Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
Department of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
Microb Pathog. 2021 May;154:104840. doi: 10.1016/j.micpath.2021.104840. Epub 2021 Mar 7.
Extensive use of antibiotics in clinical practice has been associated with increasing frequency of resistant microorganisms. So new strategy is needed to treat the resistant pathogens. Hence this study was conducted to determine the effect of Ethylenediaminetetraacetic acid (EDTA) in increasing the inhibition effect of some antibiotics on multi-drug resistant (MDR) gram-negative bacteria. For this purpose, 40 E. coli isolates, 40 K. pneumoniae isolates and 50 P. aeruginosa isolates were collected from different University's hospitals in Mansoura, Egypt. Antibacterial susceptibility pattern against 9 different antimicrobials were studied by disc diffusion method. Also the effect of two sub-inhibitory concentrations of EDTA (1 and 2 mM) on the inhibition zones of antibiotic discs against the highly multidrug resistant (MDR) isolates was determined. Checkerboard method was used for testing the activity of gentamicin/EDTA and cefotaxime/EDTA combinations on the highly MDR isolates. Additionally, the effect of EDTA on the expression of efflux pump genes was tested by real time-PCR. Most of the clinical isolates were found to be resistant to the tested antibiotics except imipenem and high prevalence of MDR isolates was recorded. 34 isolates were selected as those showed the highest multi-drug resistance and were tested to specify their MIC for EDTA as EDTA showed strong antibacterial activity with MIC ranging 4-8 mM. The addition of sub-MIC of EDTA (1or 2 mM) to the agar plate resulted in changing the 11 tested E. coli isolates from resistant to sensitive to ceftazidime, gentamicin, rifampin, ampicillin, erythromycin and vancomycin, the tested K. pneumoniae isolates were turned also from resistant to sensitive to gentamicin and ceftazidime, additionally the tested P. aeruginosa isolates became sensitive to gentamicin, ceftazidime and ciprofloxacin. Indifference to additive activity was observed for tested combinations and MIC value of cefotaxime or gentamicin in combination with EDTA was less than antibiotic alone in the most tested isolates. Moreover, significant reduction (P < 0.01) in the expression of all tested efflux pump genes in treated E. coli, K. pneumoniae and P. aeruginosa isolates with EDTA compared to untreated isolates was observed. In conclusion, these results suggest that the combination of antibiotic especially gentamicin with EDTA may be fruitful for management of resistant gram negative infections.
在临床实践中广泛使用抗生素与耐药微生物的频率增加有关。因此，需要新的策略来治疗耐药病原体。因此，本研究旨在确定乙二胺四乙酸（EDTA）在增加某些抗生素对多药耐药（MDR）革兰氏阴性菌的抑制作用方面的效果。为此，从埃及曼苏拉的不同大学医院收集了 40 株大肠杆菌、40 株肺炎克雷伯菌和 50 株铜绿假单胞菌。采用圆盘扩散法研究了 9 种不同抗菌药物的抗菌药敏模式。还测定了两种亚抑菌浓度的 EDTA（1 和 2 mM）对高度多药耐药（MDR）分离株的抗生素药敏纸片抑制区的影响。棋盘法用于测试庆大霉素/EDTA 和头孢噻肟/EDTA 组合对高度 MDR 分离株的活性。此外，通过实时 PCR 测试 EDTA 对流出泵基因表达的影响。除亚胺培南外，大多数临床分离株对测试的抗生素均表现出耐药性，并且记录了高流行的 MDR 分离株。选择 34 株作为显示出最高多药耐药性的分离株，并测试其对 EDTA 的 MIC，因为 EDTA 显示出强大的抗菌活性，其 MIC 范围为 4-8 mM。在琼脂平板中加入亚 MIC 的 EDTA（1 或 2 mM）会导致 11 株测试的大肠杆菌分离株从头孢噻肟、庆大霉素、利福平、氨苄西林、红霉素和万古霉素耐药变为敏感，测试的肺炎克雷伯菌分离株也从头孢噻肟和庆大霉素耐药变为敏感，此外，测试的铜绿假单胞菌分离株对庆大霉素、头孢噻肟和环丙沙星敏感。测试组合的活性无差异，在大多数测试的分离株中，头孢噻肟或庆大霉素与 EDTA 联合的 MIC 值小于单独使用抗生素的 MIC 值。此外，与未处理的分离株相比，用 EDTA 处理的大肠杆菌、肺炎克雷伯菌和铜绿假单胞菌分离株中所有测试的外排泵基因的表达均显著降低（P < 0.01）。总之，这些结果表明，抗生素（特别是庆大霉素）与 EDTA 的联合使用可能有助于治疗耐药革兰氏阴性感染。
