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Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii
鲍氏不动杆菌的最小抑菌浓度(MIC)测定   

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Abstract

Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an antimicrobial. Multidrug resistance Acinetobacter baumannii (A. baumannii) has emerged in recent years. Accurate determination of resistance by MIC assay is important in coping with this superbug. Here we described a protocol for determining MIC for A. baumannii in hope of assisting researchers and physicians in confirming resistance of clinical isolates correctly.

Keywords: Acinetobacter baumannii(鲍氏不动杆菌), Minimal Inhibitory concentration(最小抑菌浓度), Drug resistance(耐药性)

Materials and Reagents

  1. A. baumannii (ATCC, catalog number: 17978 )
  2. Escherichia coli (E. coli) (ATCC, catalog number: 25922 )
  3. Mueller Hinton broth (Sigma-Aldrich, catalog number: 70192 )
  4. Tigecycline (Wyeth, catalog number. 0220620-09-7 )
  5. NaCl (MDBio, catalog number: 101-1647-14-5 )
  6. KCl (Sigma-Aldrich, catalog number: P1147 )
  7. Na2HPO4 (J.T.Baker®, catalog number: 3828-01 )
  8. KH2PO4 (J.T.Baker®, catalog number: 4921-07 )
  9. HCl (J.T.Baker®, catalog number: 9535-03 )
  10. Tryptone (Pronadisa, catalog number: 1612 )
  11. Yeast extract (Pronadisa, catalog number: 1702 )
  12. Cation-adjusted Mueller-Hinton broth (CAMHB) (see Recipes)
  13. PBS (1 L) (see Recipes)
  14. Lysogeny broth (LB) (see Recipes)

Equipment

  1. 50 ml polystyrene culture tubes (sterile)
  2. Spectrophotometer to measure absorbance of cell culture (OD600)
  3. 37 °C shaking and static incubators
  4. Multichannel pipette (volume ranges 10 μl-1,000 μl)
  5. 1.5 ml Eppendorf tube
  6. A centrifuge machine
  7. 1 ml cuvette

Procedure

  1. Preparation of antibiotic stock solution and dilution range
    1. Obtain antibiotic powder from the pharmaceutical company and make a note of the relevant information, including expiry date, potency, stability and solubility.
    2. Prepare 1 ml 10 mg/ml tigecycline stock solution.
    3. Choose a suitable range of antibiotic concentrations to be tested for A. baumannii if available. If the range is not available, maximal concentration 512 µg/ml and serial diluted concentrations with CAMHB solution are used. The lowest dilution concentration is depended on the possible minimal inhibition concentration. 0.125 µg/ml is the lowest possible dilution concentration.
    4. To get different tested concentrations, solution of 10-time maximal concentration is prepared by dispensing the appropriate amount of stock solutions with micropipette and diluting with CAMHB solution.
      For example, to get 1 ml 5,120 µg/ml solution, dispense 0.512 ml stock solution and dilute with 0.488 ml CAMHB solution.

  2. Preparation of inoculum
    1. Dissolve a single colony of A. baumannii, which is picked from a LB streak plate, in 3 ml LB broth and incubate overnight at 37 °C, 220 rpm.
    2. Check OD600 (1 OD600= 109 CFU/ml) with a spectrophotometer.
    3. Dilute the bacterial solution with LB broth to get 0.1 OD600 suspension and incubate at 37 °C, 220 rpm till mid-log phase (~2 h).
    4. Put 1 ml mid-log phase bacterial solution in 1.5 ml Eppendorf tube, centrifuge at 6,000 rpm for five min, and wash with 1 ml PBS solution. Repeat the washing procedure twice.
    5. Dissolve the bacterial pellet with 1 ml CAMHB solution.
    6. Get 100 µl the above bacterial solution and mix it with 900 µl PBS, then check OD600 with a spectrophotometer. The bacterial concentration can be deduced from the measured value x 10.
    7. Adjust the bacterial concentration to 1 x 107 CFU/ml with CAMHB solution (1 OD600 ~109 CFU/ml).

  3. Inoculation and incubation
    1. Mix 50 µl adjusted A. baumannii bacterial solution (1x107 CFU/ml), 850 µl CAMHB and 100 µl solutions of 10-time serial tested antibiotic concentration. Use the E. coli ATCC25922 bacterial solution as a control.
    2. Use 900 µl CAMHB and 100 µl solutions of 10-time serial tested antibiotic concentration for OD600 measurement comparison as a negative control.
    3. Incubate at 37 °C, 220 rpm for 20-24 h.

  4. Reading and interpretation
    1. Check OD600 with a spectrophotometer.
    2. Read the MIC endpoint as the lowest concentration of antibiotic at which there is no visible growth of bacteria (no solution turbidity on naked eyes), and the difference of measured and background OD600 is less than 0.01.

Recipes

  1. CAMHB
    Dissolve 23 g Mueller Hinton broth in 0.9 L of distilled water
    Adjust pH to 7.2 using HCl
    Then fill up to 1,000 ml with distilled water
    Sterilized by autoclaving at 121 °C for 15 min
    And added
    2 ml 10 g/L Ca2+ (8.36 g MgCl22H2O in 100 ml ddH2O)
    1 ml 10 g/L Mg2+ (3.68 g CaCl26H2O in 100 ml ddH2O)
    Stored at 4 °C
  2. PBS (1 L)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve in 900 ml ddH2O
    Adjust pH to 7.2 using HCl
    Sterilized by autoclaving at 121 °C for 15 min
  3. LB
    10 g tryptone
    5 g yeast extract
    5 g NaCl
    Fill to 1 L with ddH2O
    Sterilized by autoclaving at 121 °C for 15 min

Acknowledgments

The development of this protocol was funded by a grant from the National Taiwan University Hospital, Chu-Tung Branch.

References

  1. Lin, M. F., Lin, Y. Y., Yeh, H. W. and Lan, C. Y. (2014). Role of the BaeSR two-component system in the regulation of Acinetobacter baumannii adeAB genes and its correlation with tigecycline susceptibility. BMC Microbiol 14: 119.

简介

最小抑制浓度(MIC)是可以在过夜孵育后抑制微生物可见生长的抗微生物剂的最低浓度。 MIC测定不仅用作治疗临床医生的细菌感染的诊断工具,而且用作评价抗微生物剂的功效的研究方法。 近年来出现了多药耐药性鲍曼不动杆菌(Eminetobacter baumannii)(鲍曼不动杆菌)。 通过MIC测定的抗性的精确测定对于应对该超级细菌是重要的。 这里我们描述了用于确定 A的MIC的协议。 baumannii 希望协助研究人员和医生正确地确认临床分离株的耐药性。

关键字:鲍氏不动杆菌, 最小抑菌浓度, 耐药性

材料和试剂

  1. A。 baumannii(ATCC,目录号:17978)
  2. 大肠杆菌(大肠杆菌)(ATCC,目录号:25922)
  3. Mueller Hinton肉汤(Sigma-Aldrich,目录号:70192)
  4. 替加环素(Wyeth,目录号0220620-09-7)
  5. NaCl(MDBio,目录号:101-1647-14-5)
  6. KCl(Sigma-Aldrich,目录号:P1147)

  7. ,目录号:3828-01)

  8. (J.T.Baker ®,目录号:4921-07)
  9. HCl(J.T.Baker ,目录号:9535-03)
  10. 胰蛋白胨(Pronadisa,目录号:1612)
  11. 酵母提取物(Pronadisa,目录号:1702)
  12. 阳离子调节的Mueller-Hinton肉汤(CAMHB)(参见食谱)
  13. PBS(1升)(见配方)
  14. 溶菌酶肉汤(LB)(参见食谱)

设备

  1. 50ml聚苯乙烯培养管(无菌)
  2. 分光光度计测量细胞培养物的吸光度(OD 600)
  3. 37°C摇动和静态培养箱
  4. 多通道移液器(体积范围10μl-1,000μl)
  5. 1.5 ml Eppendorf管
  6. 离心机
  7. 1 ml比色杯

程序

  1. 抗生素储备液的制备和稀释范围
    1. 从制药公司获取抗生素粉并记录   的相关信息,包括到期日期,效力,稳定性 和溶解度。
    2. 准备1毫升10mg/ml替加环素储备溶液。
    3. 选择合适的抗生素浓度范围进行测试 为 A。 baumannii (如果有的话)。 如果范围不可用,则为最大 浓度512μg/ml和用CAMHB连续稀释的浓度 溶液。 最低稀释浓度取决于 可能的最小抑制浓度。 0.125μg/ml是最低的 可能的稀释浓度
    4. 得到不同的测试 浓度,10倍最大浓度的溶液制备   用微量移液管分配适量的储备溶液 并用CAMHB溶液稀释。
      例如,为了得到1ml5120μg/ml溶液,分配0.512ml储备溶液并用0.488ml CAMHB溶液稀释。

  2. 接种物的制备
    1. 溶解单个菌落的 A。 baumannii ,从LB中挑选 条板,在3ml LB肉汤中,并在37℃,220rpm温育过夜。
    2. 使用分光光度计检查OD <600>(OD OD 600 = 10 9 CFU/ml)。
    3. 用LB肉汤稀释细菌溶液以获得0.1OD 600的悬浮液,并在37℃,220rpm温育直到对数中期(〜2小时)。
    4. 将1毫升中对数期细菌溶液放入1.5毫升Eppendorf管中, 在6,000rpm离心5分钟,并用1ml PBS溶液洗涤。 重复洗涤程序两次。
    5. 用1ml CAMHB溶液溶解细菌沉淀。
    6. 得到100微升以上细菌溶液,并与900微升PBS混合,   然后用分光光度计检查OD <600>。 细菌浓度 可以从测量值x 10推导出来。
    7. 用CAMHB溶液(1OD 600至10 10 CFU/ml)将细菌浓度调节至1×10 7 CFU/ml。

  3. 接种和孵化
    1. 混合50μl调整的鲍曼不动杆菌细菌溶液(1×10 7 CFU/ml),850 μlCAMHB和100μl10次连续测试抗生素的溶液 浓度。 使用 E。 大肠杆菌ATCC25922细菌溶液 控制
    2. 使用900微升CAMHB和100微升10时间序列的解决方案   测试的抗生素浓度用于OD 600测量比较作为a 负控制。
    3. 在37℃,220rpm孵育20-24小时。

  4. 阅读和解释
    1. 用分光光度计检查OD <600>。
    2. 读取MIC端点为 最低浓度的抗生素,其中没有可见的 细菌生长(裸眼无溶液浊度),和 测量的和背景OD 600的差异小于0.01。

食谱

  1. CAMHB
    将23g Mueller Hinton肉汤溶于0.9L蒸馏水中
    使用HCl
    调节pH至7.2 然后用蒸馏水
    填充至1,000 ml 在121℃高压灭菌15分钟,灭菌
    并添加了
    2ml 10g/L Ca 2+ 2+(8.36g MgCl 2)2·6H 2 O在100ml ddH中的溶液 2 O)
    在100ml ddH中的1ml 10g/L Mg 2+(3.68g CaCl 2)2·6H 2 O 2·6H 2 O 2 O)
    储存在4°C
  2. PBS(1 L)
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
    调节pH至7.2 在121℃高压灭菌15分钟,灭菌
  3. LB
    10g胰蛋白胨
    5g酵母提取物
    5克NaCl
    用ddH sub 2 O填充到1L 在121℃高压灭菌15分钟,灭菌

致谢

该方案的开发由国立台湾大学医院Chu-Tung分院资助。

参考文献

  1. Lin,M.F.,Lin,Y.Y.,Yeh,H.W.and Lan,C.Y。(2014)。 BaeSR双组分系统在调节鲍氏不动杆菌中的作用 adeAB基因及其与替加环素敏感性的相关性。BMC Microbiol 14:119。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lin, M., Lin, Y. and Lan, C. (2014). Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii. Bio-protocol 4(23): e1308. DOI: 10.21769/BioProtoc.1308.
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Ratcha Aunp
Thammasat University
Please explain "the difference of measured and background OD600 is less than 0.01."

What did you use for control and background?

Thanks
2/1/2017 4:57:10 AM Reply
Chung-Yu Lan
Institute of Molecular and Cellular Biology, National Tsing Hua University

2/2/2017 4:36:36 AM


Ming-Feng Lin
National Taiwan University Hospital Chu-Tung Branch

Answer: 900 µl CAMHB and 100 µl solutions of 10-time serial tested antibiotic concentration without adding bacteria was used as a negative control and its OD600 value was deemed as background value. The measured (no visible bacterial growh after the lowest concentration of antibiotics added) and background OD600 should be almost the same (difference less than 0.01).

2/2/2017 4:37:44 AM


Fahmeeda Mojan
Universiti Malaysia Terengganu
How do you obtain the volume of bacterial solution needed to get 1 x 10*7 CFU/mL (adjusted concentration) from OD600?
4/5/2016 9:42:01 AM Reply
Ming-Feng Lin
National Taiwan University Hospital Chu-Tung Branch

Answer: At first, the amount of bacteria in 1 OD600 (checked with a spectrophotometer) solution was determined. To get the result, 0.1 mL 1 OD600 bacterial solution was diluted with 10-time serial dilution for 6-7 times. Then the serial diluted bacterial solutions were inoculated and the colonies were measured after overnight culture. Next, the original amount of bacteria in these diluted solutions could be deduced by the colony number and dilution fold. Finally, we found that 1 OD600= 10*9 CFU/ml. Therefore, 0.01 OD600 = 10*7 CFU/ml.

4/8/2016 8:54:24 PM