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Jan 2021
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Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
筛选与治疗相关的噬菌体的溶原活性   

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Abstract

Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal activity; yet, these are not definitive indicators. As a result, the presence of integrase genes is often used as a hallmark for lysogenic behavior; however, the accuracy of genetic screening for lysogeny depends on the quality of the extraction, sequencing and assembly of the phage genome, and database comparison. The present protocol describes a simple phenotypic test that can be used to screen therapeutically relevant phages for lysogenic behavior. This test relies on the identification of spontaneous phage release from their lysogenized host and can be reliably used in cases where no sequencing data are available. The protocol does not require specialized equipment, is not work-intensive, and is broadly applicable to any phage with an easily culturable bacterial host, making it particularly amenable to settings with limited resources.


Graphical abstract:


Screening pipeline for lysogen activity of a given phage


Keywords: Bacteriophage (噬菌体), Phage therapy (噬菌体疗法), Lysogeny (溶原现象), Prophage integration (原噬菌体整合), Prophages (原噬菌体), Acinetobacter baumannii (鲍氏不动杆菌), Enterococcus faecium (屎肠球菌)

Background

The field of phage therapy, which is the clinical use of viruses to kill bacteria, has grown exponentially over recent decades (Gordillo Altamirano and Barr, 2019). Prior to their selection for therapeutic use, phages are typically subjected to a thorough characterization pipeline (Terwilliger et al., 2020). Lysogenic or temperate phages are those with the capacity to integrate themselves into their host’s genome, and are routinely excluded from therapeutic use due to their subpar bactericidal ability, the emergence of homoimmunity, and the potentially harmful consequences of lysogenic conversion (Fortier and Sekulovic, 2013; Hyman, 2019).


Temperate behavior is first suspected in phages that produce turbid plaques on bacterial lawns. However, plaque morphology can vary greatly depending on a variety of factors, including the growth medium used and the physiological state of the bacterial host (Ramesh et al., 2019). Delayed or substandard bactericidal activity in bacterial growth assays can also serve as an indicator, but their interpretation is not always clear-cut. Alternatively, genotypic screening for genes that are essential for lysogeny, such as integrase and excisionase enzymes, are further indicators of temperate behavior (Hyman, 2019). However, extraction and sequencing of phage genomes can be challenging, and the accuracy of homology-based analyses is highly dependent on the quality of the sequencing data, assemblies, and available databases (Russell, 2018). Importantly, the presence of a predicted integrase in the genome of a phage may not be definite confirmation of the ability to lysogenize a host in vitro or in vivo (Howard-Varona et al., 2017).


Here, we outline a simple protocol, modified from Pope et al. (2013), that evaluates the ability of a given phage to lysogenize its host in vitro. The assay can be reliably used in cases where no sequencing data are available. Furthermore, this protocol has the advantage of providing confirmation of lysogenic behavior for a given phage, instead of prediction, given by genome sequencing-based tests. It does not require specialized equipment and can complement analysis of plaque morphology and growth. Although we describe the protocol using an Acinetobacter baumannii-specific phage (Gordillo Altamirano et al., 2021), we also provide data from an Enterococcus faecium-specific phage (Figure 1), demonstrating that the methodology is broadly applicable to any phage with a bacterial host culturable on solid media. As such, incubation and media conditions may need be adapted to suit the host. Finally, in addition to excluding lysogenic phages from therapeutic use, the protocol can be used to produce lysogens (bacteria harboring a prophage) for downstream applications in phage biology and biotechnology research.


Materials and Reagents

  1. 15-ml polystyrene tubes (In Vitro Technologies, Falcon, catalog number: FAL352095)

  2. Pipettes, serological pipettes, and pipette tips

  3. 10-ml sterile rimless glass test tubes (Thermo-Fisher, Youlyy, catalog number: LBSDCT13100)

  4. Wire loop

  5. 10-ml syringes with detachable needle (McFarlane, Terumo, catalog number: 19046TE)

  6. 0.2-μm filters, attachable to syringes (Pall, catalog number: 4612)

  7. Phage to be tested, high titer lysate (≥ 106 plaque-forming units [PFU]/ml)

    Note: For a protocol on phage isolation and purification, see Bonilla et al., 2016.

  8. 1× phosphate-buffered saline (PBS) solution (Merck Australia, OmniPur, catalog number: US16506)

  9. BactoTM Tryptone (Gibco, catalog number: 211705)

  10. Granulated yeast extract (Merck Australia, Millipore, catalog number: 1037530500)

  11. NaCl (Merck Australia, Supelco, catalog number: 1064040500)

  12. Agar powder (Merck Australia, Millipore, catalog number: 05040)

  13. Overnight culture in LB of the phage’s bacterial host (see Recipes for LB medium)

  14. LB medium top agar (see Recipes)

  15. LB medium agar plates (see Recipes)

    Note: The growth media in 13-15 can be replaced to cater for the bacterial host’s growth requirements.

Equipment

  1. Microwave (Panasonic, model: 32 L stainless steel, catalog number: NN-ST67JSQPQ)

  2. 37°C incubator

  3. Shaking platform (Thermo Scientific, model: CO2-resistant shaker, catalog number: 88881102)

  4. Bunsen burner

  5. Benchtop centrifuge (Eppendorf South Pacific, model: 5810 R, catalog number: 5811000487)

  6. Autoclave (Tuttnauer, model: vertical 110L, catalog number: 5050ELV-D)

Procedure

  1. Coincubation of phages and bacterial hosts (spot assay)

    Note: The estimated time to complete this section is 45 min of benchwork followed by overnight incubation.

    1. In a sterile glass test tube, mix 1 ml bacterial culture with 3 ml molten top agar.

      Note: The top agar can be liquified using a microwave. Be careful: too hot a temperature of the top agar will kill the bacterial population, resulting in a poor lawn. The top agar should be liquid but pleasantly warm to the touch (approximately 50°C).

    2. Pour the mixture onto a room-temperature LB agar plate and allow to solidify.

    3. Prepare two or three serial dilutions (~ 1 ml) of the high-titer phage lysate in PBS.

      Note: We recommend using at least two concentrations of phage, for example 108 and 106 PFU/ml.

    4. Spot 10-μl drops of each phage lysate dilution onto the surface of the prepared plate.

      Note: Spots of further dilutions or replicates of the same dilutions can be added to the same plate.

    5. Leave the plate, lid partially on, close to a Bunsen burner until the spots are completely dry; this can take up to 30 min. Alternatively, the plates can be left to dry in a biosafety hood.

    6. Incubate at 37°C for up to 4 days, with daily checks to assess the formation of “mesas” (Figure 1A) (Pope et al., 2013).

      Note: Mesas are zones of confluent bacterial growth in the center of the lysis spots. Bacteria growing on mesas have become resistant to the phage, possibly (but not exclusively) due to homoimmunity. While checking for mesas, also look for agar dehydration due to prolonged incubation, which could hamper bacterial viability. Desiccation can be further avoided by incubating the plates in a sealed container with a wet paper towel, creating a humid chamber.

    7. Using a sterile wire loop, scrape one loopful of bacterial growth from a mesa and use it as the primary inoculum for a streak plate on fresh LB agar (see Graphical abstract). Repeat for each mesa.

    8. Incubate at 37°C overnight. Assess the growth of clearly isolated colonies.

      Note: Any of these colonies could contain the integrated prophage. We routinely test 10 colonies obtained from each of at least two mesas, for a total of at least 20 colonies.


  2. Patch plate screen

    Note: The estimated time to complete this section is 30 min of benchwork and multiple overnight incubations.

    1. Label and divide two LB agar plates into a grid with as many sections as colonies to be screened. Number the sections on both plates identically from 1 to 20.

      Note: A single plate can comfortably fit 20 colonies.

    2. Prepare one of the plates with a lawn of bacterial host following Steps A1 and A2. The other plate is used without further preparation.

    3. Using a sterile wire loop, a disposable pipette tip, or a sterile toothpick, take a random colony from the streak plate (prepared in Step A7), gently patch it onto section 1 of the LB plate, and then onto its corresponding section of the LB plate with the bacterial host lawn (Step B2).

      Note: Avoid tearing the delicate agar while performing the patches.

    4. Repeat Step B3 with as many colonies as needed.

    5. Incubate the plates at 37°C for up to 2 days, with daily checks for interpretation.

    6. To interpret the test, look closely at the plate with the host lawn. Positive colonies, those potentially harboring the prophage, will present signs of lysis (plaques, halos, loss of turbidity) on the lawn around them, which is caused by the spontaneous release of the phage (Figure 1B).

      Note: If all the screened colonies are negative for the patch test, it can be concluded that the phage is unable to lysogenize the host (see Table 1). False positive results here are possible due to carry-over of phage particles from the mesa onto the streak plate (Step A7).

    7. For each positive colony, return to the LB plate without the host lawn (prepared in Steps B3-B4), pick a section of the patch with a wire loop, and streak onto a fresh LB plate.

    8. Incubate overnight at 37°C.

    9. Pick a single colony with a wire loop and re-streak onto a fresh LB plate.

    10. Incubate overnight at 37°C.

      Note: You have now performed a double single-colony purification. Using colonies from this plate (B9), repeat Procedure B (Figure 1C) and perform tests C and D to confirm lysogenic behavior.


  3. Spontaneous phage release in liquid culture test (Supernatant assay)

    Note: The estimated time to complete this section is 45 min of benchwork followed by overnight incubation.

    1. Following purification of the colony of interest with two consecutive streak plates, inoculate the colony into a 10-ml Falcon tube containing ~4 ml LB.

    2. Incubate overnight at 37°C with aeration.

      Note: Aeration, achieved by leaving enough head space in the tube and incubating on a shaking platform at 150 rpm, is needed for optimal growth of the host used in this example. Growth conditions can be modified to cater to different hosts.

    3. Centrifuge the culture at 3,500 × g for 10 min.

    4. Collect the supernatant using a 10-ml syringe.

    5. Attach a 0.2-μm filter to the syringe and carefully depress the plunger to filter the supernatant into a fresh tube.

      Note: This step is optional. If not performed, we recommend repeating Steps C3 and C4.

    6. Perform a spot assay (Procedure A), replacing the phage lysate with the filtered supernatant from C5.

    7. For test interpretation, look for signs of lysis on the spot assay caused by the spontaneous release of phages from the lysogens into the supernatant of the liquid culture (Figure 1D).


  4. Immunity assay

    Note: The estimated time to complete this section is 15 min of benchwork followed by overnight incubation.

    1. Perform a spot assay (Procedure A) replacing the original bacterial host with the candidate lysogen purified in Step B9.

    2. For test interpretation, look for the absence of lysis caused by homoimmunity (no clearing, plaques, or loss of turbidity on the bacterial lawn) (Figure 1E).

      Note: An immunity assay showing resistance to the original phage, without positive findings of lysogeny on Procedures B and C, is suggestive of alternative mechanisms of phage resistance. Conversely, a confirmed lysogen will test positive in Procedures B, C, and D. For an expanded explanation regarding interpretation of the procedures, see Figure 1 and Table 1.



      Figure 1. Screening for lysogenic behavior of phage øCO01 (Gordillo Altamirano et al., 2021) on host strain Acinetobacter baumannii A9844 (Peleg et al., 2008) and phage øFG-VRE (unpublished) on host strain Enterococcus faecium ST796 (Buultjens et al., 2017). A. Spot assay with droplets of 108 and 106 PFU/ml phage lysate after a 48-h incubation. Black stars denote mesas. B. First patch assay using plates with and without lawns of the bacterial hosts. Black arrows (colonies 9, 13, and 15 on A. baumannii and colony 11 on E. faecium) show bacterial growth with surrounding lysis, indicating the presence of phage, possibly released from a lysogen. Zoomed sections are provided as examples of positive and negative colonies. For A. baumannii, the panel only shows half the total screened colonies (18/36). C. Second patch plate, after two rounds of single-colony purification of colonies 9, 13, and 15 (A. baumannii) and colony 11 (E. faecium), without indication of phage presence when patched onto plates containing bacterial host lawns. D. Spot assay performed with the filtered supernatant from cultures of the putative lysogenic colonies, with no indication of spontaneous phage release. E. Immunity assay of colonies 9 and 13 (A. baumannii) and colony 11 (E. faecium) indicates sensitivity to their respective phages, thereby excluding homoimmunity; for colony 15 (A. baumannii), the test demonstrates resistance to the phage, most likely due to a mechanism other than homoimmunity (see Table 1 for interpretation). Taken together, the tests demonstrate the inability of phages øCO01 and øFG-VRE to lysogenize host strains A. baumannii A9844 and E. faecium ST796, respectively. For assays with E. faecium, LB was replaced with BHI (brain heart infusion) media. Scale bars = 0.5 cm.

Data analysis

A summary of the most frequent combinations of results in each of the four described procedures and a guide to their interpretation are included in Table 1. Briefly, lysogenic behavior of a phage can be safely excluded when at least 20 host colonies from at least two different mesas are negative in a patch test (Procedure B). Additionally, for a host colony to be confirmed as harboring the prophage, it must undergo double single-colony purification and subsequently test positive in Procedures B, C, and D.


Table 1. Interpretation guide. Most commonly encountered combinations of results and their interpretation.

* At least 20 colonies screened, 10 from each of 2 different mesas. +: positive test. -: negative test.

Recipes

  1. LB medium

    10 g BactoTM Tryptone

    10 g granulated yeast extract

    5 g NaCl

    500 ml deionized water

    Note: Autoclave, cool down, and store at room temperature.

  2. LB agar plates

    7.5 g agar powder

    500 ml sterile LB medium

    Note: Autoclave, allow to cool, pour ~20 ml per petri dish, allow to solidify, and store at 4°C.

  3. LB top agar

    1.5 g agar powder

    200 ml sterile LB medium

    Note: Autoclave, cool down, and store at room temperature. When needed, warm up in a microwave for ~3 min.

Acknowledgments

Fernando L. Gordillo Altamirano acknowledges the support received from Monash University through the Monash Postgraduate Research Scholarship funding his doctoral studies. This work, including the efforts of Jeremy J. Barr, was funded by the Australian Research Council (ARC), Discovery Early Career Researcher Award (DECRA) (DE170100525), National Health and Medical Research Council (NHMRC: 1156588), and the Perpetual Trustees Australia award (2018HIG00007).

    We thank Prof Anton Y. Peleg and Prof Tim P. Stinear for providing the bacterial strains used in the present study. This protocol is derived from previous work by Broussard et al., 2013 and Pope et al., 2013 and is previously described in a summarized form by Gordillo Altamirano et al., 2021.

Competing interests

The authors declare not to have any competing interests.

References

  1. Bonilla, N., Rojas, M. I., Netto Flores Cruz, G., Hung, S. H., Rohwer, F. and Barr, J. J. (2016). Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ 4: e2261.
  2. Broussard, G. W., Oldfield, L. M., Villanueva, V. M., Lunt, B. L., Shine, E. E. and Hatfull, G. F. (2013). Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches. Mol Cell 49(2): 237-248.
  3. Buultjens, A. H., Lam, M. M., Ballard, S., Monk, I. R., Mahony, A. A., Grabsch, E. A., Grayson, M. L., Pang, S., Coombs, G. W., Robinson, J. O., Seemann, T., Johnson, P. D., Howden, B. P. and Stinear, T. P. (2017). Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium. PeerJ 5: e2916.
  4. Fortier, L. C. and Sekulovic, O. (2013). Importance of prophages to evolution and virulence of bacterial pathogens. Virulence 4(5): 354-365.
  5. Gordillo Altamirano, F. L., Forsyth, J. H., Patwa, R., Kostoulias, X., Trim, M., Subedi, D., Archer, S., Morris, F. C., Oliveira, C., Kielty, et al. (2021). Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.Nat Microbiol doi:10.1038/s41564-020-00830-7
  6. Gordillo Altamirano, F. L. and Barr, J. J. (2019). Phage Therapy in the Postantibiotic Era. Clin Microbiol Rev 32(2).
  7. Howard-Varona, C., Hargreaves, K. R., Abedon, S. T. and Sullivan, M. B. (2017). Lysogeny in nature: mechanisms, impact and ecology of temperate phages. ISME J 11(7): 1511-1520.
  8. Hyman, P. (2019). Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth. Pharmaceuticals (Basel) 12(1): 35.
  9. Peleg, A. Y., Tampakakis, E., Fuchs, B. B., Eliopoulos, G. M., Moellering, R. C., Jr. and Mylonakis, E. (2008). Prokaryote-eukaryote interactions identified by using Caenorhabditis elegans. Proc Natl Acad Sci U S A 105(38): 14585-14590.
  10. Pope, W., Sarkis, G., Hatfull, G. and Broussard, G. (2013). Lysogeny experiments. Retrieved from https://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1261&context=honors.
  11. Ramesh, N., Archana, L., Madurantakam Royam, M., Manohar, P. and Eniyan, K. (2019). Effect of various bacteriological media on the plaque morphology of Staphylococcus and Vibrio phages. Access Microbiol 1(4).
  12. Russell, D. A. (2018). Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes. Methods Mol Biol 1681: 109-125.
  13. Terwilliger, A. L., Gu Liu, C., Green, S. I., Clark, J. R., Salazar, K. C., Hernandez Santos, H., Heckmann, E. R., Trautner, B. W., Ramig, R. F. and Maresso, A. W. (2020). Tailored Antibacterials and Innovative Laboratories for Phage (Phi) Research: Personalized Infectious Disease Medicine for the Most Vulnerable At-Risk Patients. Phage 1(2): 66-74.


简介


[摘要]溶源性噬菌体可以集成到他们的细菌宿主的基因组中,有可能将任何遗传信息它们具有包括毒力或抗性基因,并且是第erefore从治疗应用常规排除。溶原行为通常见于创造混浊斑块或噬菌体具有欠佳的杀菌活性; 但是,这些并不是确定的指标。结果,整合酶基因的存在经常被用作溶原行为的标志。ħ H但是,遗传筛选的精度溶原 依赖于提取,排序的质量和组件噬菌体基因组,一个的第二数据库COMPAR ISON 。个E存在协议描述了一个简单的表型试验,可用于筛选治疗相关的噬菌体溶原性为行为。个是测试依赖于自发释放噬菌体的识别从它们的溶源化的宿主,并且可以在没有测序数据的情况下能够可靠地使用。该方案不需要专门的设备,不需要大量的劳动,并且广泛适用于具有易于培养的细菌宿主的任何噬菌体,使其特别适合于资源有限的环境。

图形摘要:

小号creen ING管道的溶源的给定噬菌体的活动

[背景]噬菌体疗法,所述的场是临床上使用的病毒来杀死细菌,已经生长成指数超过最近十年(戈迪略阿尔塔米拉诺和巴尔,2019) 。在选择其用于治疗用途之前,通常将噬菌体进行彻底的表征流水线处理(Terwilliger等,2020)。溶原性或温带型噬菌体是具有整合自身能力的宿主噬菌体,由于它们的杀菌能力低,同质免疫力的出现以及溶原性转化的潜在有害后果而被常规排除在治疗用途之外(Fortier和Sekulovic,2013 ;海曼(Hyman),2019年)。

首先怀疑在细菌性草坪上产生混浊噬菌斑的噬菌体中存在温和的行为。但是,噬菌斑形态可能会因多种因素而有很大差异,包括使用的生长培养基和细菌宿主的生理状态(Ramesh et al。,2019)。细菌生长测定中延迟或不合标准的杀菌活性也可以用作指示剂,但其解释并不总是很明确。或者,对溶原性必不可少的基因(例如整合酶和切除酶)进行基因型筛选是温带行为的进一步指标(Hyman,2019)。然而,噬菌体基因组的提取和测序可能具有挑战性,基于同源性的分析的准确性高度依赖于测序数据,程序集和可用数据库的质量(Russell,2018)。重要的是,噬菌体基因组中预测的整合酶的存在可能不能肯定地证实在体外或体内裂解宿主的能力(Howard-Varona et al。,2017)。

在这里,我们概述了从Pope等人修改而来的简单协议。(2013),其评估了给定噬菌体体外溶血其宿主的能力。在没有测序数据的情况下,该测定法可以可靠地使用。此外,该协议具有提供的优点确认对于给定的噬菌体,而不是溶原行为,预测,通过基因组sequenc给予荷兰国际集团系测试。它不需要专门的设备,并且可以补充对牙菌斑形态和生长的分析。虽然我们describ Ë协议使用的鲍曼不动杆菌-特异性噬菌体(戈迪略阿尔塔米拉诺等人,202。1 ),我们还提供数据的肠球菌,屎特异的噬菌体(图URE 1),这表明该方法广泛地适用于任何噬菌体,带有可在这种盖培养基上培养的细菌宿主。因此,可能需要调整孵育条件和培养基条件以适合宿主。最后,除了EXC泸定从治疗用途溶源性噬菌体,该协议可以用于产生细胞溶素原(细菌携带原噬菌体)用于噬菌体生物学和生物技术研究的下游应用。

关键字:噬菌体, 噬菌体疗法, 溶原现象, 原噬菌体整合, 原噬菌体, 鲍氏不动杆菌, 屎肠球菌

材料和试剂

1. 15 -毫升聚苯乙烯试管(体外技术,隼,目录号:FAL352095)     

2.移液器,血清移液管,和枪头     

3. 10 -毫升无菌无框玻璃试管(热-费希尔,Youlyy ,目录号:LBSDCT13100)     

4.线环     

5. 10 -毫升注射器用可拆卸的针(麦克法兰,泰尔茂,目录号:19046TE)     

6. 0.2 - μ米过滤器,附接至注射器(帕尔,目录号:4612)     

7.噬菌体进行测试,高滴度ř裂解物(≥ 10 6噬斑形成单位[PFU] /毫升)     

注意:有关噬菌体分离和纯化的方案,请参见Bonilla等人,2016年。


8. 1 ×磷酸盐缓冲盐水(PBS)溶液(Merck Australia,OmniPur ,目录号:US16506)     

9. Bacto TM Tryptone (Gibco ,目录号:211705)     

10. ģ ranulated Ý东提取物(Merck公司澳大利亚,Millipore公司,目录号:1037530500) 

11. NaCl(澳大利亚默克公司,Supelco,目录号:1064040500) 

12.琼脂粉(澳大利亚默克公司,密理博,目录号:05040) 

13.在噬菌体细菌宿主的LB中过夜培养(请参阅有关LB培养基的食谱) 

14. LB中顶琼脂(请参阅食谱) 

15. LB中型琼脂平板(请参阅食谱) 

注意:可以替换13-15中的生长培养基,以满足细菌宿主的生长需求。


设备


Microwav e(Panasonic,型号:32 L不锈钢,目录号:NN-ST67JSQPQ)
37 ℃下我ncubator
摇动平台(热科学,型号:CO 2 -耐振动筛,目录号:88881102)
本生灯
台式Ç entrifuge(EP pendorf南太平洋,型号:5810 R,目录号:5811000487)
高压灭菌器(Tuttnauer,型号:立式110L,货号:5050ELV-D)


程序


噬菌体和细菌宿主的共孵育(斑点测定)
注意:对E stimated时间来完成此部分是在45分钟钳工接着温育过夜。


在无菌玻璃试管中,将1 ml细菌培养物与3 ml熔融的顶部琼脂混合。
注意:顶部琼脂可以用微波炉液化。小心:太热了一顶琼脂的温度会杀死细菌群,从而在一个贫穷的草坪。顶部琼脂应为液体,但触感宜温暖(约50 °C )。


倒混合TURE到室温LB琼脂平板上,并允许以固化。
准备在PBS中的两滴或三滴高滴度噬菌体裂解液的系列稀释液(〜1 ml )。
注意:我们建议使用至少两种浓度的噬菌体,例如10 8和10 6 PFU / ml 。


点10 - μ大号每个噬菌体裂解滴稀释到所制备的板的表面上。
注意:可以将更多稀释液的斑点或相同稀释液的复制品添加到同一板中。


离开板,盖子部分上,靠近本生灯直到斑点是完全干燥; Ť他可能需要长达30分钟。可替代地,板Ç一个留在生物安全橱中干燥。
孵育在37 ℃下进行长达4天,每天检查以评估“台面”(图的形成URE 1A) (教皇等人,2013年)。
注意:台面是在裂解点中心汇合的细菌生长区域。在台地上生长的细菌可能已经(但不是唯一地)由于同种免疫而对噬菌体产生抗性。在检查台面时,还应考虑由于长时间孵育而导致琼脂脱水,这可能会阻碍细菌的生存能力。通过将板与湿纸巾在密封容器中孵育,可以进一步避免干燥,从而形成一个潮湿的腔室。


使用无菌线环,刮去一个铂环量BACT的erial生长从台面,并使用它作为在新鲜LB条纹平板琼脂初级接种物(见图形一bstract)。对每个台面重复上述步骤。
在37 °C下孵育过夜。评估明显分离的菌落的生长。
注意:这些殖民地中的任何一个都可能包含整合的预言。我们通常从至少两个台面中分别获取10个菌落,总共至少20个菌落。


隔板板筛
注意:对E stimated的时间来完成这一部分为30的分钳工和多个过夜温育。


将两个LB琼脂平板贴上标签并将其分成一个网格,该网格具有与要筛选的菌落一样多的部分。将两个板上的部分编号从1到20相同。
注意:一个平板可以舒适地容纳20个菌落。


按照步骤A1和A2,用细菌宿主的草坪准备其中一个板。无需另外准备即可使用另一块板。
使用无菌钢丝环,一次性吸管tip或无菌牙签,从条纹板中取一个随机菌落(在步骤A7中准备),轻轻地将其贴在LB板的第1部分上,然后贴在LB板的相应部分上LB板和细菌宿主草坪(步骤B2)。
注意:执行修补程序时,避免撕裂精致的琼脂。


重复S B重复B3,并根据需要添加尽可能多的菌落。
将平板在37 °C下孵育最多2天,并每天进行解释性检查。
要解释该测试,请仔细观察板与宿主草坪。阳性菌落(可能带有噬菌体的菌落)在其周围的草坪上会出现裂解迹象(噬菌斑,晕圈,浊度损失),这是由于噬菌体的自发释放引起的(图1B)。
注意:如果所有筛选的菌落均对斑片试验均呈阴性,则可以得出结论,噬菌体无法裂解宿主(见表1)。这里假阳性结果是可能的,因为结转从台面的噬菌体颗粒的直径:n要条纹板(步骤A7 )。


对于每个阳性菌落,在没有寄主草坪的情况下返回LB平板(在步骤B3-B4中准备),用金属丝环拾取一部分补片,然后将其涂在新鲜的LB平板上。
在37 °C下孵育过夜。
挑选一个带有线环的菌落,然后重新铺在新鲜的LB平板上。
在37 °C下孵育过夜。
注意:您现在已经执行了两次单菌落纯化。使用来自该板的菌落(B9),重复步骤B(图1C)并执行测试C和D以确认溶原性行为。


液体培养试验中的自发噬菌体释放(上清液测定)
注意:对E stimated时间来完成此部分是在45分钟钳工接着温育过夜。


以下PURIF的ication与两个连续的条纹感兴趣的菌落板,接种菌落成10 -毫升Falcon管中含有约4毫升LB.
于37 °C曝气下孵育过夜。
注意:通气是通过在试管中留有足够的顶部空间,并在振荡平台上以150 rpm的速度进行孵育来实现的,这是本例中所用宿主的最佳生长所必需的。可以修改生长条件以适应不同的宿主。


将培养物以3500 × g离心10分钟。
使用10收集上清液-毫升注射器。
附加一个0.2 - μ微米的过滤器到注射器并小心压下柱塞到上清液过滤到新的管中。
注意:此步骤是可选的。如果未执行ed ,我们建议重复步骤C3和C4。


执行斑点测定(P rocedure A) ,与从该C5过滤的上清液更换噬菌体裂解物。
对于测试的解释,外观为当场测定裂解的迹象引起的自发释放的噬菌体从溶源到液体培养物(图1的上清液URE 1D)。


免疫测定
注:电子stimated的时间来完成这部分是15分的钳工,然后过夜培养。


执行点检(步骤A),将原始细菌宿主替换为在步骤B9中纯化的候选溶原菌。
为了进行测试解释,请检查是否没有由于同质免疫引起的裂解(在细菌性草坪上没有清除,斑块或浑浊的损失)(图1E)。
注:表示对原始噬菌体抗性的免疫测定法中,没有阳性发现的溶原上过程B和C,则提示的噬菌体的替代机制电阻。相反,确认溶源将在程序B,C试验阳性,和D.对于一个扩展说明关于的程序解释,参见图1和表1。




图1.筛选噬菌体øCO01 (Gordillo Altamirano等,2021)对宿主菌株鲍曼不动杆菌A9844 (Peleg等,2008)和噬菌体øFG- VRE(未发表)在宿主菌株肠球菌粪肠球菌ST796 (Buultjens等)的溶原行为等人,2017)。A.斑点法用10的液滴8和10 6 PFU / ml的后噬菌体裂解一个48 -小时的温育。黑星表示台面。B.使用带有和不带有细菌宿主的板的平板进行第一次斑块测定。黑色箭头(菌落9,13 ,和15上A.杆菌和集落11上E.屎肠球菌)示出了与周围的裂解,表明噬菌体的存在,可能从释放细菌生长溶素原。变焦编部分被提供作为阳性和阴性菌落例子。为A.杆菌,面板仅示出了总筛选菌落(18/36)的一半。C.第二补丁板,经过两轮单集落的纯化的菌落9,13 ,和15(A.杆菌)和集落11(E.屎肠球菌),无噬菌体存在的指示时贴在板小号含有细菌宿主草坪s 。D.用来自假定的溶原菌落的培养物的过滤的上清液进行斑点测定,没有自发噬菌体释放的迹象。菌落9和13(的E.免疫测定法A.杆菌)和集落11(E.屎肠球菌)表示灵敏度到其各自的噬菌体,从而排除homoimmunity ; 对于第15菌落(A. baumannii ),该试验证明对噬菌体具有抗性,最有可能是由于除同质免疫以外的其他机制所致(有关解释,请参见表1)。总之,试验证明噬菌体无力øCO01和OFG -VRE到lysogenize宿主菌株A.杆菌A9844和E.屎肠球菌ST796,分别。对于测定法E.屎肠球菌,LB用BHI(脑心脏浸液)培养基替换。比例尺= 0.5厘米。


数据分析


结果的最频繁的组合的总结我n各自的四个描述的过程和引导到它们的解释被包括在表1中。简单地说,噬菌体的溶原行为可以安全地排除当从至少两个至少20个宿主菌落不同台面是负我n中的斑贴试验(方法B)。此外,要确认宿主菌群具有该噬菌体,必须对其进行两次单菌落纯化,然后在步骤B,C和D中测试呈阳性。


表1.解释指南。最常见的结果组合及其解释。


*筛选出至少20个菌落,从2个不同的台面中各筛选10个菌落。+:阳性测试。-:阴性测试。


菜谱


LB培养基
10克Bacto TM胰蛋白p


10克颗粒状酵母提取物


5克氯化钠


500毫升去离子水


注意:高压灭菌,冷却并在室温下保存。


LB琼脂板
7.5克琼脂粉


500 ml无菌LB培养基


注意:高压灭菌器,冷却,倒入每个培养皿约20 ml ,使其固化,并在4 °C下储存。


LB顶级琼脂
1.5克琼脂粉


200 ml无菌LB培养基


注意:高压灭菌,冷却并在室温下保存。W¯¯需要母鸡,在〜3分钟的微波热身。


致谢


费尔南多·戈迪略·阿尔塔米拉诺(Fernando L. Gordillo Altamirano)感谢莫纳什大学通过莫纳什研究生研究奖学金为他的博士研究提供的支持。这项工作包括杰里米·巴尔(Jeremy J. Barr)的努力,由澳大利亚研究委员会(ARC),发现早期职业研究者奖(DECRA)(DE170100525),国家卫生与医学研究委员会(NHMRC:1156588)和永久基金会资助。澳大利亚受托人奖(2018HIG00007)。


我们感谢安东教授Y.法勒和教授蒂姆·P. Stinear提供在次使用的细菌菌株E存在研究。该协议源自Broussard等人(2013年)和Pope等人(2013年)的先前工作。,2013和被预先在概括形式所描述戈迪略阿尔塔米拉诺等人。,202 1 。


利益争夺


作者宣称没有任何利益冲突。


参考


Bonilla,N.,Rojas,MI,Netto Flores Cruz,G.,Hung,SH,Rohwer ,F.和Barr,JJ(2016)。Tap噬菌体-一种快速高效的制备噬菌体实验室储备液的方案。PeerJ 4:e2261。
Broussard,GW,Oldfield,LM,Villanueva,VM,Lunt,BL,Shine,EE和Hatfull ,GF(2013)。依赖整合的噬菌体免疫力提供了对遗传开关进化的见解。摩尔电池49(2):237-248。
Buultjens ,AH,林,MM,巴拉德,S.,和尚,IR,Mahony ,AA,Grabsch ,EA,Grayson,ML,Pang,S.,Coombs,GW,Robinson,JO,Seemann ,T.,Johnson,PD ,Howden ,BP和Stinear ,TP(2017)。万古霉素耐药粪肠球菌ST796克隆的进化起源。PeerJ 5:e2916。
LC,Fortier和O.Sekulovic (2013)。噬菌体对细菌病原体的进化和毒力的重要性。毒力4(5):354-365。
戈迪略阿尔塔米拉诺,FL,福赛斯,JH,Patwa ,R.,Kostoulias ,X.,修剪,M.,苏贝迪,D.,射手,S.,莫里斯,FC,Oliveira的,C.,Kielty ,等人。(202 1 )。抵抗噬菌体的鲍曼不动杆菌对抗菌剂重新敏感。Nat Microbiol doi:10.1038 / s41564-020-00830-7
佛罗里达州的Gordillo Altamirano和JJ的Barr(2019)。抗生素时代后的噬菌体疗法。临床微生物学修订版32(2)。
Howard-瓦罗纳,C.,哈格里夫斯,KR,Abedon ,ST和Sullivan,MB(2017)。自然界的溶源性:温带噬菌体的机制,影响和生态。ISME J 11(7):1511-1520。
Hyman,P.(2019年)。用于噬菌体治疗的噬菌体:分离,鉴定和宿主范围广度。制药(巴塞尔)12(1):35。
Peleg,AY,Tampakakis ,E.,Fuchs,BB,Eliopoulos,GM,Moellering ,RC,Jr。和Mylonakis ,E。(2008)。通过使用秀丽隐杆线虫鉴定原核生物与真核生物的相互作用。PROC国家科科学院科学美国阿105(38):14585-14590。
教皇,W。萨基斯,G.Hatfull,G。和布鲁萨德,G。(2013年)。溶血原性实验。取自https://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1261&context=honors。
拉梅什,N.,阿奇纳,L.,马杜兰塔卡姆Royam ,M.,马诺哈尔,P。和Eniyan ,K。(2019)。各种细菌培养基对葡萄球菌和弧菌噬菌体的噬菌斑形态的影响。访问微生物1 (4)。
罗素,DA(2018)。测序,组装和完成完整的噬菌体基因组。方法分子生物学1681:109-125。
Terwilliger ,AL,Gu Liu,C.,Green,SI,Clark,JR,Salazar,KC,Hernandez Santos,H.,Heckmann ,ER,Trautner ,BW,Ramig ,RF和Maresso ,AW(2020)。针对噬菌体(Phi)研究的量身定制的抗菌药物和创新实验室:针对最脆弱的风险患者的个性化传染病医学。噬菌体1(2):66-74。
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引用:Gordillo Altamirano, F. L. and Barr, J. J. (2021). Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages. Bio-protocol 11(8): e3997. DOI: 10.21769/BioProtoc.3997.
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Prasanth Manohar
Zhejiang University, Zhejiang University-University of Edinburgh (ZJU-UoE)
What do you think about the addition of mitomycin c after the patch-plate assay for phage induction?
2021/4/23 19:49:57 回复
Jeremy Barr
Monash University Clayton

Hi Prasanth!
It is an interesting idea with addition of Mitomycin C for the patch plate. We've typically seen quite strong auto-induction for lysogens in this assay and haven't needed mitomycin, but it may increase sensitivity of the assay

2021/4/27 16:35:03 回复