Bajaj Ehsaas, Gupta Ayush, Purwar Shashank, Pundir Shrutika, Brar Sukhmanjit S
Microbiology, All India Institute of Medical Sciences, Bhopal, Bhopal, IND.
Cureus. 2025 Jun 4;17(6):e85321. doi: 10.7759/cureus.85321. eCollection 2025 Jun.
The emergence of infections caused by multidrug-resistant gram-negative strains has necessitated the reuse of polymyxins, which are repurposed antimicrobials due to their initial toxicity profile. However, the present antimicrobial susceptibility testing (AST) methods against them are challenging to perform or need validation. Through this study, we sought to determine how early we can differentiate between the susceptible and resistant gram-negative strains when exposed to a single breakpoint concentration of antibiotic, as expected from a microfluidics-based AST. We used time-kill assay and cell-viability assay to image the organisms and draw a parallel to the principle of microfluidics-based assay. Reference and clinical strains of Enterobacterales and of a particular minimum inhibitory concentration (MIC) value were selected for the study after initially screening by the colistin broth disk elution method and confirmed by colistin broth microdilution (BMD). For complex, only a BMD test was used to determine the MIC. Ten strains of varying MICs, ranging from ≤0.5 to ≥16 μg/mL, were selected for time-kill assay against colistin using acridine orange dye to obtain each strain's total cell count of antibiotic-exposed and non-exposed populations at different time points. These strains were then subjected to the spread-plate method for viability counts at various time points. We obtained 192 unique measurements from 48 colistin-sensitive replicates belonging to 8 strains with MICs ranging from ≤0.5 to 2 μg/mL at different time points. We found that the log count values were statistically lower in the colistin-exposed group at 2, 4, and 8 hours by both time-kill and spread-plate assays. We concluded that it is possible to get AST results against colistin as early as 2 hours if imaging techniques are used for visualizing the bacteria in a microfluidics-based AST device.
多重耐药革兰氏阴性菌引起的感染不断出现，使得多粘菌素不得不重新启用，由于其最初的毒性特征，多粘菌素属于重新利用的抗菌药物。然而，目前针对它们的抗菌药物敏感性试验（AST）方法实施起来具有挑战性，或者需要验证。通过本研究，我们试图确定，当暴露于单一断点浓度的抗生素时，我们能多早区分敏感和耐药的革兰氏阴性菌，这是基于微流控的AST所预期的。我们使用时间杀菌试验和细胞活力试验对微生物进行成像，并与基于微流控的试验原理进行对比。在最初通过黏菌素肉汤纸片洗脱法筛选并经黏菌素肉汤微量稀释法（BMD）确认后，选择了肠杆菌科的参考菌株和临床菌株以及具有特定最低抑菌浓度（MIC）值的菌株进行研究。对于铜绿假单胞菌，仅使用BMD试验来确定MIC。选择了10株MIC值不同（范围从≤0.5至≥16μg/mL）的菌株，使用吖啶橙染料对其进行针对黏菌素的时间杀菌试验，以获取每个菌株在不同时间点暴露于抗生素和未暴露群体的总细胞数。然后在不同时间点对这些菌株进行平板涂布法进行活力计数。我们从48个黏菌素敏感复制品中获得了192个独特的测量值，这些复制品属于8个菌株，MIC范围从≤0.5至2μg/mL，在不同时间点进行测量。我们发现，通过时间杀菌试验和平板涂布法，在2小时、4小时和8小时时，黏菌素暴露组的对数计数值在统计学上较低。我们得出结论，如果在基于微流控的AST装置中使用成像技术来可视化细菌，那么最早在2小时就有可能获得针对黏菌素的AST结果。
