LC-MS/MS检测水产品源致病菌和腐败菌群体感应AHLs信号分子

为分析水产品源致病菌和腐败菌中群体感应信号分子AHLs的含量和种类,实验建立液相色谱-串联质谱(LC-MS/MS)同时检测细菌群体感应11种AHLs类信号分子的技术,并与生物报告菌法比较。结果显示,报告菌紫色杆菌CV026和根癌农杆菌A136检测发现嗜水气单胞菌和2株铜绿假单胞菌是AHLs产生菌。建立的LC-MS/MS能完全分离和检测11种AHLs,并发现假单胞菌和嗜水气单胞菌产生的AHLs信号分子含量较高,其中ATCC 9027和ATCC 15692产生3-oxo-C12-HSL,嗜水气单胞菌A2产生C4-HSL,而腐败希瓦氏菌XH4分泌的C4-HSL信号分子含量较低,沙门氏菌ATCC 14028-3、大肠杆菌O157:H7和3种弧菌的AHLs很低。随着细菌浓度的增加,铜绿假单胞菌ATCC 9027产生的AHLs含量逐步增加,在12 h达到最高。研究表明,LC-MS/MS可用于多种AHLs信号分子的定性和定量检测,具有灵敏性与准确性更高的优点。在11株水产品源致病菌和腐败菌中,假单胞菌和气单胞菌分泌的AHLs含量最高。     

灿烂弧菌的疏水性和生物被膜形成能力

以仿刺参化皮病病原菌—灿烂弧菌(Vibrio splendidus)AP622为试验菌株,研究了培养材料、培养时间、培养基、葡萄糖浓度对菌株生物被膜形成能力的影响,证实鞭毛和菌毛介导的运动性,同时比较了浮游细菌与形成生物被膜的细菌对抗生素的敏感性,并研究了菌株的疏水性。结果表明,菌株AP622为高疏水性和高生物被膜形成菌株,在聚氯乙烯为培养材料、含葡萄糖浓度为0.5%、LB培养基中形成的生物被膜最多,生物被膜形成周期为24 h。菌株AP622明显表现出鞭毛介导的群集性和Ⅳ型菌毛介导的颤搐等运动力。生物被膜中的菌株AP622对抗生素的抵抗力明显强于浮游细菌,其最小抑菌浓度(MIC)是浮游细菌的32倍。综上所述,菌株AP622具有很强的疏水性和高生物被膜形成能力,判断其具有很强的黏附力,同时具有明显的抗药性。     

不同基质构建海水养殖系统硝化功能的比较分析

为比较不同基质构建海水养殖系统硝化功能的强弱,选取纤维毛球、陶粒、螺旋式生物绳等7种基质,其中陶粒、流化床填料、纤维毛球采取不同放置方式,共建立14个模拟海水养殖系统,比较不同基质硝化功能建立过程以及同种基质不同放置方式对氨氮和亚硝氮的去除效果。结果表明,单位体积珊瑚骨的氨氧化活性和亚硝酸盐氧化活性高于其他载体,在氨氮初始浓度20 mg/L条件下,氨氮和亚硝氮降解至检测不出分别需要3 d和11 d,而纤维毛球、陶粒、螺旋式生物绳、流化床填料、丝带内芯悬浮球、海绵内芯悬浮球硝化系统的建立分别需要18、26、30、25、27和22 d。纤维毛球100目筛绢悬挂、陶粒网兜悬挂、流化床填料100目筛绢悬挂优于其他放置方式,其中纤维毛球100目筛绢悬挂硝化功能建立时间为18 d,效果最优,流化床填料100目筛绢悬挂、陶粒网兜悬挂硝化系统建立分别需要21、24 d。     

Virulence regulation of cel‐EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel‐EIIB protein‐mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel‐EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel‐EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel‐EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel‐EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel‐EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel‐EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel‐EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel‐EIIB strain (p < 0.05). In conclusion, cel‐EIIB protein‐mediated cel‐PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.     

低温污水处理器中耐冷菌脱氢酶活性分析

[目的]为解决我国北方冬季污水处理困难的问题提供技术支持。[方法]运用TTc法分析了pH值、温度和时间对从低温生物膜中分离纯化鉴定得到8株耐冷菌中脱氢酶活性的影响。[结果]菌株S1、S4、S5、S7和S8在最适温度和4℃下的最适pH值都是7．5,其他3株茵在2种温度下的最适pH值不同,但都在7．5—8．5的范围内。菌株S1、S3、S4、S5和S6的最适反应温度为30℃,菌株S2、S7和S8的最适反应温度为20℃。除菌株S3外,其余菌株在4℃下的最适反应时间比最适反应温度下的长。在4℃下,菌株S3、S4和S6的最适pH值和最适反应时间分别为8．5和0．6h、7．5和12．0h、8．0和0．5h,其最高脱氢酶活性分别为7．19、6．73和7．70mg（TF）／g（SS）。[结论]菌株S3、S4和s6的脱氢酶活性较高,适合用于处理低温污水。     

海水循环水养殖系统生物膜快速挂膜试验

循环水养殖系统生物膜的挂膜成熟是一个比较耗时的过程,通常需要30～40d。为解决这一技术瓶颈,该文在一个大型海水循环水养殖厂的生物滤池内进行了生物膜快速挂膜的中试试验。该养殖厂循环水养殖系统有并联的生物滤池4个,单个为跑道式2级串联结构,总容量为800m3,采用毛刷状聚乙烯丝为生物载体。试验设计为预先培养水质净化菌的种子液,制备经200目筛绢过筛后质量比为4:1的黏土和沸石粉超细颗粒混合物悬液,然后按103cfu/mL和5g/L的浓度在两级滤池中分别加入4种不同水质净化菌的种子液和"黏土-沸石粉"混合物悬液,静水充气培养8d后,生物载体上能够形成较为牢固的生物膜。打开循环水系统运行2d后,连续5d检测生物滤池进、出水口的氨氮、硝酸盐、亚硝酸盐和COD(化学需氧量)含量,其5d平均消除率分别为:52.04%、17.24%、26.82%和62.94%。结果表明,与传统生物膜自然培养方法相比,该文所采用的挂膜方法将海水循环水养殖系统生物膜的挂膜成熟提前了20d以上,起到了增速效果,在生产上也是可行的。     

Antibiofilm potential of a tropical marine Bacillus licheniformis isolate: role in disruption of aquaculture associated biofilms

Microbial biofilms are important in aquaculture industries as they resist antibiotic treatments. In this study, we have investigated the antibiofilm potential of a tropical marine culture Bacillus licheniformis D1 (containing an antimicrobial protein BLDZ1) against two aquaculture associated pathogens namely, Vibrio harveyi and Pseudomonas aeruginosa. Both the test cultures formed biofilms on polystyrene and glass surfaces. The cell free supernatant (CFS) of B. licheniformis inhibited V. harveyi and P. aeruginosa biofilms on polystyrene surfaces up to around 80% and 78% respectively. In addition, the CFS disrupted pre‐formed biofilms of test cultures by about 73%. Fluorescence and scanning electron microscope analysis confirmed the antibiofilm potential of the CFS. The cell free supernatant displayed antiadhesive activity that inhibited the initial attachment of the bacteria during the process of biofilm formation. In addition, the CFS exhibited antimicrobial activity and mediated cell death via cytoplasmic membrane disruption.     

鸭源沙门菌江苏分离株的生物学特性研究

为了解鸭源沙门菌江苏分离株的主要生物学特性,本研究对2012年至2013年从发病或死亡鸭中分离的33株沙门菌分离株进行鉴定,即采用玻片凝集法对分离的沙门菌进行血清学分型,多重PCR方法检测17种毒力基因的分布,按照美国临床和实验室标准化研究所制定的方法进行药物敏感性检测,通过结晶紫半定量法检测分离菌株的生物被膜形成能力.33株鸭源沙门菌的血清学分型结果显示,查理沙门菌占48.5％,为优势血清型.毒力基因检测结果显示,pagC、msgA、sipB、prgH、spaN、tolC、iroN、sopB及pefA为保守基因.药物敏感性检测显示,42.4％的菌株耐受8种以上药物.生物被膜检测显示,有14株细菌生物被膜形成能力为中等以上,其中57.1％的菌株耐8种以上的药物.     

Isolation and Identification of Lytic Phages Infecting Proteus mirabilis from Canine-derived and Their Effects on Biofilm

The present study aimed to isolate phages that could lyse Proteus mirabilis from sewage in Daqing, Heilongjiang, and analyze their biological characteristics and effects on biofilm and provide the basic data for phage therapy as well as prevention. Phages were isolated and purified from sewage by double plate culture method, and their plaque morphology were observed. Morphological characteristics of purified phages were observed by transmission electron microscopy. The optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH, nucleic acids analysis and the inhibition and removal effect of biofilm were determined. Three lytic of bacteriophages of Proteus mirabilis were successfully isolated and purified, and named as vB_PmM_S, vB_PmM_W and vB_PmM_X. The phage plaques were circular, clear and transparent with smooth edge. The electron microscope observation showed that the phage particles, those head was twenty-side, head length was about (30±3) nm. The optical MOI was 0.001, the incubation period was 20 min, the burst period was 35 min, and the burst size was 70 PFU·cell^-1. The phages could withstand the temperature range from 10 to 60 ℃ and keep stable titer under pH 5.0-8.0. The phage vB_PmM_S can be stored at 4 ℃ in SM solution for a short time, and stored at -80 ℃ in glycerin (30%) or DMSO (5%) for a long time. The mixture of three bacteriophages can inhibit the biofilm formation and remove the biofilm. The results laid the foundation for the treatment of Proteus mirabilis and urinary tract infections.     

PhoR/PhoP双组份对枯草芽胞杆菌NCD-2菌株中surfactin合成的影响

 Surfactin是由芽胞杆菌类细菌产生的一种脂肽类物质,具有溶血活性和排油能力,并且与细菌生物膜形成和根际定殖能力相关。本研究分析了PhoR/PhoP双组份对生防枯草芽胞杆菌NCD-2菌株中surfactin合成的影响。在低磷环境下,同NCD-2野生型菌株相比,PhoR和PhoP突变子显著降低了NCD-2菌株的溶血活性;NCD-2菌株的排油能力很强,而PhoR和PhoP突变子均丧失排油能力;NCD-2野生型菌株的生物膜形成能力分别是PhoR和PhoP突变子的2.0和2.2倍。通过FPLC色谱分析技术证明NCD-2菌株可以产生surfactin,并发现在低磷条件中,野生型菌株NCD-2所产生的surfactin分别是PhoR和PhoP突变子的2.3和6.4倍。通过RT-qPCR技术分析了surfactin合成酶基因srfAA在不同菌株中的表达情况,结果表明在低磷环境下,PhoR和PhoP突变子中srfAA基因的表达量比NCD-2野生型菌株均降低了2.1倍。构建PhoR和PhoP突变子的互补菌株,证明互补菌株均能使突变子的性状恢复到野生型水平。以上结果证明,PhoR/PhoP双组份影响surfactin的合成。