目的 了解不同生物被膜形成能力的临床分离耐碳青霉烯类鲍曼不动杆菌菌株(CRAB)基因表达的差异.方法 选取8株临床分离CRAB,将生物被膜形成能力强的菌株4、55、78、117号作为强生物被膜(BF)组,将生物被膜形成能力弱的菌株13、177、191、196号作为弱生物被膜(WBF)组,培养两组菌株形成生物被膜,分别提取RNA进行文库构建和转录组测序(RNA-Seq),以鲍曼不动杆菌AB030(NZ_CP009257.1)的基因组作为参考基因组对测序数据进行分析,鉴定差异表达基因(DEGs),并进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析,选取8个DEGs利用实时荧光定量PCR(qRT-PCR)验证转录组测序结果.结果 RNA样品完整无污染,测序数据错误率为0.03％;BF组与WBF组相比,鉴定到171个DEGs,其中106条基因为上调表达,65条基因为下调表达;这些基因主要涉及DNA结合、膜蛋白、菌毛调控、ATP的合成和分解等;GO分析显示较多基因被注释到生物过程部分,KEGG分析显示DEGs富集到多种物质代谢途径;选取的8个DEGs利用qRT-PCR进行验证,结果与转录组测序结果趋势一致.结论 不同生物被膜形成能力的临床CRAB的基因表达存在差异,多基因和多条信号通路影响生物被膜的形成.

目的 了解临床分离的耐碳青霉烯类鲍曼不动杆菌(CRAB)生物被膜态和游离态的转录组差异,探究生物被膜形成的机制.方法 从住院病人痰液或血液样本分离CRAB 4、55、78及117菌株,挑取单菌落分别培养生物被膜态菌(A组)和游离态菌(B组),采用Trizol法提取2组细菌核糖核酸(RNA)样本进行转录组测序;使用DESeq软件进行差异表达基因(DEGs)分析,通过GOSeq R软件进行基因本体(GO)富集分析,通过KO-BAS软件进行京都基因与基因组百科全书(KEGG)通路富集分析;选择8个DEGs,利用实时荧光定量PCR(qRT-PCR)验证转录组测序分析结果.结果 RNA样品完整无污染满足测序要求,测序数据错误率不超过0.03％;A与B组相比鉴定到407个差异基因,其中212条基因为上调表达,195条基因为下调表达,涉及铁的摄取和转运、菌毛合成的调控、生化代谢酶类及转录调节因子等;GO分析显示,GO条目主要富集于分子功能类别和生物过程类别;KEGG分析显示,DEGs富集到硫代谢、ATP结合盒(ABC)转运系统、万古霉素耐药、群体感应及阳离子抗菌肽耐药等多条通路;选取的8个差异基因通过qRT-PCR进行验证,结果与转录组结果趋势一致.结论 临床分离的CRAB菌株,其生物被膜态和游离态存在基因表达差异,生物被膜的形成涉及多基因和多信号通路参与.

The drug resistance rate of Acinetobacter baumannii increases year on year, and the drugs available for the treatment of carbapenem-resistant A. baumannii (CRAB) infection are extremely limited. A. baumannii, which forms biofilms, protects itself by secreting substrates such as exopolysaccharides, allowing it to survive under adverse conditions and increasing drug resistance. Antimicrobial peptides are small molecular peptides with broad-spectrum antibacterial activity and immunomodulatory function. Previous studies have shown that the antimicrobial peptide Cec4 has a strong effect on A. baumannii, but the antibacterial and biofilm inhibition of this antimicrobial peptide on clinical carbapenem resistance A. baumannii is not thoroughly understood. In this study, it was indicated that most of the 200 strains of CRAB were susceptible to Cec4 with a MIC of 4 μg/ml. Cec4 has a strong inhibitory and eradication effect on the CRAB biofilm; the minimum biofilm inhibition concentration (MBIC) was 64-128 μg/ml, and the minimum biofilm eradication concentration (MBEC) was 256-512 μg/ml. It was observed that Cec4 disrupted the structure of the biofilm using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). A comparative transcriptome analysis of the effects of the antimicrobial peptide Cec4 on CRAB biofilm, identified 185 differentially expressed genes, including membrane proteins, bacterial resistance genes, and pilus-related genes. The results show that multiple metabolic pathways, two-component regulation systems, quorum sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were affected after Cec4 treatment. In conclusion, Cec4 may represent a new choice for the prevention and treatment of clinical infections, and may also provide a theoretical basis for the development of antimicrobial peptide drugs.

BACKGROUND:A case of Acinetobacter baumannii (A. baumannii), known as gram-negative bacteria, causes a range of nosocomial infections. Due to the continuous detection of multi-drug resistant A. baumannii in the clinic, there is an urgent need to find alternative therapies, including broad-spectrum antibacterial peptides (AMP). Recently it has been found that the peptide Cec4 has good antibacterial activity against A. baumannii, but the antibacterial mechanism remains elusive. MATERIALS AND METHODS:The basic structure of Cec4 was analyzed by circular dichroism (CD) spectroscopy, and the potential antibacterial mechanism of Cec4 was detected by flow cytometry, transmission electron microscopy, fluorescence and confocal microscopy. The minimum inhibitory concentration (MIC) of antimicrobial peptides against various A. baumannii was determinated with broth microdilution techniques. The biofilm formation and the sensitivity detection of biofilms to antimicrobial peptides were detected by crystal violet staining. RESULTS:In this study, the main secondary structure of the antibacterial peptide Cec4 is α-helix (99.7%) in the hydrophobic environment. Furthermore, after the treatment with Cec4, an amount of leakage of A. baumannii and the destruction of its cell membrane were detected. Moreover, it was observed that FITC-Cec4 can enter the cell, and more cells were held in the G1 phase with peptide Cec4. However, the DNA binding assay of the peptide Cec4 indicates that the peptide does not target DNA. In addition, peptide Cec4 was superior in reducing adherent biofilms of A. baumannii compared to conventional antibiotics and has no cytotoxicity. CONCLUSION:It is apparent that the antibacterial peptide Cec4 may achieve rapid sterilization by multi-target interaction and presents an attractive therapeutic option for the prevention and control of A. baumannii infections.

目的:探究抗菌肽Cecropin4(Cec4)分别与3种抗生素联合使用对鲍曼不动杆菌(AB)的体外抗菌效果.方法:采用微量肉汤稀释法测定抗菌肽Cec4、注射用头孢哌酮钠舒巴坦钠(SCF)、注射用亚胺培南西司他丁钠(IMP)及注射用硫酸多黏菌素B(PB)单用时对AB的最小抑菌浓度(MIC),运用棋盘法检测抗菌肽Cec4分别与这3种抗生素联用时对AB的抑菌效果,用部分抑菌浓度指数(FIC index)反映抗菌肽Cec4分别与这3种抗生素联用对AB的用药效果.结果:抗菌肽Cec4、SCF、IMP及PB对标准、多重耐药和泛耐药AB时菌株的MIC分别为4、16 ～ 64、16 ～ 64及0.625 mg/L;抗菌肽Cec4与PB联合作用于标准AB时、FIC index为0.50,抗菌肽Cec4分别与3种抗生素联合作用于多重耐药和泛耐药AB时,FIC index范围均在0.5 ～1.0.结论:抗菌肽Cec4与PB联合使用对标准AB表现为协同作用,抗菌肽Cec4分别与3种抗生素联合对多重耐药和泛耐药AB表现为相加作用.

Abstract Background It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. Methods Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. Results Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. Conclusions Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

Based on the urgent requirement for protection inshore fishing resources and rehabilitation of ecosystem, the theoretical study and practical use of artificial fish-reef (AFR) is necessary for fishery. In this paper the physical experiments and numerical simulation are carried out for hydrodynamics and ecological effects of the AFR. The most important nature hazards in China Sea-the typhoon induced extreme sea combinations ( extreme wave height with the lowest sea level) are involved to risk assessment of AFR.

This paper proposes an uncertainty analysis method of risk assessment for breakwater wave overtopping, wave forces and stability by experiments and calculation. Uncertainty analysis and multivariate joint probability study of input wave and sea level for experiments and calculation are carried out for risk analysis of wave overtopping, wave forces and stability for break-water by modeling experiments and design codes calibration. The new method involves the uncertainties and joint probabilities of input parameters and it can be used for structure reliability analysis.