Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, 310027, China.
Center of Health Science and Engineering, Hebei Key Laboratory of Biomaterials and Smart Theranostics, Hebei University of Technology, Tianjin, 300131, China.
Anal Chim Acta. 2023 Nov 22;1282:341858. doi: 10.1016/j.aca.2023.341858. Epub 2023 Oct 16.
The empirical antibiotic therapies for bacterial infections cause the emergence and propagation of multi-drug resistant bacteria, which not only impair the effectiveness of existing antibiotics but also raise healthcare costs. To reduce the empirical treatments, rapid antimicrobial susceptibility testing (AST) of causative microorganisms in clinical samples should be conducted for prescribing evidence-based antibiotics. However, most of culture-based ASTs suffer from inoculum effect and lack differentiation of target pathogen and commensals, hampering their adoption for evidence-based antibiotic prescription. Therefore, rapid ASTs which can specifically determine pathogens' susceptibilities, regardless of the bacterial load in clinical samples, are in urgent need.
We present a pathogen-specific and inoculum size-insensitive AST to achieve the reliable susceptibility determination on Escherichia coli (E. coli) in urine samples. The developed AST is featured with an 1 h sample-to-result workflow in a filter, termed on-filter AST. The AST results can be obtained by using an inducible enzymatic assay to in-situ measure the cell response of E. coli collected from urine after 20 min of antibiotic exposure. The calculated detection limit of our AST (1.95 × 10 CFU/mL) is much lower than the diagnosis threshold of urinary tract infections. The specific expression of the inducible enzyme enables on-filter AST to correctly profile the susceptibilities of target pathogen to multi-type antibiotics without the interference from commensals. We performed the on-filter AST on 1 mL urine samples with bacterial loads varying from 10 CFU/mL to 10 CFU/mL and compared the results to that of standard method, demonstrating its insensitivity to inoculum size.
The developed AST is demonstrated to be of high sensitivity, specificity, and insensitive to inoculum size. With further developments for additional bacteria and clinical validation, on-filter AST is promising as a rapid and reliable surrogate of culture-based AST to promote the evidence-based prescription at the first visit and minimize the emergency of new multi-drug resistant microorganisms.
经验性抗生素治疗细菌感染会导致多药耐药菌的出现和传播，这不仅会降低现有抗生素的疗效，还会增加医疗保健成本。为了减少经验性治疗，应对临床样本中的病原体进行快速抗菌药物敏感性测试（AST），以开出基于证据的抗生素。然而，大多数基于培养的 AST 存在接种体效应，且缺乏目标病原体和共生菌的区分，这阻碍了其在基于证据的抗生素处方中的应用。因此，急需能够特异性确定病原体敏感性的快速 AST，而无需考虑临床样本中的细菌载量。
我们提出了一种病原体特异性和接种体大小不敏感的 AST，以实现对尿液样本中大肠杆菌（E. coli）的可靠药敏测定。该开发的 AST 的特点是在过滤器中实现 1 小时的样本到结果工作流程，称为过滤器内 AST。AST 结果可以通过使用诱导酶测定法来原位测量暴露抗生素 20 分钟后从尿液中收集的大肠杆菌的细胞反应来获得。我们的 AST 的检测限（1.95×10 CFU/mL）比尿路感染的诊断阈值低得多。诱导酶的特异性表达使过滤器内 AST 能够正确分析目标病原体对多种类型抗生素的敏感性，而不受共生菌的干扰。我们在细菌载量从 10 CFU/mL 到 10 CFU/mL 的 1 mL 尿液样本上进行了过滤器内 AST，并将结果与标准方法进行了比较，证明其对接种体大小不敏感。
开发的 AST 具有高灵敏度、特异性和对接种体大小不敏感的特点。随着对其他细菌和临床验证的进一步发展，过滤器内 AST 有望成为一种快速可靠的替代基于培养的 AST 的方法，以促进首次就诊时的基于证据的处方，并最大程度地减少新的多药耐药微生物的出现。
