Department of Microbiology, School of Medicine, Kyungpook National University, 680 Gukchaebosang-Ro, Jung-gu, Daegu 41944, South Korea.
J Antimicrob Chemother. 2020 May 1;75(5):1130-1134. doi: 10.1093/jac/dkaa014.
Treatment of infections caused by Acinetobacter baumannii nosocomial strains has become increasingly problematic owing to their resistance to antibiotics. ppGpp is a secondary messenger involved in growth control and various stress responses in bacteria. The mechanism for inhibition of antibiotic resistance via ppGpp is still unidentified in various pathogenic bacteria including A. baumannii. Here, we investigated the effects of ppGpp on efflux pump (EP)-related genes in A. baumannii.
ppGpp-deficient and -complementary strains were constructed by conjugation and we confirmed (p)ppGpp measurements by thin-layer chromatography. We observed that the ppGpp-deficient strain (ΔA1S_0579) showed abnormal stretching patterns by transmission electron microscopy analysis. The MICs of antimicrobial agents for the WT A. baumannii (ATCC 17978), ppGpp-deficient and complementary strains were determined by the Etest and broth dilution assay methods. The expression levels of EP-related genes were determined by quantitative RT-PCR.
We observed morphological differences between a ppGpp-deficient strain (ΔA1S_0579) and the WT strain. Dramatic reductions of MICs in the ppGpp-deficient strain compared with the WT were observed for gentamicin (2.6-fold), tetracycline (3.9-fold), erythromycin (4-fold) and trimethoprim (>4-fold). Expression of the EP-related genes abeB (2.8-fold), tet(A) (2.3-fold), adeB (10.0-fold), adeI (9.9-fold), adeJ (11.8-fold) and adeK (14.4-fold) was also decreased in the ppGpp-deficient strain.
This study demonstrates that ppGpp regulates EP-related gene expression in A. baumannii, affecting antibiotic susceptibility. To date, treatment for MDR A. baumannii has had no new antimicrobial agents, so the A1S_0579 gene could be a novel therapeutic target for rational drug design by affecting ppGpp production.
由于对抗生素的耐药性，医院获得性鲍曼不动杆菌感染的治疗变得越来越成问题。ppGpp 是一种参与细菌生长控制和各种应激反应的次级信使。ppGpp 通过抑制抗生素耐药性的机制在包括鲍曼不动杆菌在内的各种致病菌中仍未被确定。在这里，我们研究了 ppGpp 对鲍曼不动杆菌中外排泵（EP）相关基因的影响。
通过接合构建了 ppGpp 缺陷和互补菌株，并通过薄层层析法证实了（p）ppGpp 的测量。我们观察到 ppGpp 缺陷菌株（ΔA1S_0579）通过透射电子显微镜分析显示出异常的拉伸模式。通过 Etest 和肉汤稀释法测定 WT 鲍曼不动杆菌（ATCC 17978）、ppGpp 缺陷和互补菌株的抗菌药物 MIC。通过定量 RT-PCR 测定 EP 相关基因的表达水平。
我们观察到 ppGpp 缺陷菌株（ΔA1S_0579）与 WT 菌株之间的形态差异。与 WT 相比，ppGpp 缺陷菌株的 MIC 显著降低，对庆大霉素（2.6 倍）、四环素（3.9 倍）、红霉素（4 倍）和甲氧苄啶（>4 倍）。abeeB（2.8 倍）、tetA（2.3 倍）、adeB（10.0 倍）、adeI（9.9 倍）、adeJ（11.8 倍）和 adeK（14.4 倍）的 EP 相关基因表达也在 ppGpp 缺陷菌株中降低。
本研究表明，ppGpp 调节鲍曼不动杆菌中 EP 相关基因的表达，影响抗生素敏感性。迄今为止，对多药耐药鲍曼不动杆菌的治疗没有新的抗菌药物，因此 A1S_0579 基因可能通过影响 ppGpp 的产生成为合理药物设计的新治疗靶点。
