Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant <italic style="font-style: italic">Escherichia coli</italic>

Yan SHI; Weifeng YANG; Wensheng QI

doi:10.13422/j.cnki.syfjx.20221992

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Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli

更新时间：2022-08-30

- Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 19, Pages: 73-80(2022)
- 作者机构：
  
  1.航天中心医院，北京 100049
  2.中国中医科学院医学实验中心，北京 100700
  3.中国中医科学院广安门医院，北京 100053
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221992
  CLC： R285.5;R966;R378;R978.1
- Received：29 August 2021，
  
  Published Online：23 March 2022，
  
  Published：05 October 2022
- 稿件说明：
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石岩,杨伟峰,齐文升.新加达原散联合亚胺培南西司他丁对临床分离多重耐药大肠埃希菌生物膜的影响[J].中国实验方剂学杂志,2022,28(19):73-80.

SHI Yan,YANG Weifeng,QI Wensheng.Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(19):73-80.
石岩,杨伟峰,齐文升.新加达原散联合亚胺培南西司他丁对临床分离多重耐药大肠埃希菌生物膜的影响[J].中国实验方剂学杂志,2022,28(19):73-80. DOI： 10.13422/j.cnki.syfjx.20221992.

SHI Yan,YANG Weifeng,QI Wensheng.Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(19):73-80. DOI： 10.13422/j.cnki.syfjx.20221992.

摘要

目的

通过实验筛选出产超广谱

-内酰胺酶（ESBL）且成膜能力最强的大肠埃希菌，探讨新加达原散（MDYS）联合亚胺培南西司他丁（IPM）对其生物膜状态下的干预作用。

方法

通过纸片扩散、结晶紫染色法鉴定临床分离的19株耐药大肠埃希菌产酶及生物被膜的形成能力情况，筛出产酶且成膜能力最强大肠埃希菌。通过噻唑蓝（MTT）比色法检测MDYS组和IPM组最低抑菌浓度后，四唑鎓盐（XTT）比色法检测1/2、1/4、1/8 MDYS水提取物最低抑菌浓度（MIC）与1/2、1/4、1/8 IPM MIC单独及联合用药效果筛选最佳用药浓度，BioFlux动态观察所选取最佳联合用药浓度对大肠埃希菌生物膜内细菌数量及生物膜形成的影响，并用激光共聚焦扫描显微镜观察活菌/死菌的分布。最后通过扫描电镜静态观察药物治疗后细菌的形态学变化。

结果

E5E7菌株为产ESBL且成膜能力最强大肠埃希菌。MTT比色法检测结果显示，IPM、MDYS水提物对E5E7菌株的MIC值分别为1 mg·L

-1

、250 g·L

-1

，XTT比色法检测结果显示，与空白组比较，1/2、1/4、1/8 MDYS水提物MIC及联合用药组可显著减少生物膜活菌量（

0.01），随IPM浓度减小，抑制作用减弱。与单用相同浓度的IPM组比较，联合用药组协同抑制生物膜活菌量显著增强（

0.01）。与单用相同浓度的MDYS组比较，1/2 IPM MIC联合1/2、1/4、1/8 MDYS MIC，1/4 IPM MIC联合1/2、1/4 MIC MDYS，1/8 IPM MIC联合1/2、1/4 MDYS MIC活菌量明显降低（

0.05，

0.01）。与空白组比较，1/8 IPM MIC、1/2 IPM MIC对细菌解聚解黏附作用不明显。当使用1/2、1/4 MDYS水提取物MIC时膜面积明显减小。在IPM与MDYS联合用药时，与空白组或者单用IPM组比较，膜面积变小。共聚焦结果显示，空白组活菌生力旺盛。IPM与MDYS联合用药及MDYS单独使用时可以观察到细菌大多呈红色染色，细菌代谢能力弱，为死菌；在1/2、1/8 IPM MIC时，可观察到代谢活性较强的浮游菌。扫描电镜下结果显示，与空白组和IPM组比较，IPM和MDYS联合用药作用下，分裂周期明显长于空白组，且联合用药组分裂期长度高于单独用药组。

结论

体外研究揭示了MDYS联合常用抗生素对多重耐药大肠埃希菌生物膜状态具有抑制作用，MDYS具有增敏和协同抗生素抑菌的作用。

Abstract

Objective

To screen out the extended spectrum beta-lactamase （ESBL）-producing

Escherichia coli

with the strongest biofilm-forming ability through experiments， and discuss the effect of modified Dayuansan （MDYS） combined with imipenem-cilastatin and cilastatin sodium on the biofilm of

E. coli.

Method

The paper diffusion and crystal violet staining methods were used to identify 19 clinically isolated strains of drug-resistant

E. coli

-induced enzymes and the biofilm-forming ability. The induced enzymes and the

E. coli

with the strongest biofilm-forming ability were screened out. The minimum inhibitory concentration （MIC） value of MDYS and imipenem-cilastatin and cilastatin sodium was determined by 2，3-bis（2-methoxy-4-nitro-5-sulfophenyl）-2H-tetrazolium-5-carboxamide （XTT） assay. The 1/2， 1/4， and 1/8 MIC of the water extract of MDYS， imipenem-cilastatin and cilastatin sodium alone， and MDYS combined with imipenem-cilastatin and cilastatin sodium was determined by methyl thiazolyl tetrazolium （MTT） assay to obtain the optimum concentration of drugs. BioFlux dynamically observed the effect of the optimum combined drug concentration on the number of bacteria in the biofilm and the biofilm formation of

E. coli

， and observed the distribution of live/dead bacteria with a laser confocal scanning microscope. Finally， the morphological changes in bacteria after drug treatment were observed statically by scanning electron microscopy.

Result

E5E7 strain was ESBL enzyme and the

E. coli

with the strongest biofilm-forming ability. The results of MTT assay showed that the MIC values of the water extracts of imipenem-cilastatin and cilastatin sodium and MDYS were 1 mg·L

-1

and 250 g·L

-1

， respectively. The results of XTT assay showed that compared with the blank group， the 1/2， 1/4， and 1/8 MIC MDYS groups and the combined drug groups significantly decreased the number of bacteria in the biofilm （

0.01）. The inhibitory effect diminished as the concentration of imipenem-cilastatin and cilastatin sodium decreased. Compared with the imipenem-cilastatin and cilastatin sodium group with the same concentration， the combined drug group improved the inhibitory effect on the number of bacteria in the biofilm （

0.01）. Compared with the MDYS group with the same concentration， 1/2 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2， 1/4， and 1/8 MIC MDYS， 1/4 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS， and 1/8 MIC imipenem-cilastatin and cilastatin sodium combined with 1/2 and 1/4 MIC MDYS decreased the number of bacteria （

0.05，

0.01）. The results of BioFlux showed that compared with the blank group， the 1/2 and 1/8 MIC imipenem-cilastatin and cilastatin sodium groups had an insignificant effect on the area of biofilm， whereas the 1/2 and 1/4 MIC MDYS groups significantly decreased the area of biofilm. The results under the scanning electron microscopy showed that as compared with the blank group and the imipenem-cilastatin and cilastatin sodium group， the division cycle was significantly longer under the action of MDYS combined with imipenem-cilastatin and cilastatin sodium. The length of the division cycle in the combined drug group was higher than that in drug alone group.

Conclusion

In vitro

studies reveal that MDYS combined with commonly-used antibiotics can inhibit the biofilm status of multi-drug resistant

E. coli

， and MDYS has the effect of enhancing sensitization and inhibiting bacteria with synergistic antibiotics.

关键词

Keywords

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Effect of Modified Dayuansan Combined with Imipenem-cilastatin on Biofilm of Clinically Isolated Multidrug-resistant Escherichia coli

DOI：10.13422/j.cnki.syfjx.20221992

摘要

Abstract

关键词

Keywords

references