Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, 243122, India.
ICAR-National Research Centre on Meat, Chengicherla, Telangana, 500092, India.
Probiotics Antimicrob Proteins. 2020 Jun;12(2):705-715. doi: 10.1007/s12602-019-09589-8.
The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco's modified Eagle's medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.
本研究考察了两种短链抗菌肽（AMPs），即抑菌肽和 Cecropin A（1-7）-蜂毒素（CAMA）对生物膜形成的多药耐药肠聚集性大肠杆菌（MDR-EAEC）分离株的抗生物膜功效。通过 PCR 重新验证并通过 HEp-2 细胞粘附试验确认的典型 EAEC 分离株进行抗生素敏感性测试以确认其 MDR 状态。通过刚果红结合、微量滴定板测定和疏水性指数评估 MDR-EAEC 分离株的生物膜形成能力；采用肉汤微量稀释技术测定最小抑菌浓度（MIC）和最小生物膜清除浓度（MBEC）。单独和联合使用结晶紫（CV）染色和基于共聚焦显微镜的活/死细胞定量方法，评估两种 AMP 对 MDR-EAEC 生物膜的获得 MIC 和 MBEC 值。所有三种 MDR-EAEC 菌株均显示出弱至强的生物膜形成能力，并且被发现是电子供体和弱电子受体（疏水性指数）。此外，在含有 0.45% D-葡萄糖的 Dulbecco 改良 Eagle 培养基（DMEM）中孵育 48 小时时，三种 MDR-EAEC 菌株的动态生长呈高度显著（P<0.001）时间依赖性。AMP 及其组合能够在 24 小时和 48 小时时通过 CV 染色和共聚焦定量证明抑制初始生物膜形成。此外，在 24 小时后处理时，AMP（单独和组合）对预形成的 MDR-EAEC 生物膜的应用导致高度显著的清除（P<0.001）。然而，在 AMP 处理（单独或组合）之间没有观察到显著差异。AMP 似乎是进一步研究的有效候选物，例如安全性、稳定性和适当的生物膜形成 MDR-EAEC 动物模型。
