60 (2014).
- Stewart, P. S. Antimicrobial Tolerance in Biofilms. Microbiology Spectrum. 3 (3), (2015).
- Flemming, H. C., et al. Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology. 14 (9), 563-575 (2016).
- Rumbaugh, K. P. How well are we translating biofilm research from bench-side to bedside. Biofilm. 2 (100028), (2020).
- Rumbaugh, K. P., Sauer, K. Biofilm dispersion. Nature Reviews Microbiology. 18 (10), 571-586 (2020).
- Fleming, D., Chahin, L., Rumbaugh, K. Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds. Antimicrobial Agents and Chemotherapy. 61 (2), (2017).
- Fleming, D., Rumbaugh, K. The Consequences of Biofilm Dispersal on the Host. Scientific Reports. 8 (1), 10738 (2018).
- Baker, P., et al. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms. Science Advances. 2 (5), 1501632 (2016).
- Redman, W. K., Welch, G. S., Rumbaugh, K. P. Differential Efficacy of Glycoside Hydrolases to Disperse Biofilms. Frontiers in Cellular and Infection Microbiology. 10, 379 (2020).
- Zhu, L., et al. Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms. Environmental Microbiology. 20 (6), 2026-2037 (2018).
- Fell, C. F., Rumbaugh, K. P. . Antibacterial Drug Discovery to Combat MDR. , 527-546 (2019).
- Dalton, T., et al. An in vivo polymicrobial biofilm wound infection model to study interspecies interactions. PLoS One. 6 (11), 27317 (2011).
- Wolcott, R. D., et al. Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window. Journal of Wound Care. 19 (8), 320-328 (2010).
- Korgaonkar, A., Trivedi, U., Rumbaugh, K. P., Whiteley, M. Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection. Proceedings of the National Academy of Sciences of the United States of America. 110 (3), 1059-1064 (2013).
- Watters, C., et al. Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice. Medical Microbiology and Immunology. 202 (2), 131-141 (2013).
- Watters, C., Everett, J. A., Haley, C., Clinton, A., Rumbaugh, K. P. Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms. Infection and Immunity. 82 (1), 92-100 (2014).
- Turner, K. H., Everett, J., Trivedi, U., Rumbaugh, K. P., Whiteley, M. Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection. PLOS Genetics. 10 (7), 1004518 (2014).
- Harrison, F., et al. A 1,000-Year-Old Antimicrobial Remedy with Antistaphylococcal Activity. mBio. 6 (4), 01129 (2015).
- Wolcott, R., et al. Microbiota is a primary cause of pathogenesis of chronic wounds. Journal of Wound Care. 25, 33-43 (2016).
- Ibberson, C. B., et al. Co-infecting microorganisms dramatically alter pathogen gene essentiality during polymicrobial infection. Nature Microbiology. 2, 17079 (2017).
- Fleming, D., et al. Utilizing Glycoside Hydrolases to Improve the Quantification and Visualization of Biofilm Bacteria. Biofilm. 2, (2020).
- DeLeon, S., et al. Synergistic interactions of Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro wound model. Infection and Immunity. 82 (11), 4718-4728 (2014).
- Darch, S. E., et al. Phage Inhibit Pathogen Dissemination by Targeting Bacterial Migrants in a Chronic Infection Model. mBio. 8 (2), (2017).
- Chua, S. L., et al. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles. Nature Communications. 5, 4462 (2014).
- Beitelshees, M., Hill, A., Jones, C. H., Pfeifer, B. A. Phenotypic Variation during Biofilm Formation: Implications for Anti-Biofilm Therapeutic Design. Materials (Basel). 11 (7), (2018).
- Sauer, K., et al. Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J Bacteriol. 186 (21), 7312-7326 (2004).
- Kuklin, N. A., et al. Real-time monitoring of bacterial infection in vivo: development of bioluminescent staphylococcal foreign-body and deep-thigh-wound mouse infection models. Antimicrobial Agents and Chemotherapy. 47 (9), 2740-2748 (2003).
- Francis, K. P., et al. Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Infection and Immunity. 69 (5), 3350-3358 (2001).
- Kadurugamuwa, J. L., et al. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrobial Agents and Chemotherapy. 48 (6), 2283-2287 (2004).
- Wimpenny, J., Manz, W., Szewzyk, U. Heterogeneity in biofilms. FEMS Microbiology Reviews. 24 (5), 661-671 (2000).
- Stewart, P. S., Franklin, M. J. Physiological heterogeneity in biofilms. Nature Reviews Microbiology. 6 (3), 199-210 (2008).
- Tipton, C. D., et al. Chronic wound microbiome colonization on mouse model following cryogenic preservation. PLoS One. 14 (8), 0221656 (2019).
Diesen Artikel zitieren
Redman, W. K., Welch, G. S., Rumbaugh, K. P. Assessing Biofilm Dispersal in Murine Wounds. J. Vis. Exp. (174), e62136, doi:10.3791/62136 (2021).