Relative quantification of the recA gene for antibiotic susceptibility testing in response to ciprofloxacin for pathogens of concern

Antibiotic resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antibiotic susceptibility tests (AST) are dependent on organism growth rates resulting in prolonged diagnostic answers for slow growing organisms. Changes in the cellular transcriptome can be instantaneous in the presence of stressors such as antibiotic pressure. Here, we demonstrate that relative quantification of the recA gene in response to breakpoint concentrations of ciprofloxacin is an indicator of pathogen susceptibly. For this purpose, we developed seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, as response and housekeeping genes respectively, for biothreat and ESKAPE pathogens. Surrogate biothreat agents Y. pestis and B. anthracis saw increases in relative recA gene expression, independent of growth rate after 15 minutes of exposure to ciprofloxacin with maximal expression seen after 60 minutes. Treatment with doxycycline also demonstrated an increase in relative recA fold changes compared to no treatment controls. Final evaluation of all seven duplex assays tested across 125 strains, including Tier 1 pathogens, from broth culture demonstrated an overall categorical agreement compared to gold standard microbroth dilution of 97.56% with major error (ME) rates of 1.59% and very major error (VME) rates of 3.23%. Testing on pathogen strains commonly associated with urinary tract infections in contrived clinical sample sets demonstrated an overall categorical agreement of 95.8% with a ME rate of 0.0% and VME rate of 7.69%. These data indicate that relative quantification of a single highly conserved gene accurately predicts susceptibility for multiple bacterial species in response to ciprofloxacin. Overall design: RNA sequencing was performed on bacterial samples exposed to breakpoint concentrations of ciprofloxacin across time. RNA sequencing analysis was compared between resistant and suceptable strains as well as compared across multple organisms including B. anthracis and Y. pestis.

抗生素耐药性（Antibiotic resistance, AR）是全球健康面临的最重大威胁之一，其与更高的治疗成本、更长的住院时长以及升高的死亡率紧密相关。当前主流的金标准药敏试验（Antibiotic Susceptibility Test, AST）依赖于微生物的生长速率，这使得慢生长微生物的诊断结果出具周期被显著延长。当遭遇抗生素压力等应激原时，细胞转录组的变化可在瞬时发生。本研究证实，针对环丙沙星临界浓度下的recA基因开展相对定量分析，可作为病原体药敏性的有效指示指标。
为此，我们分别以recA基因与16S rRNA基因（16S rRNA gene）作为应答基因与持家基因，开发了7种双重逆转录实时定量PCR（duplex RT-qPCR）检测方法，覆盖生物威胁病原体与ESKAPE病原体（ESKAPE pathogens）。针对替代生物威胁病原体鼠疫耶尔森菌（Y. pestis）与炭疽芽孢杆菌（B. anthracis）的实验结果显示，在暴露于环丙沙星15分钟后，二者的recA基因相对表达量即出现升高，且该变化与微生物生长速率无关；在暴露60分钟后，recA基因相对表达量达到峰值。经多西环素处理的样本，其recA基因相对表达折叠倍数同样较未处理对照组显著升高。
对125株菌株（包含一级（Tier 1）病原体）的肉汤培养物开展的全部7种双重检测方法的最终评估结果显示：与金标准微量肉汤稀释法相比，整体分类一致率为97.56%，严重错误（major error, ME）率为1.59%，极严重错误（very major error, VME）率为3.23%。在模拟临床样本集内针对常见尿路感染相关病原体的检测结果显示，整体分类一致率为95.8%，严重错误率为0.0%，极严重错误率为7.69%。
上述数据表明，对单个高度保守基因进行相对定量，可准确预测多种细菌在环丙沙星压力下的药敏性。整体实验设计：对暴露于环丙沙星临界浓度的细菌样本开展跨时间维度的RNA测序（RNA sequencing）；将耐药菌株与敏感菌株的RNA测序分析结果进行对比，并针对包括炭疽芽孢杆菌与鼠疫耶尔森菌在内的多种生物体开展跨物种对比分析。