过表达碳青霉烯酶OXA-48对菌株耐药性、适应性及宿主细胞Toll样受体信号通路的影响  

Effects of overexpression of carbapenem OXA-48 on drug resistance,adaptability of bacterial strain and Toll-like receptor signaling pathway of host cell

作　　者：潘亚菲 赵志军 党甜甜[1] 康宇婷 贾伟 PAN Yafei;ZHAO Zhijun;DANG Tiantian;KANG Yuting;JIA Wei(Laboratory Medical Center,General Hospital of Ningxia Medical University,Yinchuan 750004,the Ningxia Hui Autonomous Region,China;不详)

机构地区：[1]宁夏医科大学总医院医学实验中心,宁夏银川750004 [2]宁夏病原微生物重点实验室,宁夏银川750004 [3]宁夏医科大学总医院医学科学研究院,宁夏银川750004

出　　处：《中国生物制品学杂志》2023年第9期1032-1038,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81960386);宁夏回族自治区重点研发计划(2021BEG03090)。

摘　　要：目的探讨过表达OXA-48对菌株耐药性、适应性及宿主细胞Toll样受体(Toll-like receptor,TLR)信号通路的影响。方法将重组质粒pET32a(+)-OXA-48转化至E.coli BL21(DE3)中,获得的重组菌pET32a(+)-OXA-48-BL21(DE3)进行菌落PCR及测序鉴定。以A_(600)(0.3、0.5、0.7)、IPTG终浓度(0.4、0.6、0.8 mmol/L)及诱导时间(2、4、6 h)为变量,mRNA转录水平为响应值,设计3因素3水平正交试验,优化质粒的诱导表达条件。纸片扩散法检测重组菌pET32a(+)-OXA-48-BL21(DE3)对亚胺培南(Imipenem,IPM)、美罗培南(Meropenem,MEM)、头孢曲松(Ceftriaxone,CRO)、头孢吡肟(Cefepime,FEP)的耐药性;通过生物膜形成试验和血清抵抗力试验检测重组菌pET32a(+)-OXA-48-BL21(DE3)的适应性。将pET32a(+)-OXA-48-BL21(DE3)、pET32a(+)-BL21(DE3)和E.coli BL21(DE3)菌株分别感染小鼠肺泡巨噬细胞MH-S,qRT-PCR法检测细胞中TLR2、TLR4和NF-кB(p65)基因mRNA转录水平,ELISA法检测细胞因子白细胞介素-6(interleukin-6,IL-6)、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和转化生长因子-β(transforming growth factor-β,TGF-β)的表达情况。结果经菌落PCR及测序鉴定,重组菌pET32a(+)-OXA-48-BL21(DE3)构建正确。最佳诱导条件:A_(600)为0.3,IPTG终浓度为0.6 mmol/L,诱导时间为2 h。与pET32a(+)-BL21(DE3)菌株比较,重组菌pET32a(+)-OXA-48-BL21(DE3)对IPM、MEM、CRO和FEP 4种抗生素的耐药性明显降低(t=7.14~22.32,P均<0.05),形成的生物膜明显增多(t=15.69,P<0.05),在血清中的存活率显著升高(P<0.05);pET32a(+)-OXA-48-BL21(DE3)感染MH-S细胞24 h后,TLR2基因mRNA转录水平显著增加(t=5.77,P<0.05),感染12及24 h后,TLR4、NF-кB(p65)基因mRNA转录水平显著增加(t=3.71~10.06,P<0.05)。与正常细胞组相比较,pET32a(+)-OXA-48-BL21(DE3)感染MH-S细胞6、12和24 h后,IL-6及TNF-α的表达水平显著升高(t分别为7.90~13.44及5.40~6.32,P均<0.01),IL-10表达水平显著下降(t=3.15~4.08,P均<0.05),TGF-β的表达水平差异无统计�Objective To investigate the effects of overexpression of OXA-48 on drug resistance,adaptability of bacterial strain and Toll-like receptor(TLR)signaling pathway of host cells.Methods The recombinant plasmid pET32a(+)-OXA-48was transformed into E.coli BL21(DE3),and the recombinant strain pET32a(+)-OXA-48-BL21(DE3)was identified by colony PCR and sequencing.Taking A_(600)(0.3,0.5 and 0.7),IPTG final concentration(0.4,0.6 and 0.8mmol/L)and induction time(2,4 and 6 h)as variables and mRNA transcription level as response value,an orthogonal experiment with three factors and three levels was designed to optimize the induced expression conditions of the plasmid.The drug resistance of recombinant strain pET32a(+)-OXA-48-BL21(DE3)to Imipenem(IPM),Meropenem(MEM),Ceftriaxone(CRO)and Cefepime(FEP)was detected by disk diffusion method;The adaptability was detected by biofilm formation test and serum resistance test.Mouse alveolar macrophages(MH-S)were infected with pET32a(+)-OXA-48-BL21(DE3),pET32a(+)-BL21(DE3)and E.coli BL21(DE3)strains,respectively.The mRNA transcription levels of TLR2,TLR4 and NF-кB(p65)genes were detected by qRT-PCR method,and the expressions of Interleukin-6(IL-6),IL-10,tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β)were detected by ELISA.Results The recombinant strain pET32a(+)-OXA-48-BL21(DE3)was constructed correctly as identified by colony PCR and sequencing.The optimum induction conditions were as follows:A_(600)of 0.3,IPTG final concentration of 0.6 mmol/L and induction time of 2 h.Compared with pET32a(+)-BL21(DE3)strain,the resistance of recombinant strain pET32a(+)-OXA-48-BL21(DE3)to IPM,MEM,CRO and FEP significantly decreased(t=7.14~22.32,P<0.05),the biofilm formed significantly increased(t=15.69,P<0.05),and the survival rate in serum significantly increased(t=10.60,P<0.05);The mRNA transcription level of TLR2 gene in MH-S cells infected with pET32a(+)-OXA-48-BL21(DE3)significantly increased 24 h after infection(t=5.77,P<0.05),while the mRNA transcription level of TLR

关 键 词：碳青霉烯酶OXA-48 耐药性 适应性 TOLL样受体 细胞因子 

分 类 号：Q939.93[生物学—微生物学]