欢迎您, 登录 | 注册

首页 | English

X
加载中

Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa
从绿脓杆菌生物膜和浮游种群中分离持留细胞

微生物学 > 微生物生物膜 > 生物膜培养
作者: Cláudia N. H. Marques
Cláudia N. H. MarquesAffiliation 1: Department of Biological Sciences, Binghamton University, State university of New York at Binghamton, Binghamton, USA
Affiliation 2: Binghamton Biofilm Research Center, Binghamton University, State university of New York at Binghamton, Binghamton, USA
For correspondence: cmarques@binghamton.edu
Bio-protocol author page: a2543
Vol 5, Iss 18, 9/20/2015, 2545 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1590

[Abstract] Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).
Keywords: Persister cells(耐药株细胞), Pseudomonas aeruginossa(aeruginossa假单胞菌), Biofilms(生物膜), Tube reactors(管式反应器), Planktonic populations(浮游种群)

[Abstract]

Materials and Reagents

  1. Bacterial culture [Pseudomonas aeruginosa PA14]. This method has also been performed for Escherichia coli (BW25113) and Staphylococcus aureus (ATCC, catalog number: 6538)]
  2. LB broth Lennox (BD, catalog number: 240230 )
  3. Ciprofloxacin hydrochloride (Corning Incorporated, catalog number: 61-277-RF )
  4. Agar (BD, catalog number: 214040 )
  5. MgCl2.7H2O (VWR International, BDH®, catalog number: 0288-VBD )
  6. DI water (distilled water)
  7. Sodium chloride (VWR International, BDH®, catalog number: 0241-VBD )
  8. Spooled Masterflex peroxide-cured silicone tubing, L/S 14, 250 ft. (Cole-Parmer, catalog number: UX-96407-14 )
  9. Masterflex Norprene tubing (A60 G), L/S 13, 50 ft. (Cole-Parmer, catalog number: UX-06404-13 )
  10. PVDF barbed Y connector, 3/8" ID, 1/4", 2-3/8", 1-3/4"; Pack Of 10 (Cole-Parmer, catalog number: WU-30703-93 )
  11. Barbed fittings, Straight Connector, Clear PP,1/16" ID, 1/32", 25/32", 1/4" (Cole-Parmer, catalog number: WU-30506-00 )
  12. Barbed fittings, Reducing Connector, Clear PP,1/8" x 1/16" ID, 3/32", 13/16", 1/8" (Cole-Parmer, catalog number: WU-30506-06 )
  13. Barbed fittings, T connector, Kynar, 1/4" ID, 1/8", 1-15/16", 1-5/16"; 10/pack (Cole-Parmer, catalog number: WU-30703-75 )
  14. Sterile Petri dishes
  15. Microfuge tubes
  16. Syringe 60 ml
  17. Syringe needle (Gauge 21G)
  18. Micropipettes (P1000, P200, P20)
  19. Aluminum Foil
  20. Saline (see Recipes)
  21. Saline with Mg2Cl4.7H2O (see Recipes)
  22. LB agar with Mg2Cl4.7H2O (see Recipes)

Equipment

  1. Peristaltic pump (Cole-Parmer, catalog number: EW-07553-80 ) with an 8 channel, 6 rollers, 3-stop Ismatec minicartridge pump head (Cole-Parmer, catalog number: EW-78002-50 )
  2. Inoculation ports (VWR® Sleeve Stoppers) (VWR International, catalog number: 89097-534 )
  3. GeneMate Incubated Shakers (BioExpress, catalog number: H-2000-M )
  4. General Purpose Laboratory Incubators, SHEL LAB (BioExpress, catalog number: G-1400-1 )
  5. Nalgene® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
  6. Nalgene® Top WorksTM Aseptic Closure System, Silicone, for Bottles and Carboys, Thermo Scientific (VWR International, catalog number: 2135-8303 )
  7. Acro® 50 Vent Filters, Pall Laboratory (VWR International, catalog number: 28143-616 )
  8. Erlenmeyer Flasks (150 ml)
  9. Homogenizer (PRO SCIENTIFIC, model: Bio-Gen Pro200 )
  10. Table top refrigerated Centrifuge (Eppendorf, catalog number: 5418R )
  11. Spectrophotometer (BD, catalog number: DU700 series )

Procedure

The procedure for persister cell isolation relies on activation of the SOS response together with stringent response, through the use of ciprofloxacin as an antibiotic of choice (Keren et al., 2004; Sufya et al., 2003; Dörr et al., 2009) and saline as carrier solution (Sufya et al., 2003). The presence of ciprofloxacin activates the SOS DNA damage response via a mechanism that involves both recA and lexA and all non-persister cells are killed, while at the same time, persister cell formation occurs when a specific high or low level of SOS function is expressed (Dörr et al., 2009). The use of saline as a carrier for ciprofloxacin leads to an absence of nutrients and activates the stringent response where activation of relA and spoT results in increased levels of ppGpp that inhibit rRNA synthesis and lead to a decrease of the cell’s metabolism and a higher number of persister cells (Bernier et al., 2013, Potrykus and Cashel, 2008). Following isolation of persister cells from planktonic or biofilm cell populations, the persister cell state must be confirmed by performing the part C.

  1. Persister cell isolation from planktonic stationary phase cells.
    1. Prepare a streak plate on LB agar of a frozen stock of the bacteria of interest, and incubate under static conditions at 37 °C, for 24 h.
    2. Remove 1-2 colonies from the streak plate and inoculate a broth culture of 5 ml LB broth.
    3. Incubate at 37 °C with agitation (220 rpm) for a period of 12 h (overnight culture).
    4. Dilute the overnight culture to 1% in 50 ml of fresh LB medium in an Erlenmeyer flask. Note 2 describes problems that might arise with inoculum variability.
    5. Incubate at 37 °C with agitation (220 rpm) for a period of 24 h.
    6. Collect cells by centrifugation at 16,000 x g for 5 min, at 4 °C.
    7. Resuspend in 50 ml of cold (refrigerated and kept on ice) saline (0.85% NaCl).
    8. Repeat steps A6-7.
    9. Measure cell turbidity at a wavelength of 600 nm (OD600).
    10. Calculate how much culture (now in saline) will be needed to have a final OD600 of 0.8 in 25 ml.
      1. Example: Initial OD600 is 2.0
      2. Final OD600 needed is 0.8
      3. Final volume needed is 25 ml
      4. So, culture volume needed is
    11. In duplicate, remove 10 ml (as per example) of culture into a fresh centrifuge tube.
    12. Collect cells by centrifugation at 16,000 x g for 5 min, at 4 °C.
    13. Resuspend in 25 ml of cold (refrigerated) saline.
    14. Add ciprofloxacin at 20x the minimum inhibitory concentration (MIC) (treated sample) or saline containing acetic acid at the appropriate concentration, to account for the acetic acid present in the ciprofloxacin stock solution (control). For P. aeruginosa PA14, we used ciprofloxacin at a concentration of 20 μg/ml (original stock solution of ciprofloxacin was 4 mg/ml in saline with 0.1% acetic acid).
      Note: MIC was calculated using the methodology described by Andrews (2001). Different bacterial strains and the same strain in another laboratory can have different MICs thus, the MIC needs to be calculated for each individual bacterial strain.
    15. Incubate at 37 °C with agitation (220 rpm) for a period of 24 h.
    16. Perform sampling (500 μl) at time points 0, 1, 3, 5, 7, 9, 12, 15 and 24 h.
    17. For each time point, perform a set of 10 - Fold serial dilution series from 0 to 10-6.
    18. Drop plate 10 μl of each sample dilution onto a LB agar plate (100 x 15 mm) containing 1% MgCl2.7H2O (to neutralize ciprofloxacin) and allow the drop to slide on the agar surface, to form a line, so that the drop spreads and colonies have a higher surface area to grow. Each plate can have a maximum of 5 drops. Perform triplicate sample plates to ensure accuracy.
    19. Incubate plates at 37 °C for a period of 24-48 h.
    20. Count colonies between 10-100.
    21. Calculate the CFU/ml:
      1. Example # colonies (50) in the 10-2 dilution, the CFU/ml would be: 50 x 102/0.01 = 5 x 105 CFU/ml.
    22. Plot results as % viable cells vs time. Viable cells % is calculated based on the CFU/ml obtained for time 0, which is considered as 100% viable cells.
    23. Perform experiments in quadruplicate, this will allow for calculation of whether the results obtained are statistical significant when comparing controls to treated samples using ANOVA, followed by the Tukey’s comparison test.

  2. Persister cell isolation from biofilms
    Biofilms were cultured on tube reactors as described in the references below (Sauer et al., 2002; Davies and Marques, 2009; Marques et al., 2014), and briefly in Note 1.
    1. Repeat steps A1-5 in the above procedure for persister cell isolation from planktonic cultures.
    2. Measure the OD600 of the overnight culture, and standardize the culture to have an OD600 of 0.8 in 50 ml of LB media.
    3. Aspirate the culture with a 60 ml syringe with a needle and then cap the needle. Perform all this under aseptic conditions.
    4. Inoculate each tube reactor (Figure 2) with 2 ml of culture.
    5. Following 1 h incubation at room temperature - to allow the bacteria to attach to the silicone tubing (this time can vary, depending on the bacterial strain) - initiate the flow.
    6. Allow biofilms to develop for a period of 6 days.
    7. Expose biofilms to saline or 150 μg/ml of ciprofloxacin (150x MIC) in saline, for a period of 24 h. Monitor viability at 0, 1, 3, 5, 7, 9, 12 and 24 h.
    8. At each time point, for each sample (one control and one treated), one biofilm tube needs to be sacrificed and its contents need to be harvested using the rolling pin method (Sauer et al., 2002; Davies and Marques, 2009; Marques et al., 2014). The silicone tube containing the biofilms (Figure 2) is disconnected from the inoculation port and from the waste and placed onto a lab bench, after which, the pin is rolled on top of the tube to extrude out the liquid and the biofilm paste into centrifuge tubes containing 1 ml of saline with 1% MgCl2.7H2O (to neutralize ciprofloxacin).
    9. Homogenize samples using a homogenizer at speed 4 (approximately 20,000 rpm) for a period of 20 sec. Disinfect homogenizer between samples with 70% ethanol for a period of 1 min, followed by sterile water. Keep samples on ice during this step.
    10. Perform a set of 10 - Fold serial dilution series from 0 to 10-8.
    11. Drop plate 10 μl of sample onto a LB agar plate containing 1% MgCl2.7H2O. Each plate can have a maximum of 5 drops. Perform at least duplicate plates to ensure accuracy.
    12. Incubate plates at 37 °C for a period of 24-48 h.
    13. Count colonies between 10-100.
    14. Calculate the CFU/cm2:

      1. Example # colonies (50) in the 10-2 dilution, the CFU/ml would be: 50 x 102/0.01 = 5 x 105 CFU/ml.
      2. If sample was collected into 1 ml, then the total CFUs would be 5 x 105 CFU.
      3. If the internal volume of the tube is 2 cm2, then CFU/cm2 would be: 5 x 105/2 = 2.5 x 105 CFU/cm2.
    15. Plot results as % of viable cells vs time. Viable cells % is calculated based on the CFU/ml obtained for time 0, which is considered as 100% viable cells.
    16. Perform experiments in quadruplicate, this will allow for calculation of whether the results obtained are statistical significant, when comparing controls to treated samples using ANOVA followed by the Tukey’s comparison test.

  3. Confirmation of the persister cell state.
    1. Isolate persister cells as described above in A and B.
    2. At 18 h of ciprofloxacin exposure:
      1. In planktonic persister cell isolation experiments (Part A above), collect all cells present in the Erlenmeyer flask, for each treatment (ciprofloxacin or saline) by centrifugation at 16,000 x g for 5 min, at 4 °C and resuspend each original sample, in 12.5 ml of saline and 12.5 ml of ciprofloxacin (20 mg/L) in saline. Refer to Note 3 in regards to separation of dead cells from sample.
      2. In biofilm persister cell isolation experiments (Part B above) switch the ciprofloxacin exposure to either saline or further ciprofloxacin.
    3. Monitor viability at 0, 1, 3, 5, 7, 9, 12 and 24 h and process the data as described above.

Representative data

Using this methodology, persister cells are isolated using the SOS response, as a standard procedure (Keren et al., 2004; Dörr et al., 2009; Moker et al., 2010; Keren et al., 2004; Pan et al., 2012; Niepa et al., 2012; Hong et al., 2012). When performing persister cell isolation, either in planktonic or biofilm cultures, a typical biphasic killing curve should be observed, in the presence of ciprofloxacin (Figure 1A). Where in the first 3-6 h an exponential decrease of cell number is observed, followed by a killing plateau where no further decrease of cell viability is observed. Cells present in this plateau are considered to be the persister cell sub-population within a bacterial culture and can be from 0.0001 to 0.1% of the total population.
Once persister cells are isolated, confirmation of a persister cell state is determined by exposing the isolated cells to ciprofloxacin (20 mg/L) or saline for further 24 h. If cells are truly in a persister state, then no reduction of cell viability is observed (Figure 1B).


Figure 1. Isolation of persister cells from biofilm populations of P. aeruginosa. A. Biphasic curve observed upon exposure of P. aeruginosa biofilms to ciprofloxacin (squares) together with control, biofilms exposed to saline (open circles). B. Confirmation of persister cell state, exposure of persister cells to ciprofloxacin (black triangle) does not result in decrease of cell viability compared to control (inverted triangle). (Copyright© 2014, American Society for Microbiology ( Marques et al., 2014).

Notes

  1. In this work, biofilms were cultured in a tube reactor system (Figure 2) consisting of L/S 14 Masterflex peroxide-cured silicone tubing with 5% LB pumped through at a rate of 10.8 ml/h (Sauer et al., 2002; Davies and Marques, 2009; Marques et al., 2014). Each tube reactor was inoculated with 2 ml of a standardized overnight culture (OD600 of 0.8) and incubated, under static conditions, for a period of 1 h to facilitate cell attachment.
  2. When performing persister cell isolation in planktonic populations, the inoculum needs to be processed identically between experiments or the number of persister cells will change, creating variability. Thus, with a low bacterial cell inoculum, an absence of colonies might be present in the persister cell population, this will not be due to the absence of persister cells but to the low cell numbers and the detection levels of viable cell assessment using CFU determinations.
  3. When confirming persister cell state in planktonic populations, the cell pellet will present a gradient due to the present of dead cells. The dead cells will readily resuspend onto the liquid, and will be removed when removing the supernatant, while the live cells will remain in the pellet.

    A

    B

    Figure 2. Biofilm tube reactor system. This is a once through continuous culture system where biofilms develop inside silicone tubes. The bacterial culture is inoculated on the inoculation port and biofilms develop downstream from the pump. A. Schematic of a tube reactor system containing 4 silicone tubes. B. An assembled tube reactor system containing 16 silicone tubes where the cultures will run in tandem.

Recipes

  1. Saline
    8.5 g sodium chloride
    1 L of DI water
  2. Saline with MgCl2.7H2O
    8.5 g sodium chloride
    10 g MgCl2.7H2O
    1 L of DI water
  3. LB agar with MgCl2.7H2O
    20 g LB broth Lennox
    15 g agar
    10 g MgCl2.7H2O
    1 L of DI water

Acknowledgments

Persister isolation is a modification of previously published protocols (Keren et al., 2004; Sufya et al., 2003). This work was supported by SUNY structural funds.

References

  1. Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48 Suppl 1: 5-16.
  2. Bernier, S. P., Lebeaux, D., DeFrancesco, A. S., Valomon, A., Soubigou, G., Coppee, J. Y., Ghigo, J. M. and Beloin, C. (2013). Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin. PLoS Genet 9(1): e1003144.
  3. Davies, D. G. and Marques, C. N. (2009). A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 191(5): 1393-1403.
  4. Dörr, T., Lewis, K. and Vulic, M. (2009). SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet 5(12): e1000760.
  5. Hong, S. H., Wang, X., O'Connor, H. F., Benedik, M. J. and Wood, T. K. (2012). Bacterial persistence increases as environmental fitness decreases. Microb Biotechnol 5(4): 509-522.
  6. Keren, I., Kaldalu, N., Spoering, A., Wang, Y. and Lewis, K. (2004). Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230(1): 13-18.
  7. Keren, I., Shah, D., Spoering, A., Kaldalu, N. and Lewis, K. (2004). Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186(24): 8172-8180.
  8. Marques, C. N., Morozov, A., Planzos, P. and Zelaya, H. M. (2014). The fatty acid signaling molecule cis-2-decenoic acid increases metabolic activity and reverts persister cells to an antimicrobial-susceptible state. Appl Environ Microbiol 80(22): 6976-6991.
  9. Niepa, T. H., Gilbert, J. L. and Ren, D. (2012). Controlling Pseudomonas aeruginosa persister cells by weak electrochemical currents and synergistic effects with tobramycin. Biomaterials 33(30): 7356-7365.
  10. Pan, J., Bahar, A. A., Syed, H. and Ren, D. (2012). Reverting antibiotic tolerance of Pseudomonas aeruginosa PAO1 persister cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one. PLoS One 7(9): e45778.
  11. Potrykus, K. and Cashel, M. (2008). (p)ppGpp: still magical? Annu Rev Microbiol 62: 35-51.
  12. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W. and Davies, D. G. (2002). Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J Bacteriol 184(4): 1140-1154.
  13. Sauer, K., Cullen, M. C., Rickard, A. H., Zeef, L. A., Davies, D. G. and Gilbert, P. (2004). Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J Bacteriol 186(21): 7312-7326.
  14. Sufya, N., Allison, D. G. and Gilbert, P. (2003). Clonal variation in maximum specific growth rate and susceptibility towards antimicrobials. J Appl Microbiol 95(6): 1261-1267.

材料和试剂

  1. 细菌培养物[铜绿假单胞菌 PA14]。 对于大肠杆菌(BW25113)和金黄色葡萄球菌(ATCC,目录号:6538)也已进行了该方法。
  2. LB肉汤Lennox(BD,目录号:240230)
  3. 盐酸环丙沙星(Corning Incorporated,目录号:61-277-RF)
  4. 琼脂(BD,目录号:214040)
  5. (VWR International,BDH ,目录号:0288-VBD)
  6. 去离子水(蒸馏水)
  7. 氯化钠(VWR International,BDH ,目录号:0241-VBD)
  8. 缠绕的Masterflex过氧化物固化的硅氧烷管,L/S 14,250英尺(Cole-Parmer,目录号:UX-96407-14)
  9. Masterflex Norprene管(A60G),L/S 13,50英尺(Cole-Parmer,目录号:UX-06404-13)
  10. PVDF有倒钩的Y型连接器,3/8"ID,1/4",2-3/8",1-3/4" Pack Of 10(Cole-Parmer,目录号:WU-30703-93)
  11. 直型连接器,清除PP,1/16"ID,1/32",25/32",1/4"(Cole-Parmer,目录号:WU-30506-00)
  12. 倒钩连接器,清除PP,1/8"x 1/16"ID,3/32",13/16",1/8"(Cole-Parmer,目录号:WU-30506-06)
  13. 倒钩接头,T型连接器,Kynar,1/4"ID,1/8",1-15/16",1-5/16" 10/pack(Cole-Parmer,目录号:WU-30703-75)
  14. 无菌培养皿
  15. Microfuge管
  16. 注射器60 ml
  17. 注射器针(量规21G)
  18. 微量移液器(P1000,P200,P20)
  19. 铝箔
  20. 盐水(见配方)
  21. 盐水与Mg 2+ sub 4+ 7H O(参见配方)
  22. LB琼脂,其中Mg 2 Cl 4 sub 7H O(参见配方)

设备

  1. 蠕动泵(Cole-Parmer,目录号:EW-07553-80),带有8通道,6个辊,3挡Ismatec小型泵头(Cole-Parmer,目录号:EW-78002-50)
  2. 接口(VWR ®套筒塞)(VWR International,目录号:89097-534)
  3. GeneMate Incubated Shakers(BioExpress,目录号:H-2000-M)
  4. 通用实验室培养箱,SHEL LAB(BioExpress,目录号:G-1400-1)
  5. Nalgene ® Carboys with Handles,Polypropylene,Thermo Scientific(VWR International,目录号:16101-084)
  6. Nalgene ® Top Works TM 无菌封闭系统,硅胶,用于瓶和瓶,Thermo Scientific(VWR国际,目录号:2135-8303)
  7. Acro ® 50通风过滤器,Pall Laboratory(VWR国际,目录号:28143-616)
  8. 锥形烧瓶(150ml)
  9. 均质器(PRO SCIENTIFIC,型号:Bio-Gen Pro200)
  10. 台式冷冻离心机(Eppendorf,目录号:5418R)
  11. 分光光度计(BD,目录号:DU700系列)

程序

持续细胞分离的程序依赖于通过使用环丙沙星作为选择的抗生素的SOS反应的激活以及严格反应(Keren等人,2004; Sufya et al。 ,2003; Dör等人,2009)和盐水作为载体溶液(Sufya等人,2003)。环丙沙星的存在通过涉及recA 和 lexA 的机制激活SOS DNA损伤反应,并且所有非持续细胞被杀死,而同时,持续细胞当表达特定的高或低水平的SOS功能时形成发生(Dör等人,2009)。使用盐水作为环丙沙星的载体导致营养物质的缺乏并激活严格的反应,其中relA 和 spoT 的激活导致抑制rRNA合成的ppGpp水平增加并导致细胞代谢的减少和更多数量的持续细胞(Bernier等人,2013,Potrykus和Cashel,2008)。在从浮游生物或生物膜细胞群中分离持续细胞后,必须通过执行部分C来确认持续细胞状态。

  1. 将浮游细胞与浮游性固定期细胞隔离
    1. 在细菌的冷冻原液的LB琼脂上制备条板 感兴趣,并在静态条件下在37℃孵育24小时
    2. 从条纹板上除去1-2个菌落,并接种5ml LB肉汤的肉汤培养物
    3. 在37℃下搅拌(220rpm)孵育12小时(过夜培养)
    4. 在50ml的新鲜LB培养基中稀释过夜培养物至1%   锥形瓶。 注2描述了可能出现的问题 接种变异性
    5. 在37℃下搅拌(220rpm)孵育24小时
    6. 通过在4℃下以16,000×g离心5分钟收集细胞
    7. 重悬于50ml冷(冷藏并保持在冰上)盐水(0.85%NaCl)
    8. 重复步骤A6-7。
    9. 在600nm波长(OD 600)测量细胞浊度。
    10. 计算需要多少培养物(现在在盐水中)在25ml中具有0.8的最终OD <600> 。
      1. 示例:初始OD <600> 为2.0
      2. 最终OD <600>需要的是0.8 /
      3. 所需的最终体积为25ml
      4. 所以,所需的文化体积是
    11. 一式两份,取出10毫升(按实例)的培养物到新鲜的离心管中
    12. 通过在4℃下以16,000×g离心5分钟收集细胞
    13. 重悬于25 ml冷(冷藏)盐水中。
    14. 以20倍的最小抑制浓度(MIC)加入环丙沙星   (处理的样品)或含有乙酸的盐水 浓度,以解释存在于中的乙酸 环丙沙星储备溶液(对照)。 对于 p。 铜绿 PA14,我们使用 浓度为20μg/ml的环丙沙星(原始储备溶液   环丙沙星在含0.1%乙酸的盐水中为4mg/ml)。
      注意: 使用Andrews(2001)描述的方法计算MIC。 不同的菌株和相同的菌株在另一个实验室 可以具有不同的MIC,因此需要对每个MIC进行计算 个别细菌菌株。
    15. 在37℃下搅拌(220rpm)孵育24小时
    16. 在时间点0,1,3,5,7,9,12,15和24小时进行取样(500μl)。
    17. 对于每个时间点,执行从0到10 -6 的一组10 - 系列稀释系列。
    18. 将每个样品稀释液倒入LB琼脂平板(100×   15mm),其含有1%MgCl 2·7H 2 O·7H 2 O(以中和环丙沙星),并允许   使液滴在琼脂表面滑动,形成一条线,使液滴   传播和殖民地具有更高的生长表面积。 每块板可以   有最多5滴。 进行三次重复的样品板以确保 精度。
    19. 在37℃孵育平板24-48小时。
    20. 计数菌落在10-100之间。
    21. 计算CFU/ml:
      1. 10 10稀释液中的实施例#集落(50),CFU/ml将是:50×10 2 /0.01=5×10 5/sup> CFU/ml。
    22. 绘制结果为活细胞%对时间。 活细胞% 基于为时间0获得的CFU/ml计算,其被考虑 作为100%活细胞
    23. 进行四次实验,这   将允许计算所获得的结果是否 当使用时比较对照和处理的样品时统计学显着   ANOVA,随后进行Tukey比较检验。

  2. 持续细胞与生物膜隔离
    如下文参考文献(Sauer等人,2002; Davies和Marques,2009; Marques等人,2014)中所述在管式反应器上培养生物膜,并简要地 在注1中
    1. 重复上述步骤中的步骤A1-5,用于使浮游细胞与浮游培养物分离。
    2. 测量过夜培养物的OD 600,并在50ml LB培养基中使培养物标准化以具有0.8的OD 600.
    3. 用60毫升注射器用针吸出培养物,然后盖针。 在无菌条件下执行所有这些操作。
    4. 用2ml培养物接种每个管反应器(图2)
    5. 在室温下孵育1小时 - 以允许细菌   以连接到硅胶管(这个时间可以变化,取决于 细菌菌株) - 启动流动。
    6. 允许生物膜发展6天的时间。
    7. 将生物膜暴露于盐水或150μg/ml环丙沙星(150x MIC) 在盐水中,持续24小时。 在0,1,3,5,7,9, 12和24小时。
    8. 在每个时间点,对于每个样品(一个对照 和一个处理),需要牺牲一个生物膜管和其 内容物需要使用滚针法收获(Sauer等人, ,2002; Davies和Marques,2009; Marques等人,2014)。 硅胶   包含生物膜的管(图2)从 接种端口并从废物中放置到实验台上,之后 其中,销在管的顶部上滚动以挤出液体 并将生物膜糊剂倒入含有1ml盐水的离心管中 与1%MgCl 2 2·7H 2 O(以中和环丙沙星)。
    9. 均质化 样品使用匀浆器以速度4(约20,000rpm)进行 期间20秒。 用70%乙醇在样品之间消毒匀浆器   持续1分钟,然后加入无菌水。 将样品保存在冰上 在此步骤中。
    10. 执行从0到10-8的一组10 - 系列稀释系列。
    11. 将10μl样品滴在含有1% MgCl 2 2 7H 2 O。 每个板最多可以有5滴。 至少执行 复制板以确保准确性
    12. 在37℃孵育平板24-48小时。
    13. 计数菌落在10-100之间。
    14. 计算CFU/cm 2

      1. 10 10稀释液中的实施例#集落(50),CFU/ml将是:50×10 2 /0.01=5×10 5/sup> CFU/ml
      2. 如果将样品收集到1ml中,则总CFU为5×10 5 CFU。
      3. 如果管的内部体积为2cm 2,那么CFU/cm 2 将为:5×10 5/s/2 = 2.5× 10 CFU/cm 2 。
    15. 将结果绘制为活细胞的%对时间。 活细胞% 基于为时间0获得的CFU/ml计算,其被考虑 作为100%活细胞
    16. 进行四次实验,这   将允许计算所获得的结果是否 当将对照与处理的样品比较时,统计学显着 使用ANOVA,然后进行Tukey比较检验。

  3. 确认持续细胞状态。
    1. 如上所述在A和B中分离持续细胞。
    2. 在环丙沙星暴露18小时:
      1. 在浮游留菌细胞分离实验(上述A部分)中, 收集存在于锥形瓶中的所有细胞,用于每次处理 (环丙沙星或盐水)通过在16,000×g离心5分钟,在4   并将每个原始样品重悬于12.5ml盐水和12.5ml中 的环丙沙星(20mg/L)。 参见注3 从样品中分离死细胞
      2. 在生物膜持续细胞 分离实验(上面B部分)切换环丙沙星暴露 到盐水或另外的环丙沙星。
    3. 在0,1,3,5,7,9,12和24小时监测活力,并如上所述处理数据。

代表数据

使用该方法,使用SOS响应作为标准程序分离持续细胞(Keren等人,2004;Dör等人,2009; Moker等人, et al。,2010; Keren et al。,2004; Pan et al。,2012; Niepa et al。, 2012; Hong 等人,2012)。当在浮游生物或生物膜培养物中进行持久细胞分离时,在环丙沙星存在下应观察到典型的双相杀伤曲线(图1A)。其中在前3-6小时观察到细胞数的指数减少,随后是杀死平台,其中没有观察到细胞活力的进一步降低。存在于该平台中的细胞被认为是细菌培养物中的持续细胞亚群,并且可以是总群体的0.0001至0.1%。
一旦分离出持续细胞,通过将分离的细胞暴露于环丙沙星(20mg/L)或盐水另外24小时来确定持续细胞状态。如果细胞真正处于持续状态,则没有观察到细胞活力的降低(图1B)

图1.来自p的生物膜群体的持续细胞的分离。铜绿假单胞菌。 A.在暴露于P时观察到的双相曲线。铜绿假单胞菌生物膜与环丙沙星(正方形)以及对照,暴露于盐水的生物膜(空心圆圈)。 B.与对照(倒三角形)相比,持续细胞状态的确认,持续细胞暴露于环丙沙星(黑色三角形)不会导致细胞活力的降低。 (版权© 2014,美国微生物学会(Marques ,2014)。

笔记

  1. 在这项工作中,生物膜在管式反应器系统(图2)中培养,所述管式反应器系统由L/S 14 Masterflex过氧化物固化的有机硅管道组成,其中5%的LB以10.8ml/h的速率泵送通过(Sauer等, ,2002; Davies 和Marques,2009; Marques等人,2014)。每个管反应器用2ml标准化的过夜培养物(OD 600为0.8)接种,并在静态条件下培养1小时以促进细胞附着。
  2. 当在浮游群体中进行持续细胞分离时,接种物需要在实验之间相同地处理或持续细胞的数量将改变,产生变异性。因此,对于低细菌细胞接种物,持续细胞群中可能存在集落不存在,这不是由于持续细胞的缺乏,而是由于低细胞数目和使用CFU测定的活细胞评估的检测水平。
  3. 当确认浮游细胞群体中的持续细胞状态时,细胞沉淀将由于存在死细胞而呈现梯度。死细胞将容易地重悬在液体上,并且当去除上清液时将被去除,而活细胞将保留在沉淀中。

    A

    B

    图2.生物膜管反应器系统。这是一个通过连续培养系统,其中生物膜在硅胶管内发展。细菌培养物接种在接种口上,生物膜从泵的下游发育。 A.含有4根硅胶管的管式反应器系统示意图。 B.组装的管式反应器系统,其包含16个硅树脂管,其中培养物将串联运行

食谱

  1. 盐水
    8.5克氯化钠
    1升去离子水
  2. 具有MgCl 2盐水的盐水 7H 2 O 8.5克氯化钠
    10g MgCl 2 7H O
    1升去离子水
  3. 具有MgCl 2的LB琼脂。 O
    20克LB肉汤Lennox
    15克琼脂
    10g MgCl 2 7H O
    1升去离子水

致谢

持久性分离是对先前公布的方案的修改(Keren等人,2004; Sufya等人,2003)。 这项工作得到了SUNY结构基金的支持。

参考文献

  1. Andrews,J.M。(2001)。 最小抑制浓度的测定 J Antimicrob Chemother 48 Suppl 1:5-16。
  2. Bernier,S.P.,Lebeaux,D.,DeFrancesco,A.S.,Valomon,A.,Soubigou,G.,Coppee,J.Y.,Ghigo,J.M.and Beloin,C。(2013)。 饥饿,连同SOS反应,介导对氟喹诺酮氧氟沙星的高生物膜特异性耐受。 a> PLoS Genet 9(1):e1003144。
  3. Davies,D.G.and Marques,C.N。(2009)。 脂肪酸信使负责诱导微生物生物膜中的分散。 J Bacteriol 191(5):1393-1403
  4. Dör,T.,Lewis,K。和Vulic,M。(2009)。 SOS响应在大肠杆菌中诱导氟喹诺酮持续存在。 PLoS Genet 5(12):e1000760。
  5. Hong,S.H.,Wang,X.,O'Connor,H.F.,Benedik,M.J.and Wood,T.K。(2012)。 细菌持续性随着环境适应性降低而增加。 Microb Biotechnol 5(4):509-522。
  6. Keren,I.,Kaldalu,N.,Spoering,A.,Wang,Y.and Lewis,K。(2004)。 保留细胞和耐受抗生素。 FEMS Microbiol Lett 230(1):13-18
  7. Keren,I.,Shah,D.,Spoering,A.,Kaldalu,N.and Lewis,K。(2004)。 专门的持续性细胞和大肠杆菌中的多药耐药机制。 J Bacteriol 186(24):8172-8180。
  8. Marques,C.N.,Morozov,A.,Planzos,P.and Zelaya,H.M。(2014)。 脂肪酸信号分子顺式-2-癸烯酸增加代谢活性并将持续细胞恢复到抗微生物敏感状态。 Appl Environ Microbiol 80(22):6976-6991。
  9. Niepa,T.H.,Gilbert,J.L.and Ren,D。(2012)。 通过弱电化学电流和妥布霉素的协同效应控制铜绿假单胞菌持续细胞。 Biomaterials 33(30):7356-7365。
  10. Pan,J.,Bahar,A.A。,Syed,H。和Ren,D。(2012)。 绿脓杆菌PAO1持续细胞的抗生素耐受性由(Z)-4-溴-5-(溴甲基)-3-甲基呋喃-2(5H) - 酮。 PLoS One 7(9):e45778。
  11. Potrykus,K.和Cashel,M。(2008)。 (p)ppGpp:仍然不可思议? Annu Rev Microbiol 62:35-51。
  12. Sauer,K.,Camper,A.K.,Ehrlich,G.D.,Costerton,J.W.and Davies,D.G。(2002)。 铜绿假单胞菌(Pseudomonas aeruginosa)在发育期间显示多种表型作为生物膜。 184(4):1140-1154。
  13. Sauer,K.,Cullen,M.C.,Rickard,A.H.,Zeef,L.A.,Davies,D.G。和Gilbert,P。(2004)。 在绿脓假单胞菌中的营养诱导分散的表征 PAO1生物膜。 a> J Bacteriol 186(21):7312-7326。
  14. Sufya,N.,Allison,D.G。和Gilbert,P。(2003)。 最大比生长速率和抗微生物药物敏感性的克隆变异。 Microbiol 95(6):1261-1267
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Marques, C. N. (2015). Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa. Bio-protocol 5(18): e1590. DOI: 10.21769/BioProtoc.1590; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook
Cláudia N. H. Marques的其他实验方案(1)