临床分离肺炎克雷伯菌生物膜形成能力及基因相关性研究
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摘要
目的研究肺炎克雷伯菌生物膜形成能力及其与luxS、oqxB、rarA等基因的相关性。方法通过96孔板结晶紫染色法测定200株对亚胺培南、头孢他啶、环丙沙星和庆大霉素敏感的肺炎克雷伯菌(敏感组)和300株对上述药物耐药的肺炎克雷伯菌(耐药组)体外生物膜形成能力;采用扫描电镜观察不同生物膜形成能力肺炎克雷伯菌的生物膜形态特点;通过实时荧光定量PCR分析生物膜形成相关基因(luxS、oqxB、rarA、acrB、ramA、ompK36和micF)表达量。结果强生物膜形成能力组的平均生物膜形成量是弱形成能力组的16.8倍,敏感组和耐药组肺炎克雷伯菌生物膜OD590的平均值分别为0.783±0.642和0.672±0.534,差异无统计学意义;扫描电镜显示生物膜形成能力强的菌株团块内包含大量的生物膜,形态完整且细菌密集排列;强生物膜形成能力组中luxS表达水平分别为中等和弱形成能力组的5.98倍和3.37倍;外排泵、外膜蛋白编码基因及其调控基因的表达水平有统计学差异。结论肺炎克雷伯菌生物膜形成能力与luxS基因的表达量有关,luxS作为一个高阶调控基因,其与ramA、ompK36和micF等基因构成错综复杂的关系网,共同影响生物膜的形成。
Objective To investigate the relationship between the biofilm formation ability and luxS, oqxB, rarA and other related genes in Klebsiella pneumoniae. Methods The biofilm formation ability was detected by 96-well crystal violet staining method in 200 clinical isolates of Klebsiella pneumoniae which were sensitive to imipenem, ceftazidime, ciprofloxacin and gentamycin(sensitive group) and 300 isolated of Klebsiella pneumoniae which were resistant to those antimicrobials(resistant group); the biofilm features of Klebsiella pneumoniae was ocserved with scanning electron microscopy(SEM); the expression levels of biofilm-related genes(luxS, oqxB, rarA, acrB, ramA, ompK36 and micF) were analyzed by qRT-PCR. Results The average capacity of biofilm formation in the strong biofilm formation group was 16.8 times than that in the weak formation group, however,no significant difference was obtained between the sensitive group(0.783±0.642) and the resistant group(0.672±0.534) for biofilm formation ability. The SEM showed that quantity of the strains with strong ability of biofilm formation contained a large number of biofilms in their clumps, the morphology of the strains was integrated and the arrangement was dense. The level of luxS expression in the strong biofilm forming group was 5.98 and 3.37 times higher than that in the medium and weak forming groups.The expression levels of efflux pump and outer membrane protein coding genes and their regulatory genes were also different.Conclusion The biofilm formation ability is associated with the gene expression level of luxS in Klebsiella pneumoniae. As a high-order regulatory gene, luxS is involved in the complex regulatory network with ramA, ompK36 and micF, which affects the biofilm formation simultaneously.
Objective To investigate the relationship between the biofilm formation ability and luxS, oqxB, rarA and other related genes in Klebsiella pneumoniae. Methods The biofilm formation ability was detected by 96-well crystal violet staining method in 200 clinical isolates of Klebsiella pneumoniae which were sensitive to imipenem, ceftazidime, ciprofloxacin and gentamycin(sensitive group) and 300 isolated of Klebsiella pneumoniae which were resistant to those antimicrobials(resistant group); the biofilm features of Klebsiella pneumoniae was ocserved with scanning electron microscopy(SEM); the expression levels of biofilm-related genes(luxS, oqxB, rarA, acrB, ramA, ompK36 and micF) were analyzed by qRT-PCR. Results The average capacity of biofilm formation in the strong biofilm formation group was 16.8 times than that in the weak formation group, however,no significant difference was obtained between the sensitive group(0.783±0.642) and the resistant group(0.672±0.534) for biofilm formation ability. The SEM showed that quantity of the strains with strong ability of biofilm formation contained a large number of biofilms in their clumps, the morphology of the strains was integrated and the arrangement was dense. The level of luxS expression in the strong biofilm forming group was 5.98 and 3.37 times higher than that in the medium and weak forming groups.The expression levels of efflux pump and outer membrane protein coding genes and their regulatory genes were also different.Conclusion The biofilm formation ability is associated with the gene expression level of luxS in Klebsiella pneumoniae. As a high-order regulatory gene, luxS is involved in the complex regulatory network with ramA, ompK36 and micF, which affects the biofilm formation simultaneously.
引文
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[13]Vuotto C,Longo F,Pascolini C,et al.Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains[J].J Appl Microbiol,2017,123(4):1003-1018.DOI:10.1111/jam.13533.
[2]Ivin M,Dumigan A,de Vasconcelos FN,et al.Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection[J].PLo S Pathog,2017,13(11):e1006696.DOI:10.1371/journal.ppat.1006696.
[3]Flemming HC,Wingender J,Szewzyk U,et al.Biofilms:an emergent form of bacterial life[J].Nat Rev Microbiol,2016,14(9):563-575.DOI:10.1038/nrmicro.2016.94.
[4]Benthall G,Touzel RE,Hind CK,et al.Evaluation of antibiotic efficacy against infections caused by planktonic or biofilm cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae in Galleria mellonella[J].Int J Antimicrob Agents,2015,46(5):538-545.DOI:10.1016/j.ijantimicag.2015.07.014.
[5]张洪,邱晓沛,蒋天伦,等.细菌生物膜清除策略研究进展[J].中华微生物学和免疫学杂志,2014,34(6):487-490.DOI:10.3760/cma.j.issn.0254-5101.2014.06.016.
[6]Niemirowicz K,Piktel E,Wilczewska AZ,et al.Core-shell magnetic nanoparticles display synergistic antibacterial effects against Pseudomonas aeruginosa and Staphylococcus aureus when combined with cathelicidin LL-37 or selected ceragenins[J].Int JNanomedicine,2016,11:5443-5455.DOI:10.2147/IJN.S113706.
[7]Murphy CN,Clegg S.Klebsiella pneumoniae and type 3 fimbriae:nosocomial infection,regulation and biofilm formation[J].Future Microbiology,2012,7:991-1002.DOI:10.2217/fmb.12.74.
[8]Qu Y,Locock K,Verma-Gaur J,et al.Searching for new strategies against polymicrobial biofilm infections:guanylated polymethacrylates kill mixed fungal/bacterial biofilms[J].J Antimicrob Chemother,2016,71(2):413-421.DOI:10.1093/jac/dkv334.
[9]Aguila-Arcos S,Alvarez-Rodriguez I,Garaiyurrebaso O,et al.Biofilm-Forming Clinical Staphylococcus Isolates Harbor Horizontal Transfer and AntibioticResistance Genes[J].Front Microbiol,2017,8:2018.DOI:10.3389/fmicb.2017.02018.
[10]Lin TH,Tseng CY,Lai YC,et al.IscR Regulation of Type 3 Fimbriae Expression in Klebsiella pneumoniae CG43[J].Front Microbiol,2017,8:1984.DOI:10.3389/fmicb.2017.01984.
[11]Zuberi A,Misba L,Khan AU.CRISPR Interference(CRISPRi)Inhibition of luxS Gene Expression in E.coli:An Approach to Inhibit Biofilm[J].Front Cell Infect Microbiol,2017,7:214.DOI:10.3389/fcimb.2017.00214.
[12]Wu X,Jacobs NT,Bozio C,et al.Competitive Dominance within Biofilm Consortia Regulates the Relative Distribution of Pneumococcal Nasopharyngeal Density[J].Appl Environ Microbiol,2017,83(16):e00953-17.DOI:10.1128/AEM.00953-17.
[13]Vuotto C,Longo F,Pascolini C,et al.Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains[J].J Appl Microbiol,2017,123(4):1003-1018.DOI:10.1111/jam.13533.