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Multidrug resistance Acinetobacter baumannii (A. baumannii) (MDRAB) has emerged as a serious threat in hospitals in recent years. Currently, there are few antibiotics, including tigecycline, available to treat infections caused by MDRAB effectively. Both tigecycline-resistant and tigecycline-susceptible isogenic strains of MDRAB are valuable in understanding the mechanisms underlying tigecycline resistance. To get the isogenic strains in the laboratory, we describe a protocol for induction of tigecycline resistance in A. buamannii by serial passage to plates with tigecycline of different concentrations. The minimal inhibitory concentration of A. baumannii by tigecycline was determined according to the protocol “Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii” (Lin et al., 2014b).

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Induction of Tigecycline Resistance in Acinetobacter baumannii
鲍氏不动杆菌中替加环素抗性的诱导

微生物学 > 抗微生物试验 > 抗细菌试验
作者: Ming-Feng Lin
Ming-Feng LinAffiliation 1: Department of Medicine, National Taiwan University Hospital Chu-Tung Branch, Taiwan
Affiliation 2: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
Bio-protocol author page: a1836
Yun-You Lin
Yun-You LinAffiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
Bio-protocol author page: a1837
 and Chung-Yu Lan
Chung-Yu LanAffiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Taiwan
For correspondence: cylan@life.nthu.edu.tw
Bio-protocol author page: a1838
Vol 4, Iss 23, 12/5/2014, 2429 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1307

[Abstract] Multidrug resistance Acinetobacter baumannii (A. baumannii) (MDRAB) has emerged as a serious threat in hospitals in recent years. Currently, there are few antibiotics, including tigecycline, available to treat infections caused by MDRAB effectively. Both tigecycline-resistant and tigecycline-susceptible isogenic strains of MDRAB are valuable in understanding the mechanisms underlying tigecycline resistance. To get the isogenic strains in the laboratory, we describe a protocol for induction of tigecycline resistance in A. buamannii by serial passage to plates with tigecycline of different concentrations. The minimal inhibitory concentration of A. baumannii by tigecycline was determined according to the protocol “Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii” (Lin et al., 2014b).
Keywords: Acinetobacter baumannii(鲍氏不动杆菌), Tigecycline(替加环素), Drug resistance(耐药性), Serial passages(串行通道)

[Abstract]

Materials and Reagents

  1. A. baumannii (ATCC, catalog number: 17978 )
  2. Tigecycline (Wyeth, catalog number. 0220620-09-7 )
  3. Tryptone (Pronadisa, catalog number: 1612 )
  4. Yeast extract (Pronadisa, catalog number: 1702 )
  5. NaCl (MDBio, catalog number: 101-1647-14-5 )
  6. 99% glycerol (Honeywell International, Riedel-deHaen, catalog number: 5523 )
  7. Lysogeny broth (LB) (see Recipes)
  8. 20% glycerol (see Recipes)

Equipment

  1. 50 ml polystyrene culture tubes (sterile)
  2. 37 °C shaking and static incubators
  3. Multichannel pipette (volume ranges 10 μl-1,000 μl)

Procedure

  1. Fetch the A. baumannii ATCC 17978 stock stored at -80 °C, scrape the surface of the frozen stock, and plate it on a LB agar.
  2. Put the LB agar in the 37 °C incubator overnight.
  3. Dissolve a single colony of A. baumannii from the overnight culture in 3 ml LB broth and incubate overnight at 37 °C, 220 rpm.
  4. On day 1, 3 ml of LB broth containing tigecycline at the MIC, which is determined by the protocol “Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii” (Lin et al., 2014b), is inoculated with 30 μl broth containing A. baumannii (step 1), and the cultures are incubated at 37 °C with shaking (220 rpm) overnight.
    Note: The choice of the volumes 30 μl and 3 ml is aimed to keep 1:100 dilutions. If other volumes or dilution ratios are used, it may lead to the change of tigecycline resistance induction time.
  5. On day 3, 30 μl of the culture is transferred to 3 ml of LB broth containing tigecycline at 8x MIC (step 2), and the cultures are again incubated at 37 °C with shaking (220 rpm). If no bacterial growth is noted from step 1, lower the tigecycline concentration to half of the 8x MIC, then repeat step 1.
  6. On day 5, 30 μl of the culture is transferred into LB broth containing tigecycline at 16x MIC (step 3), and the cultures are again incubated at 37 °C with shaking (220 rpm).
  7. This passaging is repeated on day 7 (step 4).
  8. On day 9, aliquots (0.5 ml) of the cultures are mixed with 0.5 ml 20% glycerol and stored at -80 °C until use.
  9. Daily passaging in tigecycline-free LB is conducted for 30 days after the tigecycline resistant strain is obtained to make sure the induction of tigecycline resistance is successful and long-lasting.

Recipes

  1. 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
  2. 20% glycerol
    20 ml 99% glycerol fill to 100 ml 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. (2014a). 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.
  2. Lin, M., Lin, Y. and Lan, C. (2014b). Minimal inhibitory concentration (MIC) assay for Acinetobacter baumannii. Bio-protocol 4(23): e1308.

材料和试剂

  1. A。 baumannii(ATCC,目录号:17978)
  2. 替加环素(Wyeth,目录号0220620-09-7)
  3. 胰蛋白胨(Pronadisa,目录号:1612)
  4. 酵母提取物(Pronadisa,目录号:1702)
  5. NaCl(MDBio,目录号:101-1647-14-5)
  6. 99%甘油(Honeywell International,Riedel-deHen,目录号:5523)
  7. 溶菌酶肉汤(LB)(参见食谱)
  8. 20%甘油(见配方)

设备

  1. 50ml聚苯乙烯培养管(无菌)
  2. 37°C摇动和静态培养箱
  3. 多通道移液器(体积范围10μl-1,000μl)

程序

  1. 获取 A。 baumannii ATCC 17978储存于-80℃,刮取冷冻储存物的表面,并将其铺在LB琼脂上。
  2. 将LB琼脂在37℃培养箱中过夜。
  3. 溶解单个菌落的 A。 鲍曼不动杆菌3ml LB肉汤中的过夜培养物中,并在37℃,220rpm下培养过夜。
  4. 在第1天,将3ml含有替加环素的LB肉汤(MIC),其通过方案"最小抑制浓度使用含有鲍曼不动杆菌的30μl肉汤(步骤1)接种鲍曼不动杆菌的细菌(MIC)测定(Lin等人,2014b)并将培养物在37℃下振荡(220rpm)孵育过夜。
    注意:体积30μl和3 ml的选择目的是保持1:100稀释。如果使用其他体积或稀释比,可能导致替加环素耐药诱导时间的变化。
  5. 在第3天,将30μl培养物转移到3ml含有8×MIC的替加环素的LB肉汤(步骤2),并将培养物再次在37℃下振荡(220rpm)孵育。如果从步骤1未注意到细菌生长,将替加环素浓度降低至8x MIC的一半,然后重复步骤1.
  6. 在第5天,将30μl培养物转移到含有16x MIC的替加环素的LB肉汤中(步骤3),并将培养物再次在37℃下振荡(220rpm)孵育。
  7. 在第7天重复该传代(步骤4)。
  8. 在第9天,将等份(0.5ml)的培养物与0.5ml 20%甘油混合,并储存在-80℃直至使用。
  9. 在获得替加环素抗性菌株后,进行不含替加环素的LB的日常传代30天,以确保替加环素抗性的诱导成功并持久。

食谱

  1. 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
  2. 20%甘油 将20ml 99%甘油填充至100ml,使用ddH 2 sub 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。(2014a)。 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.
  2. Lin,M.,Lin,Y.and Lan,C.(2014b)。 鲍曼不动杆菌的最小抑制浓度(MIC)测定 生物协议 4(23):e1308。
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How to cite this protocol: Lin, M., Lin, Y. and Lan, C. (2014). Induction of Tigecycline Resistance in Acinetobacter baumannii. Bio-protocol 4(23): e1307. DOI: 10.21769/BioProtoc.1307; Full Text



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