Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.     

Genome Sequences of Three Live Attenuated Vaccine Strains of Brucella Species and Implications for Pathogenesis and Differential Diagnosis

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.     

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.     

LuxS-Dependent AI-2 Regulates Versatile Functions in Enterococcus faecalis V583

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.     

Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells

~~Screening and identification of Shigella flexneri 2a virulence-related genes induced after invasion of epithelial cells1. Jin, Q., Yuan, Z., Xu, J., Wang, Y., Shen, Y., Lu, W., Wang, J., Liu, H., Yang, J., Yang, P., Zhang, X., Zhang, J., Yang, G, Wu, H., Qu, D., Dong, J., Sun, L., Xue, Y, Zhao, A., Gao, Y., Zhu, J., Kan, B., Ding, K.. Chen, S., Cheng, H., Yao, Z., He, B., Chen, R., Ma, D., Qiang, B., Wen, Y, Hou, Y., Yu, J., Genome sequence of Shigella flexneri 2…     

布鲁氏菌BP26基因标记疫苗株的构建及鉴别PCR方法的建立

[目的]由于现有的减毒活疫苗仍存在较强的毒力,因抗原与毒株的差异不大而很难区分疫苗免疫和自然感染等缺点,限制了现有布鲁氏菌减毒活疫苗的广泛应用.本文拟对布鲁氏菌的减毒活疫苗株M5进行遗传改造,克服这些缺点.[方法]本研究利用同源重组的方法,用卡那抗性基因替换了布鲁氏菌减毒疫苗株M5的BP26基因,得到了新的标记疫苗株M5△BP26.分别用标记疫苗株和野生株侵染巨噬细胞和感染小鼠,比较分析标记株在细胞内和小鼠体内的存活能力.根据种特异性保守基因dnaK和缺失的BP26基因设计引物,建立双重PCR,用于区分标记株与野生株.[结果]成功构建了.BP26基因标记疫苗株,细胞实验和动物实验结果表明,标记株仍能在胞内和小鼠内存活,具备作为减毒活疫苗的特性.小鼠实验结果显示,感染后两周野生株的细菌数为1022.9 ,而突变株为101.1 (P<0.01),至第3周野生株的细菌数为102.2 ,而突变株未能检出,表明与原疫苗株相比,标记株的感染力进一步减弱.根据DNA序列的差异,建立了能够区分标记疫苗株与野生株的双重PCR方法,标记株因只能扩增出一条带而能与野生株和毒株相区分,从而可以区分自然感染和疫苗免疫.[结论]基因标记疫苗株的构建及鉴别PCR方法的建立,为布鲁氏菌疫苗的进一步研发奠定了基础.     

InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin

Internalization of Listeria monocytogenes into non-phagocytic cells is tightly controlled by host cell actin dynamics and cell membrane alterations. However, knowledge about the impact of phosphatidylcholine cleavage driven by host cell phospholipase D (PLD) on Listeria internalization into epithelial cells is limited. Here, we report that L. monocytogenes activates PLD in Vero cells during the internalization. With immunostaining it was shown that both PLD1 and PLD2 surrounded partially or completely the phagocytic cup of most L. monocytogenes. Either up- or down-regulation of PLD expression (activity) diminished Listeria internalization. Both PLD1 and PLD2 in Vero cells were required for efficient Listeria internalization, and could substitute for each other in the regulation of Listeria internalization. Further, exogenous InlB activated host cell PLD1 and PLD2 via the Met receptor, and restored host PLD activation by InlB-deficient L. monocytogenes. InlB-induced PLD activation and Listeria internalization were tightly controlled by phospho-cycling of cofilin. PLD1, but not PLD2, was involved in cofilin-mediated PLD activation and Listeria internalization. These data indicate that cofilin-dependent PLD activation induced by InlB may represent a novel regulation mechanism for efficient Listeria internalization into epithelial cells.     

微生物功能基因组学研究

自从1995年流感嗜血杆菌的基因组序列测定完成之后[1],目前已有75种(株)微生物的基因组完成测序,160多种(株)微生物的基因组测序正在进行中[2]。随着各种微生物基因组测序工作的不断完成和序列信息的积累,微生物基因组学研究的重点已由结构基因组学向功能基因组学转移。微生物功能基因组学研究不仅要阐明微生物基因组内每个基因的作用或功能,还要研究基因的调节及表达谱,进而从整个基因组及其全套蛋白质产物的结构、功能、机理的高度去了解微生物生命活动的全貌,揭示微生物世界的各种前所未知的规律,并使之为人类和社会服务。与真核生物相比,虽然微生物的基因组相对简单,但微生物基因组学研究仍具有重大的科学和经济意义。在细菌基因组中,既有编码在极端环境下起催化作用的酶的基因,也有编码分解化学污染物的酶的基因,这些基因在真核细胞是不存在的。通过微生物功能基因组学研究,还能发现药物靶位和疫苗抗原。微生物基因的功能及表达研究结果也能为研究复杂生物的基因功能提供参考。近些年微生物功能基因组学研究受到了普遍重视。日本组织了十几所大学和研究机构,计划用5年时间完成大肠杆菌的功能基因组研究[3]。日本还与欧洲联合正在开展枯草杆菌功能基因组学研究[4]。其它微生物的功能基因组学研究也在进行中。由于微生物的种类繁多,功能基因组研究的内容又较丰富,要全面介绍微生物功能基因组学研究是困难的。本文仅从未知功能基因的鉴定、药物靶位及疫苗抗原研究、致病机制研究、生物功能图谱研究4个方面进行简要的评述。     

Genome Sequence of Brucella melitensis S66, an Isolate of Sequence Type 8, Prevalent in China

Brucella melitensis is the most-represented Brucella species causing human brucellosis in China. Here we report the complete genome sequence of B. melitensis strain S66, a representative strain of sequence type 8 (ST8), which is prevalent in China, making it possible to compare the genome sequences of isolates from different countries.