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大肠埃希菌质粒编码的blaKPC-2基因介导碳青霉烯类药物低水平耐药的分子机制

白栓成, 杨澜, 罗湘, 黄丽雅, 王雨欣, 王熔, 廖凤婷, 廖晓萍

白栓成, 杨澜, 罗湘, 等. 大肠埃希菌质粒编码的blaKPC-2基因介导碳青霉烯类药物低水平耐药的分子机制[J]. 华南农业大学学报, 2025, 46(3): 326-335. DOI: 10.7671/j.issn.1001-411X.202411006
引用本文: 白栓成, 杨澜, 罗湘, 等. 大肠埃希菌质粒编码的blaKPC-2基因介导碳青霉烯类药物低水平耐药的分子机制[J]. 华南农业大学学报, 2025, 46(3): 326-335. DOI: 10.7671/j.issn.1001-411X.202411006
BAI Shuancheng, YANG Lan, LUO Xiang, et al. Molecular mechanism of plasmid-encoded blaKPC-2 gene mediating low-level resistance to carbapenems in Escherichia coli[J]. Journal of South China Agricultural University, 2025, 46(3): 326-335. DOI: 10.7671/j.issn.1001-411X.202411006
Citation: BAI Shuancheng, YANG Lan, LUO Xiang, et al. Molecular mechanism of plasmid-encoded blaKPC-2 gene mediating low-level resistance to carbapenems in Escherichia coli[J]. Journal of South China Agricultural University, 2025, 46(3): 326-335. DOI: 10.7671/j.issn.1001-411X.202411006

大肠埃希菌质粒编码的blaKPC-2基因介导碳青霉烯类药物低水平耐药的分子机制

基金项目: 

玉林师范学院高层次人才科研启动基金(G2024ZK13);国家重点研发计划(2023YFD1800100);广东省珠江人才计划本地创新研究团队项目(2019BT02N054)

详细信息
    作者简介:

    白栓成,主要从事兽医病原微生物耐药性的风险评估与控制研究,E-mail: 1556042576@qq.com

  • 中图分类号: S859

Molecular mechanism of plasmid-encoded blaKPC-2 gene mediating low-level resistance to carbapenems in Escherichia coli

  • 摘要:
    目的 

    明确碳青霉烯酶编码质粒p21QH43K-KPC从肺炎克雷菌Klebsiella pneumoniae转移到大肠埃希菌Escherichia coli后介导碳青霉烯类药物低水平耐药的分子机制。

    方法 

    采用构建EZ-Tn5转座子突变文库、基因敲除、转录组测序和全基因组测序(WGS)等手段,探究blaKPC-2基因在大肠埃希菌中对美罗培南产生低水平耐药的分子机制。

    结果 

    通过构建接合子EC600/p21QH43K-KPC的EZ-Tn5转座子突变文库,获得1株对美罗培南耐药水平增强的转座子突变株EC600/p21QH43K-KPC-130(MIC=8.000 mg/L)。WGS结果和Mauve分析显示,该转座子插入到染色体ompR基因,同时发现了2个点突变基因ltrAiron。基因敲除发现,只有ompR缺失突变体对碳青霉烯的敏感性降低,并且可以通过基因回补恢复这一表型。

    结论 

    质粒编码的blaKPC-2基因在大肠埃希菌中介导碳青霉烯类药物低水平耐药与ompRblaKPC-2基因的调控有关。

    Abstract:
    Objective 

    To elucidate the molecular mechanism by which the carbapenemase-encoding plasmid p21QH43K-KPC is transferred from Klebsiella pneumoniae to Escherichia coli, and mediates low-level resistance to carbapenem drugs in E. coli.

    Method 

    The construction of EZ-Tn5 transposon mutation library, CRISPR-Cas9-mediated gene knockout, transcriptome sequencing and whole genome sequencing (WGS) were used to determine the regulatory mechanism of low-level resistance to meropenem mediated by the blaKPC-2 gene in E. coli.

    Result 

    A transposon mutant EC600/p21QH43K-KPC-130 with enhanced meropenem resistance (MIC=8.000 mg/L) was obtained by constructing the EZ-Tn5 transposon mutation library of the EC600/p21QH43K-KPC conjugon. WGS and Mauve analysis revealed that the transposon had inserted into one chromosomal gene ompR, and two point mutation genes of iron and ltrA were found. However, through gene knockout, only ompR deletion mutants exhibited reduced sensitivity to carbapenem that could be restored by gene complementation.

    Conclusion 

    The molecular mechanism of the blaKPC-2 gene encoded on the plasmid mediating low-level resistance to carbapenems in E. coli is related to the regulation of blaKPC-2 gene by ompR.

  • 猪格拉瑟病是由副猪格拉瑟菌Glaesserella parasuis引起的以猪的多发性浆膜炎、脑膜炎、关节炎、心包炎为特征的细菌性传染病[1]G. parasuis可感染断奶至屠宰任何阶段的猪群,并且与多种细菌、病毒、支原体等病原的混合感染广泛发生,给养猪业带来严重损失[2-3]G. parasuis具有很强的基因变异性[4],目前已知有15种血清型,不同血清型在基因型构成、毒力方面差异较大,这极大地制约了人们对G. parasuis感染的控制[5-7],使得该病成为世界范围内引起仔猪死亡的重要因素之一。在临床治疗中,抗生素的广泛使用加速了G. parasuis耐药性的产生,为该病的防控带来巨大挑战。疫苗免疫是G. parasuis防控的主要手段,当前中国市场上的G. parasuis可用商品化疫苗主要是本地分离株灭活苗类。但灭活苗的保护效果有限,不同血清型之间缺少交叉保护作用[8],而不同猪群流行的G. parasuis血清型可能不同,甚至同一头病猪的不同部位也可能存在不止一种血清型[9]。因此,研究具有交叉保护力的新型疫苗成为G. parasuis防控研究的重点[10]

    磷酸甘油醛脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase,GAPDH)是广泛存在于真核生物以及原核生物中的一种酶,可与免疫细胞表面的受体结合激活相关信号通路。研究发现,GAPDH是G. parasuis的新型免疫原[11],Fu等[12]通过基因测序与生物信息学分析发现,GAPDH广泛存在于各种血清型的G. parasuis中并且高度保守。但GAPDH作为G. parasuis普遍存在的免疫原性蛋白,却较少有针对该蛋白重组疫苗株的研究。外膜蛋白(Outer membrane proteins,OMPs)是细菌表面重要的抗原成分,能够参与决定宿主的特异性免疫应答。有学者发现在针对G. parasuis的免疫应答中存在OMPs的抗体,但没有脂多糖等成分的抗体,这表明OMPs比该生物体其他成分或许更具有免疫原性[13]。多个团队的研究者[14-16]G. parasuis基因组中筛选出不同的OMPs作为潜在毒力相关因素,并证实其在豚鼠、小鼠模型中对致死量G. parasuis的攻击具有显著的保护作用。已有研究[17]显示,编码G. parasuis GAPDH和OmP26蛋白的基因在G. parasuis不同血清型菌株中普遍存在,且能够介导免疫反应,这意味着它们有可能成为广谱的免疫原。

    理想的疫苗载体应具备多种优良特性,包括安全性、免疫原性等。赵战勤等[18]通过基因工程方法构建猪霍乱沙门氏菌Salmonella choleraesuis asd缺失株C500Δasd,并证明该缺失株相较亲本菌株C500毒力降低;同时具有良好的生长特性、稳定的遗传背景及高效的表达能力。随后,徐引弟等[19]和赵战勤等[20]对C500Δasd缺失株展开更进一步研究,证实在C500Δasd缺失株中插入不同外源抗原如支气管败血波氏杆菌免疫原性基因、巴氏杆菌毒素基因后重组菌株仍具有良好的遗传稳定性,并高效表达外源抗原,将重组菌株通过口服免疫小鼠和仔猪,外源抗原有良好的表达活性,且接种动物无任何不良反应。上述研究成果表明,C500Δasd缺失株是构建重组菌株的有力候选载体。

    因此,本研究选择GAPDH基因和OmP26基因作为免疫原性基因,将上述基因同时导入S. choleraesuis C500Δasd缺失株,以求构建能够高效表达G. parasuis免疫基因,同时能有效刺激机体产生针对S. choleraesuis特异性免疫保护作用的重组菌株,并对其基本生物学特性及免疫效果进行评估;旨在探索一种更为有效的猪格拉瑟病防控策略,同时为G. parasuisS. choleraesuis混合感染的防控提供新的思路和方法,以应对这2种疾病给养猪业带来的挑战。

    S. choleraesuis C500弱毒疫苗株的asd基因缺失株、大肠埃希菌Escherichia coli x6097及无抗性原核表达质粒pYA3493由华南农业大学兽医学院动物传染病教研室保存;限制性内切酶EcoR I + Sal I、Sal I + Hind III等各种内切酶购自New England Biolabs公司;T4 DNA连接酶购自TaKaRa公司;His标签蛋白纯化试剂盒(P2226)购自碧云天公司;4周龄雌性BALB/c小鼠购自广东省实验动物中心。

    参考Genebank中G. parasuis血清5型SH02165菌株的膜蛋白基因GAPDH(GenBank ID:72785966)和外膜蛋白基因OmP26(GenBank ID:219691637)的序列,利用Primer 5.1设计引物(GAPDH-F:CGGAATTCATGGCAATTAAAATTG,GAPDH-R:GCGTCGACTTAGCCTTTGTAGTTG;OmP26-F:GCGTCGACATGAAAAATTTATTTAAACTTGC,OmP26-R:CCAAGCTTTTATTTTTTCACTTCTTCTGG),并以G. parasuis SH02165菌株的基因组为模板扩增引物。

    分别利用限制性内切酶EcoR I + Sal I、Sal I + Hind III对“1.2”回收获得的基因片段GAPDHOmP26进行双酶切,原核表达质粒pYA3493使用对应限制性内切酶进行双酶切,然后经琼脂糖凝胶电泳回收GAPDHOmP26基因片段与相应的线性质粒pYA3493,以T4 DNA连接酶进行连接。将质粒转入x6097,涂布在无抗性培养基上,挑取单菌落,在LB液体培养基中于37 ℃培养12 h后进行质粒小提,以酶切鉴定方法获取阳性克隆pYA-GAPDH和pYA-OmP26。用Sal I + Hind III双酶切基因片段OmP26与pYA-GAPDH载体,回收、纯化、连接后转入x6097,重复上述步骤,获得pYA-GAPDH-OmP26质粒,并将其送至上海生工生物工程有限公司进行测序鉴定。

    将重组质粒pYA-GAPDH、pYA-OmP26和pYA-GAPDH-OmP26电转入C501,完成电击后迅速加入37 ℃预热培养基,并将菌液转移到无菌管中;于37 ℃、120 r/min震荡1 h培养后离心收集菌体,重悬于单纯培养基并涂布于LB平皿表面。使用目的基因引物对平皿表面生长的菌落进行扩增鉴定。

    重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)及C501(pYA-GAPDH-OmP26)在无抗性LB液体培养基中连续传代培养,每25代进行PCR鉴定,测定其遗传特性。于37 ℃条件下培养重组菌株,每隔1 h测定D600 nm,以菌液培养时间为横坐标,菌液D600 nm为纵坐标绘制重组菌株的生长曲线。

    将重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)及C501(pYA-GAPDH-OmP26)培养于无抗性LB液体培养基中,转接至以阿拉伯糖、乳糖、棉子糖、山梨醇、淀粉、半乳糖、葡萄糖、甘露糖、果糖、鼠李糖和麦芽糖等为唯一碳源的细菌微量生化鉴定管进行生化试验。对重组菌株全菌裂解物进行Western-Blot鉴定。

    选取40只4周龄雌性BALB/c小鼠,分3组:C501(pYA-GAPDH-OmP26)组(20只小鼠)、C501(pYA3493)组(10只小鼠)、PBS阴性对照组(10只小鼠),按每只5×108 CFU/mL剂量皮下多点位注射。首次免疫后第14天进行加强免疫,操作程序同首次免疫。加强免疫14 d后各组分别按照每只2.5×109 CFU/mL剂量腹腔攻毒G. parasuis 5型强毒株SH0165和每只5×107 CFU/mL剂量灌胃攻毒S. choleraesuis强毒株C78-1,各小组中2种菌株攻毒相同数量小鼠,连续观察14 d,记录发病及死亡情况。

    目的基因GAPDHOmP26扩增结果分别为1 020、798 bp,与预期片段长度一致,测序结果正确(图1)。重组质粒pYA-GAPDH、pYA-OmP26酶切鉴定均能成功扩增出约3 000 bp的外源基因片段(图2)。pYA-GAPDH、pYA-OmP26和pYA-GAPDH-OmP26测序结果均符合预期。重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)及C501(pYA-GAPDH-OmP26)用GAPDH、OmP26引物扩增,测序结果与目的外源基因一致。

    图  1  目的基因PCR扩增片段
    Figure  1.  PCR amplified fragments of target genes
    M: DNA Marker, 1: GAPDH, 2: OmP26.
    图  2  重组质粒pYA-GAPDH和pYA-OmP26的酶切鉴定
    Figure  2.  Enzymatic identification of recombinant plasmids pYA-GAPDH and pYA-OmP26
    M: DNA Marker, 1: pYA-GAPDH, 2: pYA-OmP26.

    重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)及C501(pYA-GAPDH-OmP26)连续传代100代,每25代进行1次PCR鉴定,结果显示,不同代次均能扩增出外源基因条带(图3),表明重组质粒pYA-GAPDH、pYA-OmP26和pYA-GAPDH-OmP26均能在宿主菌菌株C501中稳定遗传。重组菌株的生长曲线与对照菌株C501(pYA3493)非常接近(图4),表明外源基因基本不影响宿主菌株生长趋势。

    图  3  重组菌株遗传稳定性
    M:DNA Marker;1~4:C501(pYA-GAPDH) 25、50、75、100代;6~9:C501(pYA-OmP26) 25、50、75、100代;11~14:C501(pYA-GAPDH-OmP26) 25、50、75、100代;5、10:pYA3493。
    Figure  3.  Genetic stability of recombinant strains
    M: DNA Marker; 1−4: C501(pYA-GAPDH) 25, 50, 75, 100 generations; 6−9: C501(pYA-OmP26) 25, 50, 75, 100 generations; 11−14: C501(pYA-GAPDH-OmP26) 25, 50, 75, 100 generations; 5, 10: pYA3493.
    图  4  重组菌株C501(pYA-GAPDH-OmP26)与对照菌株C501(pYA3493)的生长曲线
    Figure  4.  Growth curves of recombinant strain C501 (pYA-GAPDH-OmP26) and control strain C501 (pYA3493)

    重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)及C501(pYA-GAPDH-OmP26)与对照菌株C501(pYA3493)均不能利用阿拉伯糖、乳糖、棉子糖、山梨醇、淀粉和半乳糖,但可以利用葡萄糖、甘露醇、果糖、麦芽糖和鼠李糖(表1),表明外源片段引入不影响Salmonella自身的生化特性。

    表  1  重组沙门氏菌对不同碳源的利用情况1)
    Table  1.  Utilization of different carbon sources by recombinant Salmonella spp.
    菌株
    Strain
    阿拉伯糖Arabinose 乳糖Lactose 棉子糖Raffinose 山梨醇Sorbitol 淀粉Starch 半乳糖Galactose 葡萄糖Glucose 甘露醇Mannitol 果糖Fructose 鼠李糖Rhamnose 麦芽糖Maltose
    C501(pYA3493) + + + + +
    C501(pYA-GAPDH) + + + + +
    C501(pYA-OmP26) + + + + +
    C501(pYA-GAPDH-OmP26) + + + + +
     1) −:阴性;+:阳性。
     1) −: Negative; +: Positive.
    下载: 导出CSV 
    | 显示表格

    重组菌株C501(pYA-GAPDH)、C501(pYA-OmP26)全菌裂解物Western-Blot结果显示,与空质粒对照相比,重组疫苗菌株能够正确表达外源蛋白,蛋白相对分子质量分别为37 400、37 000(图5)。

    图  5  重组菌株Western-Blot分析
    Mr:相对分子质量,M:Marker,1:C501(pYA3493),2:C501(pYA-GAPDH),3:C501(pYA-OmP26)。
    Figure  5.  Western-Blot analysis of recombinant strains
    Mr: Relative molecular mass, M: Marker, 1: C501(pYA3493), 2: C501(pYA-GAPDH), 3: C501(pYA-OmP26).

    C501(pYA-GAPDH-OmP26)组免疫小鼠对S. choleraesuis强毒株C78-1攻毒的保护率为62.5%,对G. parasuis 5型强毒株SH0165攻毒的保护率为50.0%;C501(pYA3493)组、PBS对照组对G. parasuis 5型强毒株SH0165攻毒的保护率均为0(图6)。

    图  6  免疫小鼠的攻毒保护率
    Figure  6.  Attack protection rate of immunized mice

    接种疫苗是预防猪格拉瑟病的有效措施。但现有的商业疫苗对异源菌株的交叉保护作用有限,不能提供广泛的异源保护[21]。如基于血清型2和5组合、血清型1和6组合的市售疫苗,对其他血清型甚至同一血清型不同菌株的保护力常常无法达到预期[22]。因此,研发对不同血清型具有交叉保护作用的新型疫苗对该病的防治具有重要意义。诸多研究表明,S. choleraesuis C500Δasd缺失株作为活疫苗载体能够同时表达多种不同病原的抗原,且具有极高的安全性和可行性,作为胞内侵袭细菌,Salmonella能够有效递呈抗原,激发机体产生抗Salmonella免疫反应,同时诱导产生针对外源基因的特异性细胞免疫和体液免疫[23-24]S. choleraesuisC500Δasd缺失株导入含asd质粒后才能在无外源二氨基庚二酸条件下存活,这使得质粒携带的外源基因不易丢失,同时避免Salmonella过度繁殖[25-26]。本研究选取的外源抗原OmP26是G. parasuis的外膜蛋白,本身具有良好的免疫活性;GAPDH作为G. parasuis多种血清型普遍存在的蛋白,具有免疫调节功能。将二者结合同时克隆,可增加机体可识别的抗原位点,从而提高机体免疫应答的效率。同时,GAPDH和OmP26二者同时表达可能存在协同刺激免疫系统的效果,产生更强的免疫反应。此外,同时包含GAPDHOmP26基因的表达载体对于血清型众多的G. parasuis而言,能够提供更广泛的保护。

    基于此,本研究从G. parasuis中筛选出具有良好免疫效应的蛋白GAPDH、OmP26,将其同时插入S. choleraesuis C500Δasd缺失株,成功构建二联基因工程弱毒株C501(pYA-GAPDH-OmP26)。试验结果显示,C501(pYA-GAPDH-OmP26)能稳定携带、表达外源基因,并与对照菌株C501(pYA3493)具有相近的生物学特性,表明外源片段的插入基本不影响宿主菌株的生长与代谢。通过皮下注射C501(pYA-GAPDH-OmP26)免疫的小鼠对致死剂量的G. parasuis强毒株表现出50.0%的保护率,与对照组C501(pYA3493)组、PBS组(保护率为0)均存在明显差异。同时,C501(pYA-GAPDH-OmP26)、C501(pYA3493)对S. choleraesuis C78-1的保护率分别为62.5%、50.0%,说明外源GAPDH和OmP26抗原的表达没有降低C500Δasd缺失株针对Salmonella感染的免疫保护力。但该重组菌株在小鼠与在猪体内引发的免疫反应可能存在差异,目前针对G. parasuis重组疫苗菌株的研究也大都在小鼠试验模型上进行,少有在猪模型上检验[27]。在后续研究中,有必要在本体动物仔猪动物模型中进一步评估该重组菌株的免疫效力。

    本研究选取G. parasuis的GAPDH和OmP26为外源性抗原并成功研制G. parasuis-S. choleraesuis二联基因工程弱毒株C501(pYA-GAPDH-OmP26),与空质粒组C501(pYA3493)相比,该重组菌株对G. parasuis 5型强毒株SH0165的保护效果更好且存在明显差异,同时保留了对S. choleraesuis C78-1的高效保护,为进一步研发G. parasuis新型疫苗提供了参考。

  • 图  1   基于转座子突变文库筛选目标基因

    Figure  1.   Screening of the target gene based on transposon mutation library

    图  2   ompR缺失对大肠埃希菌EC600/p21QH43K-KPC转录组谱的影响

    a:差异表达基因火山图,由t检验确定相对于EC600/p21QH43K菌株的差异显著性; b:上调和下调的Top 10显著差异表达基因;c:采用RT-qPCR检测blaKPC-2的转录本水平,该基因在EC600/p21QH43K菌株中的转录水平设为1, 误差条表示3个独立试验的标准差。

    Figure  2.   Effect of ompR deletion on the transcriptome profile in stain EC600/p21QH43K-KPC

    a: Volcano map of differentially expressed genes, the significance of the difference relative to the EC600/p21QH43K strain was determined by t-test; b: Top 10 up- and down-regulated significantly differentially expressed genes; c: The transcript levels of blaKPC-2 determined by RT-qPCR, the transcriptional level of this gene in the EC600/p21QH43K strain was set as 1, and the error bars represent the standard deviation of three independent experiments.

    表  1   本研究使用的菌株和质粒

    Table  1   Strains and plasmids used in this study

    类型 Type名称 Name用途 Purpose
    菌株 Strain21QH43K临床菌 21QH43K
    ATCC700603肺炎克雷伯菌工程菌
    ∆ompRompR基因敲除菌
    ∆ompCompC基因敲除菌
    ∆ompFompF基因敲除菌
    blaKPC-2blaKPC-2基因敲除菌
    ∆ltrAltrA基因敲除菌
    ∆ironiron基因敲除菌
    E. coli DH5α用于构建质粒的菌株
    WM3064用于基因敲除的工程菌
    E. coli 600用于接合转移的工程菌
    MG1655用于接合转移的工程菌
    BL21用于蛋白表达的菌株
    质粒 PlasmidpSGKP-tet用于基因敲除的工程质粒
    pCasKP-APR用于基因敲除的工程质粒
    pSZU-Apr用于基因敲除的工程质粒
    pluxCDABE用于验证blaKPC基因启动子活性的工程质粒
    pGEN-APR用于克隆blaKPC基因的工程质粒
    pET28a (+)-kan用于OmpR蛋白表达的工程质粒
    下载: 导出CSV

    表  2   本研究使用的PCR引物

    Table  2   PCR primers used in this study

    试验 Test 引物 Primer 引物序列 (5'→3') Primer sequence
    RT-qPCR KPC-qPCR-F ccactgggcgcgcacctatt
    KPC-qPCR-R tgttaggcgcccgggtgtag
    16S-F cctacgggaggcagcag
    16S-R attaccgcggctgctgg
    敲除blaKPC-2 KPC-N20-F ttgggcgtcaacgggcagtagttttagagctagaaatagcaagtt
    Knock out blaKPC-2 KPC-N20-R tactgcccgttgacgcccaaactagtattatacctaggactgagc
    KPC-500-F1 ttttttgatatcgaattcctgcagcccggattgaaaccatgaccgaac
    KPC-500-R1 gcattgaccttggcatcttc
    KPC-500-F2 gaagatgccaaggtcaatgcggtatccatcgcgtacacac
    KPC-500-R2 ggccgctctagaagtagtggatccccccgtcaagatctacaaccacagc
    pSGKPtestF tctcgtttggattgcaactg
    pSGKPtestR tgcttccggctcgtatgttg
    敲除ompR ompR-N20-F agtggaccgtatcgtaggccgttttagagctagaaatagcaagtt
    Knock out ompR ompR-N20-R ggcctacgatacggtccactactagtattatacctaggactgagc
    ompR-500-F1 atatcgaattcctgcagcccgcattaacataccagctcgc
    ompR-500-R1 gtgcgcattatcaaacagac
    ompR-500-F2 gtctgtttgataatgcgcacctcatcgtcaccttgctg
    ompR-500-R2 ctagaagtagtggatcccccgttcgagatcgaccaacg
    pSGKPtestF tctcgtttggattgcaactg
    pSGKPtestR tgcttccggctcgtatgttg
    ompR基因回补 ompR-F gcggatcccggtaccaagcttgacaccctcgttgattccctttgtct
    ompR gene complementation ompR-R agctaactcacattaattgcgttgcgccgtacgggcaaatgaacttc
    pGEN-F ggtgtcaagcttggtacc
    pGEN-R gcgcaacgcaattaatgtga
    pGENtestF ggaacgaagccgccttaacc
    pGENtestR cttggagcgaactgcctacc
    下载: 导出CSV

    表  3   携带blaKPC-2的质粒p21QH43K-KPC在不同遗传背景菌株中介导对碳青霉烯类药物的耐药表型

    Table  3   Carbapenems resistance conferred by blaKPC-2-carrrying plasmid p21QH43K-KPC in strains with different genetic backgrounds

    菌株 Strain 最小抑菌浓度/(mg·L−1) Minimal inhibitory concentration
    美罗培南 Meropenem 亚胺培南 Imipenem 厄他培南 Ertapenem
    21QH43K 64.000 16.000 >64.000
    ATCC 700603 0.064 0.032 0.064
    ATCC 700603/p21QH43K-KPC 4.000 4.000 8.000
    EC600 0.016 0.125 0.008
    EC600/p21QH43K-KPC 0.125 0.500 0.250
    MG1655 0.016 0.125 0.002
    MG1655/p21QH43K 0.125 0.500 0.250
    EC600/p21QH43K-KPC-130 8.000 4.000 4.000
    下载: 导出CSV

    表  4   ompRironltrA基因缺失后大肠埃希菌对受试抗菌药的耐药性

    Table  4   Resistance of Escherichia coli to the tested drugs after the deletion of the ompR, iron and ltrA genes

    菌株 Strain
    最小抑菌浓度/(mg·L−1) Minimal inhibitory concentration
    美罗培南 Meropenem 亚胺培南 Imipenem 厄他培南 Ertapenem
    EC600/p21QH43K-KPC 0.125 0.500 0.250
    EC600ΔompR/p21QH43K-KPC 8.000 16.000 32.000
    EC600ΔompR::ompR/p21QH43K-KPC 1.000 0.250 0.500
    EC600 0.016 0.125 0.008
    EC600ΔompR 0.064 0.125 0.064
    EC600Δiron/p21QH43K-KPC 0.250 0.500 0.250
    EC600ΔltrA/p21QH43K-KPC 0.125 0.500 0.250
    下载: 导出CSV

    表  5   ompR基因缺失后大肠埃希菌对受试抗菌药的耐药性1)

    Table  5   Resistance of Escherichia coli to the tested drugs after the deletion of the ompR gene

    菌株 Strain 最小抑菌浓度/(mg·L−1) Minimal inhibitory concentration
    FOS AMK CTX FOX AMP CAZ TET ATM CIP
    EC600/p21QH43K-KPC 256 >256 16 16 >256 64 1 16 0.032
    EC600ΔompR/p21QH43K-KPC 8 >256 16 64 >256 256 1 32 0.125
    EC600ΔompR::ompR/p21QH43K-KPC >256 >256 32 64 >256 128 1 64 0.125
     1) FOS:磷霉素,AMK:阿米卡星,CTX:头孢噻肟,FOX:头孢西丁,AMP:氨苄西林,CAZ:头孢他啶,TET:四环素,ATM:氨曲南,CIP:环丙沙星。
     1) FOS: Fosfomycin, AMK: Amikacin, CTX: Cefotaxime, FOX: Cefoxetine, AMP: Ampicillin, CAZ: Ceftazidime, TET: Tetracycline, ATM: Aztreonam, CIP: Ciprofloxacin.
    下载: 导出CSV

    表  6   ompC/ompF基因缺失后大肠埃希菌对碳青霉烯类药物的耐药性

    Table  6   Carbapenems resistance of Escherichia coli after the deletion of the ompC/ompF gene

    菌株 Strain 最小抑菌浓度/(mg·L−1) Minimal inhibitory concentration
    美罗培南 Meropenem 亚胺培南 Imipenem 厄他培南 Ertapenem
    EC600/pQH43K-KPC 0.125 0.500 0.250
    EC600ΔompC/p21QH43K-KPC 0.500 1.000 0.500
    EC600ΔompF/p21QH43K-KPC 0.500 1.000 0.500
    EC600 0.016 0.125 0.008
    EC600ΔompC 0.016 0.064 0.008
    EC600ΔompF 0.016 0.250 0.008
    下载: 导出CSV
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  • 收稿日期:  2024-11-04
  • 网络出版日期:  2025-03-13
  • 发布日期:  2025-03-26
  • 刊出日期:  2025-05-09

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