临床分离肺炎克雷伯菌Ⅰ整合子分布及其与qnr基因关系研究

目的 了解临床分离肺炎克雷伯菌中qnr基因和Ⅰ类整合子基因的分布及其耐药特征.方法 采用PCR法对45株耐环丙沙星肺炎克雷伯菌进行qnrA、qnrB、qnrS基因筛查并测序,用PCR法检测qnr阳性菌株Ⅰ类整合子基因,并采用SPSS 13.0和Whonet 5.4软件分析药敏结果及比较.结果 45株肺炎克雷伯菌中,24株(51.1％)细菌检出qnrS基因,未检出qnrA和qnrB基因.20株qnr阳性菌株同时携带Ⅰ类整合子基因.qnr阳性菌株Ⅰ类整合子基因携带率显著高于阴性菌株,qnr阳性菌株对阿米卡星、妥布霉素、亚胺培南、哌拉西林/他唑巴坦及头孢哌酮舒巴坦的敏感性较高.结论 肺炎克雷伯菌对氟喹诺酮类抗菌药物耐药主要由qnrS引起,qnr阳性株同时携带Ⅰ类整合子,导致呈现多重耐药性,加强临床耐药监测对控制多重耐药传播有着重要的意义.     

产超广谱β- 内酰胺酶肺炎克雷伯菌耐药性及其基因分型分析

目的:了解新疆独山子地区肺炎克雷伯菌超广谱β-内酰胺酶(ESBLs)的发生率、作为指示剂的五种抗生素的检出情况及ESBLs主要基因型。方法:收集临床分离的148株肺炎克雷伯菌,采用双纸片协同筛选法、NCCLS推荐的表型筛选和确证试验对细菌进行ESBLs产酶株的识别;耐药基因的质粒重组、转化,聚合酶链反应(PCR)扩增阳性产物测序,通过GenBank对序确定基因型。结果:本地区肺炎克雷伯菌ESBLs的分离率达31.1%,头孢曲松(CRO)安曲南(ATM)和头孢噻肟(CTX)头孢泊肟(CPD)、头孢他定(CAZ)作为指示剂检出率分别为97.8%、95.6%、93.4%、76.0%、65.2%;本地区产ESBLs菌株耐药基因型CTX-M-22占54.3%,CTX-M-18占41.3%,TEM61.8%,52.1%的产ESBLs肺炎克雷伯菌SHV耐药基因阳性。结论:CRO、ATM和CTX对检测ESBLs阳性率较高;CTX-M-22、CTX-M-18是本地区产ESBLs菌株的主要基因型。     

Antibacterial effect of extracts of Hermetia illucens (Diptera: Stratiomyidae) larvae against Gram‐negative bacteria

Larvae of the black soldier fly, Hermetia illucens are well‐known fly larvae that inhabit many countries around the world. Antimicrobial agents derived from the larvae may be among the substances that are produced in the body for their survival. This study was carried out to identify the antimicrobial effects of H. illucens larvae that commonly inhabit animal waste and food waste. To evaluate the pharmacological effects of H. illucens larvae extracts, the larvae were extracted by various organic solvents, and their antibacterial effects were determined by antimicrobial methods, such as agar disk diffusion and turbidometric assays. The methanol extracts (ME) indicated antibacterial effects against the proliferation of Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei. However, antibacterial effects were not induced in Gram‐positive bacteria such as Bacillus subtilis, Streptococcus mutans and Sarcina lutea. The bacterial growth treated with ME was strongly inhibited from 20 mg/mL in a dose‐dependent manner compared with other extracts, and antibacterial activity gradually decreased after 24 h. Moreover, the minimal inhibitory concentration (MIC) values of ME against Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei for 12 h were measured as 44.74 mg/mL, 43.98 mg/mL and 43.96 mg/mL, respectively. These results demonstrate that ME of H. illucens larvae not only has antibacterial activity which strongly inhibits the growth and proliferation of the bacteria but also unique properties which effectively block the viability of the bacteria.     

Characterization and identification of virulent Klebsiella oxytoca isolated from abalone (Haliotis diversicolor supertexta) postlarvae with mass mortality in Fujian, China

An epidemic of mass mortality of abalone (Haliotis diversicolor supertexta) postlarvae aged 40 days or less has existed across south coast of China since the second half of 2002. Among 20 bacterial strains isolated from diseased abalone postlarvae on 2216E marine agar plates during an outbreak of postlarval disease in August 2005, a predominant strain (designated strain 20) was demonstrated to be virulent to postlarvae with an LD(50) value of 1.0x10(5) colony forming units (CFUml(-1)) on day 4, while the other 19 strains were either avirulent (16 strains) or weakly virulent (3 strains). The same bacterium could be re-isolated from postlarvae after bacterial challenge using 2216E marine agar plates. Preliminary toxicity tests of ECPs of strain 20 revealed that at 2.77mgproteinml(-1), crude ECPs completely liquefied postlarvae within 24h, leaving only shells. API 20E analysis identified strain 20 as Klebsiella oxytoca. 16S and ITS rDNA sequencing and phylogenetic analyses further confirmed this identification. Antibiotic susceptibility tests showed that strain 20 exhibited 94% of susceptibility to 16 various antibiotics tested and only showed resistance to streptomycin. Results of this work demonstrated that K. oxytoca is also linked to this epidemic in Fujian, China. This is considered to be the first report regarding K. oxytoca involved in the mass mortality of postlarval abalone in south China and the world.     

耐高糖高产2,3-丁二醇产酸克雷伯氏杆菌的选育

以产酸克雷伯氏杆菌(Klebsiella oxytoca) ME-UD-3为出发菌株,经紫外线及硫酸二乙酯复合诱变后分别在葡萄糖浓度逐渐提高的液体培养基中进行富集培养,筛选获得了一株耐高糖的2,3-丁二醇高产突变菌株K. oxytoca ME-UD-3-4;该菌株的初始葡萄糖耐受浓度从出发菌株的120g/L提高到300g/L以上,在初始葡萄糖浓度为95 g/L的条件下发酵培养,与出发菌株相比发酵时间缩短了8h,2,3-丁二醇的产量由原来的38.5g/L提高到43.0g/L,生产强度从0.80 g/L·h提高到1.08 g/L·h,转化率达到了理论值的91%。     

克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究

以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶（GDH）的基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coliBL21（DE3）中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH),纯化后比酶活达到156U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质,酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25～45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。     

能利用五碳糖和六碳糖生产乙醇的基因工程菌菌株的构建

燃料乙醇是一种极具前景的燃油代用品,近年来发展尤为迅速,为了推广这种能源和满足日益增长的需求,我们有必要开发更为高效的生产工艺和寻找更为廉价的原料。解决此问题的关键在于获得高效的工程菌,使其能利用木质纤维素水解液中的五碳糖和六碳糖发酵生产乙醇。通过代谢工程的研究和基因重组技术,几种重组细菌显示出良好的开发前景,它们是运动发酵单胞菌、大肠杆菌、产酸克雷伯氏菌和菊欧文氏菌。本文就这四种细菌的研究进展以及基因重组过程进行了介绍和评价。     

以gfp为报告基因的肺炎链球菌启动子诱捕文库的构建与分析

首先构建一个以gfp(green fluorescence protein)为报告基因的自杀质粒pEVP3-SDGFP,将肺炎链球菌基因组DNA的随机酶切片段(200bp～800 bp)克隆到该质粒gfp基因上游的多克隆位点,得到约58000个含有肺炎链球茵基因组DNA随机酶切片段的重组子,提取质粒即为质粒库,该库大约覆盖肺炎链球菌基因组全长的5倍,插入率达到90%以上,且有较强的随机性,质量较高.将该质粒库转化入肺炎链球菌TIGR4菌株,带有随机片段的报告质粒通过同源重组的方式将gfp基因融合于细菌染色体上该随机片段之后,利用质粒的抗生素抗性基因筛选出重组菌株,从而构建出相应的菌株库,共获得包含约500000个肺炎链球菌转化子的菌株库,经体内、外实验表明,其包含插入了S.pn体内、外表达基因片段的细菌,可以报告特定条件下的基因表达,并可通过流式细胞仪识别、分选.该文库的构建为进一步利用差异荧光诱导技术筛选肺炎链球菌体内诱导基因奠定了基础.     

利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

摘氢酶是生物制氢的关键酶,大多数氢酶因对氧极敏感而易失活,因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1％甲基磺酸乙酯对Klebsiella oxytoca HPl进行了两轮诱变,经40mmol／L甲硝唑和21％氧联合处理1h（第一轮诱变）或2h（第二轮诱变）进行筛选。所得突变菌株经产氢测试,结果在15％氧浓度条件下,第一代突变菌株HPl-A15产氢活性为出发菌株Klebsiella oxytoca HPl的3.70倍,在21％氧浓度条件下第二代突变菌株HPAl5-37产氢活性为HPl-A15菌株的2.75倍,是出发菌株的11倍。突变菌株HPl-A15和HPAl5-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.