A Real-Time PCR Antibiogram for Drug-Resistant Sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.

当前用于败血症药敏检测的分子诊断技术，均依赖于对已知耐药基因盒（resistance cassettes）存在情况的基因分型。该技术存在固有局限性，无法检出新发耐药基因。传统表型药敏检测虽一直是检测耐药性的更优方法，但依赖长达数日的培养周期以确定抗菌药物使用方案，会危及患者生存。上述因素导致广谱抗菌药物被广泛且不合理地使用。本文所述的实时PCR（real-time PCR）药敏谱，将通用型表型药敏检测与PCR的快速诊断能力相结合。本研究开发了一种检测方法：对暴露于不同抗菌药物的血液样本，以高度保守的16S rRNA基因（16S rRNA gene）作为标记物监测病原体载量，以此判断药敏性。该优化后的实验流程可去除血液中的血红素与人类背景DNA，使得标准实时PCR检测系统能够以高灵敏度（<100 CFU/mL，菌落形成单位每毫升）开展检测。研究将3株大肠杆菌（其中2株为耐药株）接种至全血样本中，并分别使其暴露于3种不同的抗菌药物。在通过实时PCR完成病原体载量测定后，未处理组与处理组样本间的ΔCt值小于3.0即可判定为抗菌药物耐药（P<0.01）。本研究还针对敏感菌完成了最低抑菌浓度（minimum inhibitory concentration, MIC）的测定，并通过3株革兰氏阴性菌与2株革兰氏阳性菌验证了泛细菌检测能力。物种鉴定则通过分析高可变区扩增子完成。综上，本研究开发了一种通用型诊断表型检测技术，可借助PCR的快速性实现耐药败血症的药敏检测。该实时PCR药敏谱可在24小时内完成病原体检测、药敏试验、最低抑菌浓度测定以及物种鉴定。