基于构建微生物传感器的甲基对硫磷降解菌的分离鉴定及其降解特性研究

有机磷农药是目前环境中残留量最多的农药之一,对其残留量的检测及降解机制的研究对于环境污染及生态修复具有重要意义。微生物传感器由生物学元件与换能器构成,因具有成本低廉、易于微型化及选择性高等特点而被广泛应用于各种生化物质的分析和检测。本文从长期受农药污染的土壤中分离出4株能以甲基对硫磷为碳源生长的菌株,根据形态特征和16S r RNA基因序列同源性分析,对4株降解菌进行鉴定,利用高效液相色谱测定降解率,选取降解率最高的1株菌进行降解机制研究,以期将其应用于测定环境中甲基对硫磷残留的电位型微生物传感器的构建。结果表明,在甲基对硫磷初始浓度50 mg·L-1、30℃、p H 7.0的培养条件下培养7 d,4株菌对甲基对硫磷的降解率均在78%以上,其中1株菌的降解效率可达100%。16S r RNA基因序列测定表明,该菌株属于克雷伯氏菌属,命名为Klebsiella sp.MP-6。利用液相色谱-质谱联用对其降解产物的研究表明,菌株MP-6水解甲基对硫磷主要产生二甲基硫代磷酸(dimethyl thiophosphoric acid,DMTP)和对硝基苯酚(p-nitrophenol,PNP),极少部分PNP通过产生4-硝基邻苯二酚(4-nitrocatechol,4-NC)和1,2,4-苯三酚(1,2,4-BT)进一步代谢。结果表明,基于测定中间产物对硝基苯酚(p-nitrophenol,PNP)的电位响应信号,该菌株适用于构建测定海水及土壤等环境中有机磷农药的微生物传感器。     

河南驻马店地区猪源肺炎克雷伯氏菌分离菌株生物学特性

为了分析驻马店地区猪源肺炎克雷伯氏菌分离菌株生物学特性,从驻马店地区不同养殖场中采集患呼吸道疾病猪肺脏、鼻拭子等病料组织74份中分离得到了43株肺炎克雷伯氏菌。采用人工感染小鼠致病性试验、PCR方法和K-B药敏纸片分别检测49株肺炎克雷伯氏菌的致病性、血清型及耐药性。结果显示,30株肺炎克雷伯氏菌对小鼠具有很强的致病性;43株分离菌株以K5、K20为主要流行血清型,分别占分离菌株的37.2%、27.9%;43株分离菌株对氨苄西林、阿莫西林、大观霉素等7种药物耐药性较高,耐药率在51.2%以上,对其他药物的耐药率在18.6%~234.9%之间,且呈现多重耐药性,耐10、9种药物分离菌株最多,分别占分离菌株的25.6%、27.9%。本试验为该地区猪源肺炎克雷伯氏菌病的防治提供理论基础。     

固氮肺炎克氏杆菌的产酸和解磷能力

本文报道了549、258属于肺炎克氏杆菌的这一类联合固氮细菌具有产酸解磷的能力.549在培养过程中能产生多种有机酸成分,使无机磷的有效性提高.它还能利用卵磷脂这种有机磷作为自己的磷源,在其培养过程中产酸量有一定限度,达到一定酸度时,培养环境中的酸基本上就不再增加,维持在一个相对稳定的水平.549、258产酸解磷的结果能够改善植物的磷素营养,从非豆科植物根际联合固氮的研究角度考虑,可以认为产酸的联合固氮细菌具有固氮和解磷的双重性.     

猪臭鼻克雷伯氏菌的分离与鉴定

从呼吸系统症状明显、高热、耳朵等薄皮处发绀的病猪血与病死猪的心血、肝、脾中分离到1株细菌，经形态学观察、生化试验鉴定为臭鼻克雷伯氏菌；经人工感染小白鼠试验，表明具有较强的毒性。选用药敏试验筛选的敏感药物——头孢曲松钠治愈了病猪，控制了疫情。     

Changes of IFN-γ, IL-6 and TNF-α levels in rats with severe pneumonia

AIM:To study the variety of cytokines in severe bacterial pneumonia of Sprague Dawleg (SD) rats. METHODS:A total of 60 SD rats were randomly divided into three groups: model Ⅰ group (n=24), model Ⅱ group (n=24), and control group (n=12). Rats in the model Ⅰ group and the model Ⅱ group were intratracheally instilled with suspension of klebsiella pneumoniae at different doses. Rats in the control group were intratracheally injected with 1 mL saline. On the 2nd, 4th and 6th day after intratracheal instillation, 1/3 rats in each group were killed to determine the concentration of IFN-γ, IL-6 and TNF-α in blood. RESULTS:The levels of IL-6 and TNF-α in model groups were higher than those in control group, while the level of IFN-γ was lower. The change of cytokines was more significant in the model Ⅱ group (severe pneumonia) than those in the model Ⅰ group. CONCLUSION:The cytokines we studied may play an important role in the pathogenesis of severe pneumonia. The change of cytokines is more significant in severe pneumonia than those in common pneumonia.     

2,3-丁二醇脱氢酶基因(BudC)过表达菌株的构建和初步鉴定

旨在过表达BudC进而上调2,3-丁二醇（2,3-Butanediol, 2,3-BD）的合成,通过基因工程手段构建整合载体pR1SW-BudC,经PCR和酶切双重验证后将其电转化至产酸克雷伯氏菌(Klebsiella oxytoca) HD79。结果经卡那霉素筛选,获得一株菌株K. oxytoca HD79-01。以原始菌株为对照,摇瓶发酵检测2,3-BD含量和相关酶活,q RT-PCR检测BudC表达差异,最终BudC在HD79-01中转录水平的表达量为HD79的45.25倍（p<0.01）。摇瓶发酵显示2,3-BD的最高产量、转化率和生产强度分别提高了24.54%、10.97%、45.08%（分别为30.818 ± 0.003 g/L、0.253 ± 0.013 g/g、0.43 ± 0.01 g/(L·h)）,酶活提高了3.61倍(0.563 ± 0.003 U/mg)。因此,2,3-丁二醇脱氢酶基因(BudC)过表达菌株构建不仅成功的提高了2,3-BD的产量也为实现2,3-BD的工业化生产奠定基础。     

苄嘧磺隆降解菌的分离及生长降解特性研究

苄嘧磺隆是一种磺酰脲类除草剂,被广泛应用于稻田土壤中防除一年生和多年生阔叶杂草。在土壤中的持效期较长,大量施用后易对后茬敏感作物产生药害,微生物降解是土壤中苄嘧磺隆转化的主要方式。本研究从长期施用该除草剂的稻田土壤中分离筛选到1株苄嘧磺隆降解菌株75B,经形态学及生理生化特征、16S rRNA基因扩增测序构建系统发育树分析,鉴定为Klebsiella pneumoniae(肺炎克雷伯氏菌)。75B菌株对环境的适应能力较强,在p H 5.0!9.0、0.5!5.0%Na Cl浓度下、培养温度为26!38℃的范围内生长良好。将该菌株按5%接种量置于p H 7.0、以苄嘧磺隆为唯一碳源的无机盐培养基(2.0%Na Cl浓度)中、34℃条件下培养,对50 mg/L苄嘧磺隆的5 d降解率为74.11%,培养至10 d时的降解率为97.65%。结果表明75B菌株可以有效地降解苄嘧磺隆,具有应用于该除草剂污染环境修复的潜力。     

克雷伯氏肺炎杆菌群体感应系统缺失对合成2,3丁二醇的影响

通过同源重组技术敲除克雷伯氏肺炎杆菌的luxS基因,使其群体感应系统失活,研究突变株在2,3-丁二醇发酵培养基中的生长状况及对2,3-丁二醇合成的影响;同时检测对其他副产物合成的影响,通过对野生株和luxS突变株发酵合成2,3-丁二醇的结果进行研究发现,与野生株相比,失去了群体感应功能的luxS突变株利用同样质量浓度的葡萄糖合成2,3-丁二醇的能力提高了47.75%,同时琥珀酸、乳酸和乙醇等主要副产物的质量浓度大幅降低。     

氟磺胺草醚降解菌F-12的分离鉴定及降解特性,

为了研究氟磺胺草醚污染土壤的生物修复机理,利用富集培养技术从长期施用氟磺胺草醚的土壤中分离得到1株能够以氟磺胺草醚为唯一碳源生长的细菌,命名为F-12。通过菌落形态、生理生化特性和16SrDNA基因序列分析,初步鉴定菌株F-12为克雷伯氏菌属（Klebsiella sp.）。并分析了氟磺胺草醚的初始浓度、接种量、温度和pH值对菌株F-12降解氟磺胺草醚效果的影响,确定了最佳降解条件。结果显示,该菌在氟磺胺草醚浓度为100 mg/L、接种量为15%、pH为6.0、温度35℃条件下,培养2 d后对氟磺胺草醚的降解效率达到80%以上。具有应用到氟磺胺草醚污染土壤生物修复的能力。     

Piscidin is Highly Active against Carbapenem-Resistant Acinetobacter baumannii and NDM-1-Producing Klebsiella pneumonia in a Systemic Septicaemia Infection Mouse Model

This study was designed to investigate the antimicrobial activity of two synthetic antimicrobial peptides from an aquatic organism, tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4), in vitro and in a murine sepsis model, as compared with ampicillin, tigecycline, and imipenem. Mice were infected with (NDM-1)-producing K. pneumonia and multi-drug resistant Acinetobacter baumannii, and subsequently treated with TP3, TP4, or antibiotics for different periods of time (up to 168 h). Mouse survival and bacterial colony forming units (CFU) in various organs were measured after each treatment. Toxicity was determined based on observation of behavior and measurement of biochemical parameters. TP3 and TP4 exhibited strong activity against K. pneumonia and A. baumannii in vitro. Administration of TP3 (150 μg/mouse) or TP4 (50 μg/mouse) 30 min after infection with K. pneumonia or A. baumannii significantly increased survival in mice. TP4 was more effective than tigecycline at reducing CFU counts in several organs. TP3 and TP4 were shown to be non-toxic, and did not affect mouse behavior. TP3 and TP4 are able at potentiate anti-Acinetobacter baumannii or anti-Klebsiella pneumonia drug activity, reduce bacterial load, and prevent drug resistance, indicating their potential for use in combating multidrug-resistant bacteria.