抗菌药对鲍曼不动杆菌外膜囊泡产量及主要生物学特性的影响

Antimicrobials affect the production and characteristics of outer membrane vesicles of Acinetobacter baumannii

摘要:

【背景】细菌耐药性已成为全球健康卫生和经济发展的巨大威胁。替加环素是治疗多重耐药肠杆菌所致严重感染的主要药物之一，但在2019年发现了可介导其高水平耐药的可转移替加环素耐药基因tet（X3）。外膜囊泡作为介导水平基因转移的新型方式，在介导tet（X3）水平转移中的作用目前尚无报道。【目的】以tet（X3）阳性替加环素耐药鲍曼不动杆菌34AB为对象，探究不同抗菌药物对其外膜囊泡产量及主要生物学特性的影响。【方法】采用微量肉汤稀释法测定细菌药物敏感性，超速离心法提取细菌外膜囊泡，BCA法测定外膜囊泡产量，使用马尔文纳米粒度电位仪测定外膜囊泡的粒径与电位，PCR法（定性）及RT-qPCR法（定量）检测外膜囊泡中携带的tet（X3）基因。【结果】相较于无抗生素对照组[（0.64±0.04） mg/mL]，在不同抗菌药物亚抑菌浓度（1/2 MIC和1/4 MIC）处理后，34AB外膜囊泡的产量均有所增加，以头孢他啶[1/2 MIC，（2.83±0.57） mg/mL；1/4 MIC，（2.38±0.29） mg/mL]和美罗培南[1/2 MIC，（2.19±0.11） mg/mL；1/4 MIC，（1.96±0.37） mg/mL]作用最为显著（p<0.01）。同时抗菌药物作用后，各组外膜囊泡粒径和电位均有所降低，而携带的tet（X3）基因拷贝数均有所上升（2.80×10⁴-2.63×10⁷copies/μL）。【结论】抗菌药物的临床应用可能会导致耐药细菌外膜囊泡产量及携带的耐药基因丰度增加，进而增强其作为水平基因转移载体传播耐药基因的风险。

Abstract:

[Background] Antimicrobial resistance poses a huge threat to global health and economic development. Tigecycline is one of the important drugs for treating severe infections caused by multidrug-resistant Enterobacteriaceae. However, the mobile resistance gene tet(X3), which could mediate high-level tigecycline resistance, was discovered in 2019. Outer membrane vesicles have been recognized as a new route for horizontal gene transfer, while little is known about the role of outer membrane vesicles in the disseminating of tet(X3). [Objective] To investigate the effects of different antimicrobials on the production and characteristics of outer membrane vesicles of tet(X3)-positive tigecycline-resistant Acinetobacter baumannii strain 34AB. [Methods] The antimicrobial susceptibility was determined by the broth micro-dilution method. The bacterial outer membrane vesicles were extracted by ultracentrifugation. The protein concentration of the outer membrane vesicles was determined by the BCA method, and the size and potential tested by Zeta-sizer nano-ZS. The copy number of tet(X3) within the outer membrane vesicles was determined by RT-qPCR. [Results] Compared with the control group without antibiotic treatment [(0.64±0.04) mg/mL], antimicrobials at 1/2 MIC or 1/4 MIC increased the production of outer membrane vesicles, with the effects of ceftazidime [1/2 MIC, (2.83±0.57) mg/mL; 1/4 MIC, (2.38±0.29) mg/mL] and meropenem [1/2 MIC, (2.19±0.11) mg/mL; 1/4 MIC, (1.96±0.37) mg/mL] being the most significant (P<0.01). Meanwhile, the antimicrobial treatments decreased the size and potential of outer membrane vesicles and increased the copy number of tet(X3) (2.80×10⁴-2.63×10⁷copies/μL). [Conclusion] The clinical application of antimicrobials may increase the production and the antimicrobial resistance gene abundance of outer membrane vesicles, thereby increasing the risk of them as vectors for disseminating of antimicrobial resistance genes.