Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China.
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, China.
Microb Pathog. 2021 Mar;152:104743. doi: 10.1016/j.micpath.2021.104743. Epub 2021 Jan 21.
To analyze the difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution and to further explore the function of mrkH for biofilm formation from the perspective of gene regulation.
40 imipenem-resistant strains and 40 imipenem-sensitive strains were selected to conduct experiments. Carbapenem (imipenem) susceptibility test was performed by the agar-dilution method. bla resistance gene, type 3 fimbriae-related coding genes (mrkA and mrkD) and regulation gene (mrkH) were screened by PCR. Biofilm formation assay was performed using crystal violet staining method in MHB. The relative expression of genes that critically involved in biofilm formation (mrkA, luxS, pgaA) and carbapenem resistance (ompk35, ompk36, acrB) were measured by quantitative real-time PCR (qRT-PCR). Furthermore, the mrkH cassette was cloned into pGEM-T Easy plasmid to yield pGEM:pmrkH and expressed in Escherichia coli DH5α and K. pneumoniae FK1911, and the biofilm formation assay after transformation was further tested.
The MICs of imipenem were all more than 16 μg/mL in 40 imipenem-resistant strains and ranged from 0.125 μg/mL to 0.5 μg/mL in 40 imipenem-sensitive strains. Moreover, the bla was identified in the 40 imipenem-resistant K. pneumoniae strains. All 80 K. pneumoniae strains were found to carry mrkA and mrkD genes. Interestingly, the mrkH gene was detected in 43 strains, of which 32 were carbapenem-sensitive strains. The biofilm formation capacity of strains carried mrkH cassette was significantly higher than other 37 strains in MHB media. The relative expression of mrkA in K. pneumoniae carrying mrkH gene was significantly up-regulated. Importantly, the biofilm formation ability of FK1911-pGEM:pmrkH strain was more higher than the strain of FK1911 in MHB medium.
Our data demonstrated that MrkH played a crucial role in the regulation of biofilm formation by K. pneumoniae. In contrast to carbapenem-sensitive K. pneumoniae, carbapenem-resistant K. pneumoniae was less likely to have strong biofilm-forming capacity because it does not carry the mrkH gene.
基于 mrkH 分布分析，比较耐碳青霉烯类和碳青霉烯敏感肺炎克雷伯菌生物膜形成的差异，并从基因调控角度进一步探讨 mrkH 对生物膜形成的功能。
选择 40 株亚胺培南耐药株和 40 株亚胺培南敏感株进行实验。采用琼脂稀释法进行碳青霉烯（亚胺培南）药敏试验。采用 PCR 筛选 bla 耐药基因、III 型菌毛相关编码基因（mrkA 和 mrkD）和调控基因（mrkH）。采用结晶紫染色法在 MHB 中进行生物膜形成试验。采用实时荧光定量 PCR（qRT-PCR）检测生物膜形成相关关键基因（mrkA、luxS、pgaA）和碳青霉烯类耐药基因（ompk35、ompk36、acrB）的相对表达水平。进一步将 mrkH 盒克隆到 pGEM-T Easy 质粒中，得到 pGEM：pmrkH，并在大肠杆菌 DH5α 和肺炎克雷伯菌 FK1911 中表达，然后进行转化后的生物膜形成试验。
40 株亚胺培南耐药株的亚胺培南 MIC 值均>16μg/mL，40 株亚胺培南敏感株的亚胺培南 MIC 值为 0.125μg/mL 至 0.5μg/mL。此外，40 株亚胺培南耐药肺炎克雷伯菌均检出 bla 基因。80 株肺炎克雷伯菌均携带 mrkA 和 mrkD 基因。有趣的是，43 株菌检测到 mrkH 基因，其中 32 株为亚胺培南敏感株。在 MHB 培养基中，携带 mrkH 盒的菌株生物膜形成能力明显高于其他 37 株。携带 mrkH 基因的肺炎克雷伯菌 mrkA 基因的相对表达水平明显上调。重要的是，FK1911-pGEM：pmrkH 菌株在 MHB 培养基中的生物膜形成能力明显高于 FK1911 菌株。
本研究数据表明，MrkH 对肺炎克雷伯菌生物膜形成的调控起着关键作用。与亚胺培南敏感肺炎克雷伯菌相比，耐碳青霉烯类肺炎克雷伯菌由于不携带 mrkH 基因，其生物膜形成能力较弱。
