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Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules
采用大分子生物合成法评估抗菌剂对大分子合成的影响   

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Abstract

One of the most compelling approaches in the discovery of novel antimicrobials is screening of natural sources. In our publication we report on the activity of a compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. We evaluate its applicability for treatment of implant-associated infections. A comprehensive analysis of the mechanism of action of EN4 against staphylococci revealed its membranolytic properties and a general inhibition of macromolecular biosynthesis, which was confirmed in a macromolecular biosynthesis assay and suggested a multitarget activity. The method used to investigate an influence of EN4 on the synthesis of peptidoglycan, RNA, DNA and proteins is based on precipitation of macromolecules with trichloroacetic acid. These macromolecules are synthesised from respective [3H]-labelled precursors. The incorporated radioactivity with and without an antimicrobial is measured and it reflects the mode of action of the tested compound. Antibiotics with known mechanisms of action are used as controls.

Keywords: Macromolecular synthesis(大分子合成), Mechanism of action(作用机制), Antibiotic(抗生素), Antimicrobial(抗菌), Bacterial biosynthesis(细菌的生物合成)

Materials and Reagents

  1. [3H] N-acetylglucosamine (Hartmann Analytic, catalog number: ART 0101 )
  2. [3H] Uridine (Hartmann Analytic, catalog number: MT-602 )
  3. [3H] Thymidine (PerkinElmer, catalog number: NET35500 )
  4. [3H] Leucine (Hartmann Analytic, catalog number: MT-672 )
  5. Triptic soy broth (TSB) (Becton Dickinson, catalog number: 211825 )
  6. Control antimicrobials for the influence on synthesis of:
    1. Peptidoglycan: vancomycin (Vancocin 500 mg) (Teva Pharma)
    2. RNA: actinomycin D (Sigma-Aldrich, catalog number: A1410
    3. DNA: ciprofloxacin (Ciproxin Infusion 0.2 g) (Bayer) 
    4. Proteins: chloramphenicol (Applichem, catalog number: A1806 )
    5. All macromolecules (antiseptic activity): chlorhexidine dihydrochloride (Sigma-Aldrich, catalog number: C8527
      Note: All substances prepared according to the manufacturer’s instructions; minimal inhibitory concentration (MIC) of each antimicrobial can be determined according to Clinical and Laboratory Standards Institute guidelines (see Reference 1) (The following MICs were determined for S. aureus WSPPA: MICvancomycin = 2 μg/ml, MICactinomycin D = 6.25 μg/ml, MICciprofloxacin = 2 μg/ml, MICchloramphenicol = 8 μg/ml, MICchlorhexidine = 0.78 μg/ml)
  7. Scintillation cocktail (Ultima Gold, catalog number: 6013321 ).
  8. Phosphate-buffered saline (PBS) (Reagens, catalog number: 9007695 ).
  9. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  10. KH2PO4 (Sigma-Aldrich, catalog number: P8416 )
  11. MgSO4.7H2O (Fluka, catalog number: 63138 )
  12. NH4Cl (Sigma-Aldrich, catalog number: A9434 )
  13. NaCl (Sigma-Aldrich, catalog number: S3014 )
  14. Sodium citrate tribasic dihydrate (Sigma-Aldrich, catalog number: S4641 )
  15. Glucose (B. Braun Medical, catalog number: 395176 )
  16. Various amino acids (i.e., L-alanine (Sigma-Aldrich, catalog number: A7469 )
  17. L-Valine (Sigma-Aldrich, catalog number: V0513 )
  18. L-Isoleucine (Sigma-Aldrich, catalog number: I7403 )
  19. L-Aspartic acid (Sigma-Aldrich, catalog number: A7219 )
  20. L-Glutamic acid (Sigma-Aldrich, catalog number: G8415 )
  21. L-Serine (Sigma-Aldrich, catalog number: S4311 )
  22. L-Threonine (Sigma-Aldrich, catalog number: T8441 )
  23. L-Cysteine hydrochloride (Sigma-Aldrich, catalog number: C6852 )
  24. L-Arginine (Sigma-Aldrich, catalog number: A8094 )
  25. L-Leucine (Sigma-Aldrich, catalog number: L8912 )
  26. L-Lysine (Sigma-Aldrich, catalog number: L9037 )
  27. L-Proline (Sigma-Aldrich, catalog number: P5607 )
  28. L-Phenylalanine (Sigma-Aldrich, catalog number: P5482 )
  29. L-Tryptophan (Sigma-Aldrich, catalog number: T8941 )
  30. L-Histidine monohydrochloride (Sigma-Aldrich, catalog number: H5659
  31. Glycine (Sigma-Aldrich, catalog number: G7126 )
  32. Cyanocobalamine (Sigma-Aldrich, catalog number: V2876 )
  33. p-Aminobenzoate (Fluka, catalog number: 06940 )
  34. Biotin (Sigma-Aldrich, catalog number: B3399 )
  35. Nicotinic acid (Sigma-Aldrich, catalog number: N0761 )
  36. D-pantothenic acid hemicalcium salt (Sigma-Aldrich, catalog number: P5155 )
  37. Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P6280 )
  38. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
  39. Riboflavin (Sigma-Aldrich, catalog number: R9504 )
  40. ZnCl2 (Sigma-Aldrich, catalog number: 208086
  41. MnCl2.4H2O (Sigma-Aldrich, catalog number: M5005 )
  42. BH3O3 (Fluka, catalog number: 15665 )
  43. CoCl2.6H2O (Sigma-Aldrich, catalog number: C8661 )
  44. CuCl2.2H2O (Sigma-Aldrich, catalog number: C3279 )
  45. NiCl2.6H2O (Sigma-Aldrich, catalog number: 223387 )
  46. Na2MoO4.2H2O (Sigma-Aldrich, catalog number: M1003 )
  47. FeCl2.4H2O (Fluka, catalog number: 44939 )
  48. NaOH (Sigma-Aldrich, catalog number: S5881 )
  49. Uracil (Sigma-Aldrich, catalog number: U0750 )
  50. Cytosine (Sigma-Aldrich, catalog number: C3506 )
  51. Adenine (Sigma-Aldrich, catalog number: A2786 )
  52. Guanine (Sigma-Aldrich, catalog number: G11950 )
  53. Sodium dodecyl sulphate (SDS) (Sigma-Aldrich, catalog number: L4390 )
  54. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T9159 )
  55. 15 ml TPP centrifuge tubes (TPP 91015 )
  56. 10% TCA, 5% TCA, 5% TCA/1.5 M NaCl (see Recipes)
  57. Completely defined medium (CDM) (see Recipes)
    Note: This medium has been optimised for Staphylococcus (S.) aureus and may require further optimisation if other bacteria are investigated. 

Equipment

  1. 37 °C bacterial culture incubator
  2. Sterile capped glass tubes (to reduce bacterial adherence) (GlasKeller, catalog number: 2613111 )
  3. Ultracentrifuge tubes (2 ml) (Eppendorf, catalog number: 0030120.094 )
  4. Scintillation tubes (PerkinElmer, catalog number: 6000288 )
  5. Benchtop centrifuge
  6. Vacuum pump
  7. Liquid scintillation analyser (Tri-CARB, catalog number: 1900TR )

Procedure

Note: The glass tubes for incubation must be pre-warmed and the reagents and tubes for precipitation must be pre-cooled; to assess the interference with biosynthesis of one macromolecule prepare 14 pre-warmed glass tube (untreated control, investigated antimicrobial and five control antimicrobials, in duplicates).

  1. Overnight bacterial culture (for S. aureus WSPPA approximately 1.5 x 108 CFU/ml) prepared in TSB is diluted 1:100 in 10 ml CDM and, for [3H] Leucine incorporation, in 10 ml CDM-Leu.
    Note: For S. aureus WSPPA the overnight and logarithmic-phase cultures are prepared in 15 ml TPP centrifuge tubes, without shaking.
  2. Incubation: 5 h, 37 °C.
    Note: The incubation time must be adjusted respectively to the strain in order to reach the logarithmic growth phase.
  3. The log-phase culture (0.9 ml for S. aureus WSPPA, which corresponds to approximately 2 x 107 CFU) is transferred in duplicates to pre-warmed glass tubes and all antimicrobials at concentration of 4x MIC are added; untreated controls are incubated with an adequate volume of a solvent used to prepare solution of investigated antimicrobial.
    Note: Untreated control must be prepared in exactly the same solvent as used for the antimicrobial of interest to control for the effect of solvent on bacterial biosynthesis. It is therefore recommended to dilute all antimicrobials to the concentrations of 4x MIC in the same solvent, which additionally does not interfere with bacterial viability (e.g. PBS). If due to its chemical properties any substance needs to be diluted in a harsh solvent then a concentrated stock solution is prepared in the harsh solvent followed by diluting in a mild solvent to the 4x MIC to reduce the concentration of the first solvent below a level affecting bacteria. The untreated controls are prepared in exactly the same way for incorporation of each of the precursors.
  4. The samples are mixed thoroughly and [3H]-labelled precursors are immediately transferred to separate tubes:
    [3H] N-acetylglucosamine up to a concentration of 0.1 μCi per ml (for peptidoglycan synthesis)
    [3H] Uridine up to 1 μCi per ml (for RNA synthesis)
    [3H] Thymidine up to 1 μCi per ml (for DNA synthesis)
    [3H] Leucine up to 3 μCi per ml (for protein synthesis)
  5. Incubation: 37 °C.
    At time points of interest 0.5 ml aliquots are transferred into 2 ml ultracentrifuge tubes containing 1 ml of ice-cold 10% TCA, mixed thoroughly and placed on ice for at least 1.5 h to facilitate the precipitation.
    Note: Time points of interest can be determined in the time-killing study according to Clinical and Laboratory Standards Institute guidelines (see Reference 1). For S. aureus WSPPA time point of 1 h was chosen.
  6. The precipitates are washed one time with 0.5 ml of 5% TCA/1.5 M NaCl followed by one-time washing with 0.5 ml of 5% TCA (16,100 x g, 10 min, 20 °C), supernatants are removed using vacuum pump.
  7. After the second wash samples are solubilised with 0.5 ml of 0.1% SDS/0.1 M NaOH by vortexing at room temperature.
  8. The solubilised precipitates are transferred into scintillation tubes and thoroughly mixed with 2 ml of scintillation cocktail.
  9. The incorporated radioactivity is measured in counts per minute using liquid scintillation analyser and the results are expressed as percentage of untreated control (Figure 1A - 1D).



    Figure 1. Inhibition of biosynthesis of macromolecules by EN4. Incorporation of [3H] N-acetylglucosamine (A), [3H] Uridine (B), [3H] Thymidine (C) and [3H] Leucine (D) by WSPPA treated for 1 h with EN4, vancomycin (VAN), actinomycin D (ActD), ciprofloxacin (CIP), chloramphenicol (CHL) or chlorhexidine (CHX) at 4x MIC, was expressed as percentage of untreated control (for peptidoglycan = 3444 ± 1212 cpm, RNA = 115538 ± 19533 cpm, DNA = 13862 ± 762 cpm, protein = 7065 ± 323 cpm); values shown are the means of at least two independent experiments prepared in duplicates ± SDs; dotted lines represent 100% incorporation. Significant reduction of biosynthesis as compared to results for the untreated control is indicated; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA (Kruskal-Wallis test) with a Dunns post test. (Nowakowska et al., 2013)

Recipes

  1. CDM (1,000 ml)
    Mix 1.77 g of Na2HPO4, 1.36 g of KH2PO4, 0.2 g of MgSO4.7H2O, 0.5 g of NH4Cl, 0.5 g of NaCl, 294.1 g of sodium citrate tribasic dihydrate, 3.75 ml of 40% glucose, 160 mg each of various amino acids, L-Valine, L-Isoleucine, L-Aspartic acid, L-Glutamic acid, L-Serine, L-Threonine, L-Cysteine hydrochloride, L-Arginine, L-Leucine, L-Lysine, L-Proline, L-Phenylalanine, L-Tryptophan, L-Histidine, monohydrochloride, and 1.6 g of glycine, 0.05 mg of cyanocobalamine, 0.04 mg of p-aminobenzoate, 0.01 mg of biotin, 0.1 mg of nicotinic acid, 0.1 mg of D-pantothenic acid hemicalcium salt, 0.15 mg of pyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride, 0.1 mg of riboflavin, 69.5 μg of ZnCl2, 0.1 μg of MnCl2.4H2O, 6 μg of BH3O3, 0.347 mg of CoCl2.6H2O, 2.6 μg of CuCl2.2H2O, 24 μg of NiCl2.6H2O, 36 μg of Na2MoO4.2H2O, 0.15 mg of FeCl2.4H2O, 120 mg of NaOH, 5 mg of uracil, 5 mg of cytosine, 5 mg of adenine, and 5 mg of guanine with 1,000 ml sterile dH2O;
    The concentration of L-Leucine in CDM-Leu was 22.5 mg/L instead of 160 mg/L;
    Filter-sterilise (0.22 μm)
    Store at  4 °C.
  2. 5% (10%) TCA (1,000 ml)
    Mix 50 g (100 g) of TCA with 1,000 ml dH2O
    Store at  4 °C.
  3. 5% TCA/1.5 M NaCl (100 ml)
    Mix 8.8 g NaCl with 100 ml of 5% TCA
    Store at  4 °C.
  4. 0.1% SDS/0.1M NaOH (100 ml)
    Gently mix by rotating 0.1 g sodium dodecyl sulphate (SDS) (w/v) and 0.6 g NaOH (w/v) with 100 ml dH2O
    Store at room temperature.

Acknowledgments

This protocol has been adapted from Nowakowska et al. (2013). The study was supported by the CCMX Competence Centre for Materials Science and Technology, Lausanne, Switzerland.

References

  1. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th ed. CLSI document M7-A7 (ISBN 1-56238-587-9). Clinical and Laboratory Standards Institute. Wayne, PA, 2006.
  2. Nowakowska, J., Griesser. H. J., Textor, M., Landmann, R., Khanna, N. (2013) Antimicrobial Properties of 8-Hydroxyserrulat-14-en-19-oic Acid for Treatment of Implant-Associated Infections. Antimicrob Agents Chemother 57(1): 333-42.

简介

在发现新的抗微生物剂中最引人注目的方法之一是筛选天然来源。在我们的出版物中,我们报告了一种化合物8-羟基胆固醇-14-烯-19-酸(EN4)(一种从澳大利亚植物中分离的二萜烯)的活性。我们评估其适用于植入物相关感染的治疗。对EN4对葡萄球菌的作用机制的综合分析揭示其膜分解性质和大分子生物合成的一般抑制,其在大分子生物合成测定中被证实并提出多靶点活性。用于研究EN4对肽聚糖,RNA,DNA和蛋白质的合成的影响的方法是基于大分子与三氯乙酸的沉淀。这些大分子由相应的[3 H] - 标记的前体合成。测量掺入和不掺入抗微生物剂的放射性,并反映测试化合物的作用模式。使用具有已知作用机制的抗生素作为对照。

关键字:大分子合成, 作用机制, 抗生素, 抗菌, 细菌的生物合成

材料和试剂

  1. [H] 3 H] N-乙酰葡糖胺(Hartmann Analytic,目录号:ART 0101)
  2. [H 3 H]尿苷(Hartmann Analytic,目录号:MT-602)
  3. [suph 3 H]胸苷(PerkinElmer,目录号:NET35500)
  4. [H 3 H]亮氨酸(Hartmann Analytic,目录号:MT-672)
  5. Triptic大豆肉汤(TSB)(Becton Dickinson,目录号:211825)
  6. 控制抗菌素对合成的影响:
    1. 肽聚糖:万古霉素(Vancocin 500mg)(Teva Pharma)
    2. RNA:放线菌素D(Sigma-Aldrich,目录号:A1410)
    3. DNA:环丙沙星(Ciproxin Infusion 0.2g)(Bayer)
    4. 蛋白质:氯霉素(Applichem,目录号:A1806)
    5. 所有大分子(抗菌活性):氯己定二盐酸盐(Sigma-Aldrich,目录号:C8527)
      注意:根据制造商的说明制备的所有物质;每种抗微生物剂的最小抑制浓度(MIC)可以根据临床和实验室标准实验指南(参考文献1)来确定。 aure = 6.25μg/ ml MIC环丙沙星=2μg/ hloramphenicol = 8μg/ > )
  7. 闪烁鸡尾酒(Ultima Gold,目录号:6013321)。
  8. 磷酸盐缓冲盐水(PBS)(Reagens,目录号:9007695)。
  9. Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  10. KH sub 2 PO 4(Sigma-Aldrich,目录号:P8416)
  11. MgSO 4·7H 2 O(Fluka,目录号:63138)。
  12. NH 4 Cl(Sigma-Aldrich,目录号:A9434)
  13. NaCl(Sigma-Aldrich,目录号:S3014)
  14. 柠檬酸三钠二水合物(Sigma-Aldrich,目录号:S4641)
  15. 葡萄糖(B.Braun Medical,目录号:395176)
  16. 各种氨基酸(即L-丙氨酸)(Sigma-Aldrich,目录号:A7469)
  17. L-缬氨酸(Sigma-Aldrich,目录号:V0513)
  18. L-异亮氨酸(Sigma-Aldrich,目录号:I7403)
  19. L-天冬氨酸(Sigma-Aldrich,目录号:A7219)
  20. L-谷氨酸(Sigma-Aldrich,目录号:G8415)
  21. L-丝氨酸(Sigma-Aldrich,目录号:S4311)
  22. L-苏氨酸(Sigma-Aldrich,目录号:T8441)
  23. L-半胱氨酸盐酸盐(Sigma-Aldrich,目录号:C6852)
  24. L-精氨酸(Sigma-Aldrich,目录号:A8094)
  25. L-亮氨酸(Sigma-Aldrich,目录号:L8912)
  26. L-赖氨酸(Sigma-Aldrich,目录号:L9037)
  27. L-脯氨酸(Sigma-Aldrich,目录号:P5607)
  28. L-苯丙氨酸(Sigma-Aldrich,目录号:P5482)
  29. L-色氨酸(Sigma-Aldrich,目录号:T8941)
  30. L-组氨酸单盐酸盐(Sigma-Aldrich,目录号:H5659)
  31. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  32. 氰基钴胺(Sigma-Aldrich,目录号:V2876)
  33. 对 - 氨基苯甲酸酯(Fluka,目录号:06940)
  34. 生物素(Sigma-Aldrich,目录号:B3399)
  35. 烟酸(Sigma-Aldrich,目录号:N0761)
  36. D-泛酸半钙盐(Sigma-Aldrich,目录号:P5155)
  37. 盐酸吡哆醇(Sigma-Aldrich,目录号:P6280)
  38. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  39. 核黄素(Sigma-Aldrich,目录号:R9504)
  40. ZnCl 2(Sigma-Aldrich,目录号:208086)
  41. (Sigma-Aldrich,目录号:M5005)。

  42. (Fluka,目录号:15665)。
  43. (Sigma-Aldrich,目录号:C8661)。
  44. CuCl 2 2H 2 O(Sigma-Aldrich,目录号:C3279)
  45. (Sigma-Aldrich,目录号:223387)。
  46. (Sigma-Aldrich,目录号:M1003)。
  47. (Fluka,目录号:44939)
  48. NaOH(Sigma-Aldrich,目录号:S5881)
  49. 尿嘧啶(Sigma-Aldrich,目录号:U0750)
  50. 胞嘧啶(Sigma-Aldrich,目录号:C3506)
  51. 腺嘌呤(Sigma-Aldrich,目录号:A2786)
  52. 鸟嘌呤(Sigma-Aldrich,目录号:G11950)
  53. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4390)
  54. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T9159)
  55. 15ml TPP离心管(TPP 91015)
  56. 10%TCA,5%TCA,5%TCA/1.5M NaCl(参见配方)
  57. 完全定义的介质(CDM)(参见配方)
    注意:该培养基已经针对金黄色葡萄球菌(Staphylococcus(S.)aureus)进行了优化,并且如果其他细菌被调查,则可能需要进一步优化

设备

  1. 37℃细菌培养箱
  2. 无菌封盖玻璃管(以减少细菌粘附)(GlasKeller,目录号:2613111)
  3. 超速离心管(2ml)(Eppendorf,目录号:0030120.094)
  4. 闪烁管(PerkinElmer,目录号:6000288)
  5. 台式离心机
  6. 真空泵
  7. 液体闪烁分析仪(Tri-CARB,目录号:1900TR)

程序

注意:用于孵育的玻璃管必须预热,用于沉淀的试剂和管必须预冷却;评估一个大分子的生物合成的干扰制备14预热的玻璃管(未处理的对照,研究的抗微生物和五个对照抗微生物,一式两份)。

  1. 在TSB中制备的过夜细菌培养物(用于金黄色葡萄球菌WSPPA约1.5×10 8 CFU/ml)在10ml CDM中稀释1:100,对于[ 3 H]亮氨酸掺入到10ml CDM-Leu中 注意:对于金黄色葡萄球菌WSPPA,在15ml TPP离心管中制备过夜和对数期培养物,不摇动。
  2. 孵育:5小时,37℃ 注意:孵育时间必须分别调整到应变以达到对数生长期。
  3. 将对数期培养物(对于金黄色葡萄球菌WSPPA为0.9ml,对应于约2×10 7 CFU)一式两份转移到预热的玻璃管中,加入浓度为4x MIC的抗微生物剂;未处理的对照与适当体积的用于制备所研究的抗微生物剂的溶液的溶剂一起温育 注意:未处理的对照必须在与用于感兴趣的抗微生物剂完全相同的溶剂中制备,以控制溶剂对细菌生物合成的影响。因此,建议将所有抗微生物剂稀释至在相同溶剂中的4x MIC的浓度,其另外不干扰细菌活力(例如PBS)。如果由于其化学性质,任何物质需要在苛刻的溶剂中稀释,则在苛性溶剂中制备浓缩储备溶液,随后在温和溶剂中稀释至4x MIC,以将第一溶剂的浓度降低至影响菌。以完全相同的方式制备未处理的对照以掺入每种前体。
  4. 将样品彻底混合,并将[3 H] - 标记的前体立即转移到单独的管中:
    [用于肽聚糖合成]的浓度为0.1μCi/ml的[3 H] N-乙酰葡萄糖胺。 [/sup] H]尿苷高达1μCi/ml(用于RNA合成)
    [高达3 H]胸苷高达1μCi/ml(用于DNA合成)
    [/sup> H]亮氨酸高达3μCi/ml(用于蛋白质合成)
  5. 温育:37℃ 在感兴趣的时间点,将0.5ml等分试样转移到含有1ml冰冷的10%TCA的2ml超速离心管中,充分混合并置于冰上至少1.5小时以促进沉淀。
    注意:根据临床和实验室标准协会指南(参见参考文献1),可以在时间消耗研究中确定感兴趣的时间点。对于金黄色葡萄球菌,选择WSPPA时间点为1小时。
  6. 沉淀物用0.5ml 5%TCA/1.5M NaCl洗涤一次,然后用0.5ml 5%TCA(16,100×g,10分钟,20℃)一次洗涤,上清液使用真空泵除去
  7. 第二次洗涤后,样品在室温下涡旋振荡,用0.5ml 0.1%SDS/0.1M NaOH溶解。
  8. 将溶解的沉淀转移到闪烁管中并与2ml闪烁混合物充分混合
  9. 使用液体闪烁分析仪以每分钟计数测量结合的放射性,结果表示为未处理对照的百分比(图1A-1D)。



    图1. EN4对大分子生物合成的抑制将[3 H] N-乙酰葡糖胺(A),[3 H]尿苷通过用EN4,万古霉素(VAN),放线菌素处理1小时的WSPPA处理[B],[3 H]胸苷(C)和[3 H]亮氨酸(对于肽聚糖= 3444±1212cpm,RNA = 115538±19533cpm,DNA = 13862±2150cpm)的百分比表示为在4x MIC时的D(ActD),环丙沙星(CIP),氯霉素(CHL)或氯己定762cpm,蛋白质= 7065±323cpm);显示的值是以重复±SDs制备的至少两个独立实验的平均值;虚线表示100%掺入。指示与未处理对照的结果相比生物合成的显着减少; *,P < 0.05; **,P < 0.01; ***,P < 0.001,通过单因素方差分析(Kruskal-Wallis检验)。 (Nowakowska等人,2013)

食谱

  1. CDM(1,000 ml)
    将1.77g的Na 2 HPO 4,1.36g的KH 2 PO 4,0.2g的MgSO 4, 7H 2 O,0.5g NH 4 Cl,0.5g NaCl,294.1g柠檬酸钠三元二水合物,3.75ml 40%葡萄糖,160mg各种氨基酸,L-缬氨酸,L-异亮氨酸,L-天冬氨酸,L-谷氨酸,L-丝氨酸,L-苏氨酸,L-半胱氨酸盐酸盐,L - 精氨酸,L-亮氨酸,L-赖氨酸,L-脯氨酸,L-苯丙氨酸,L-色氨酸,L-组氨酸,一盐酸盐和1.6g甘氨酸,0.05mg氰基钴胺,0.04mg对氨基苯甲酸盐,0.01mg生物素,0.1mg烟酸,0.1mg D-泛酸半钙盐,0.15mg盐酸吡哆醇,0.1mg盐酸硫胺素,0.1mg核黄素,69.5μgZnCl 2,0.1μg的MnCl 2·6H 2 O,4H 2·6H 2 O,6μg的BH 3 O 3·6H 2 O, ,0.347mg的CoCl 2·6H 2 O·6H 2 O,2.6μg的CuCl 2·6H 2 O。 24μg的NiCl 2·6H 2 O·6H 2 O,36μg的Na 2 2 O 2 MoO 4+,0.15mg FeCl 2 Sub 2 O 3,0.15mg FeCl 2·6H 2 O。 4H 2 O 2,120mg NaOH,5mg尿嘧啶,5mg胞嘧啶,5mg腺嘌呤和5mg鸟嘌呤的DMEM培养基(用1000ml无菌dH 2 SO 4,/sub> O;
    CDM-Leu中L-亮氨酸的浓度为22.5mg/L而不是160mg/L;
    过滤灭菌(0.22μm)
    存储在  4℃。
  2. 5%(10%)TCA(1,000ml) 将50g(100g)TCA与1,000ml dH 2 O混合 存储在  4℃。
  3. 5%TCA/1.5M NaCl(100ml) 将8.8g NaCl与100ml 5%TCA混合 存储在  4℃。
  4. 0.1%SDS/0.1M NaOH(100ml) 通过旋转0.1g十二烷基硫酸钠(SDS)(w/v)和0.6g NaOH(w/v)与100ml dH 2 O缓冲混合 在室温下储存。

致谢

该协议已经从Nowakowska等人修改(2013)。 该研究得到了CCMX材料科学和技术能力中心,瑞士洛桑的支持。

参考文献

  1. 临床和实验室标准研究所。 稀释对有氧生长的细菌的抗微生物敏感性试验的方法; 批准标准,第7版。 CLSI文件M7-A7(ISBN 1-56238-587-9)。 临床和实验室标准研究所。 Wayne,PA,2006.
  2. Nowakowska,J.,Griesser。 HJ,Textor,M.,Landmann,R.,Khanna,N.(2013)。 Antimicrob Agents Chemother 57(1):333-42。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nowakowska, J., Khanna, N. and Landmann, R. (2013). Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules. Bio-protocol 3(12): e798. DOI: 10.21769/BioProtoc.798.
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