黄单胞菌的筛选及其降解烷烃性能

从受石油污染的海水筛选了一株能降解烃类的菌株黄单胞菌(Pseudomonas Xanthomonsa)。并实验研究了它的降解性能。结果表明，该菌株能降解庚烷和甲苯，在48h内对1．5％庚烷的利用率达到30．06％，且能在氯化钠浓度为10～90g／L的培养基中生长。     

eDNA,Amyloid Fibers and Membrane Vesicles Identified in Pseudomonas fluorescens SBW25 Biofilms

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.     

Serratiopeptidase Affects the Physiology of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is frequently involved in cystic fibrosis (CF) airway infections. Biofilm, motility, production of toxins and the invasion of host cells are different factors that increase P. aeruginosa’s virulence. The sessile phenotype offers protection to bacterial cells and resistance to antimicrobials and host immune attacks. Motility also contributes to bacterial colonization of surfaces and, consequently, to biofilm formation. Furthermore, the ability to adhere is the prelude for the internalization into lung cells, a common immune evasion mechanism used by most intracellular bacteria, such as P. aeruginosa. In previous studies we evaluated the activity of metalloprotease serratiopeptidase (SPEP) in impairing virulence-related properties in Gram-positive bacteria. This work aimed to investigate SPEP’s effects on different physiological aspects related to the virulence of P. aeruginosa isolated from CF patients, such as biofilm production, pyoverdine and pyocyanin production and invasion in alveolar epithelial cells. Obtained results showed that SPEP was able to impair the attachment to inert surfaces as well as adhesion/invasion of eukaryotic cells. Conversely, SPEP’s effect on pyocyanin and pyoverdine production was strongly strain-dependent, with an increase and/or a decrease of their production. Moreover, SPEP seemed to increase swarming motility and staphylolytic protease production. Our results suggest that a large number of clinical strains should be studied in-depth before drawing definitive conclusions. Why different strains sometimes react in opposing ways to a specific treatment is of great interest and will be the object of future studies. Therefore, SPEP affects P. aeruginosa’s physiology by differently acting on several bacterial factors related to its virulence.     

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 Star^TM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC₅₀ value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.     

基于预测微生物学的冷却牛肉货架期预测模型的建立

为实时监测冷却牛肉贮藏期间的品质变化与货架期,分别测定冷却牛肉0、4、7、10 ℃贮藏过程中假单胞菌菌数、总挥发性盐基氮含量和感官评分。利用修正的Gompertz方程建立不同贮藏温度下假单胞菌生长的动力学模型,以Belehradek方程为二级模型,描述贮藏温度对假单胞菌生长的影响,并利用2、5、8 ℃条件下贮藏的冷却牛肉验证货架期预测模型的准确性。结果表明：所建立的Gompertz模型拟合相关系数均在0.99以上,预测结果准确度在1.013～1.126之间,偏差度在0.926～1.057之间,贮藏温度与μmax 1/2和（1/λ）1/2均呈良好的线性关系,说明建立的一级和二级模型能够真实、有效地预测0～10 ℃贮藏条件下冷却牛肉中假单胞菌的生长情况;货架期的预测值与实测值相对误差在±10%以内,该货架期模型可有效预测冷却牛肉在0～10 ℃贮藏条件下任意温度下的货架期。     

Growth, survival and inactivation of Pseudomonas aeruginosa and Staphylococcus aureus strains of various origin in the presence of ethanol

The influence of ethanol on the behaviour of Pseudomonas aeruginosa and Staphylococcus aureus strains was evaluated throughout this study. Strains of different origin were used: collection, clinical and industrial strains were selected. Concentrations of ethanol from 0 to 20% (v/v) were evaluated by automated optical density measurements and by enumeration. When growth conditions were observed, predictive microbiology models were used to assess quantitatively for the ethanol effect. Primary modelling of kinetics was performed to determine growth rate values; secondary modelling was performed on these growth rates as influenced by ethanol, and minimum inhibitory concentrations of ethanol were determined for each strain. Staphylococcus aureus strains were more resistant to ethanol than P. aeruginosa strains, in growth conditions as well as in inactivation conditions. Furthermore, clinical S. aureus strains were more resistant than the collection strain. The method was promising for management of microbiological safety in cosmetics.     

包装饮用水中铜绿假单胞菌的耐药分析及同源性研究

采用国标方法对湖北省地区15家包装饮用水生产企业的水源水、各个生产环节水样及包装容器等共116份样品进行铜绿假单胞菌的分离和鉴定,将分离得到的48株阳性菌株通过梅里埃药敏鉴定卡进行耐药性分析,同时利用基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF MS）进行同源性研究。结果表明,48株水源性铜绿假单胞菌中有6株耐药菌株,耐药水平总体较低,而同一来源的铜绿假单胞菌亲缘关系较近,提示铜绿假单胞菌的污染主要来自于水源。     

食品中椰毒假单胞菌酵米亚种实时荧光PCR检测的研究

目的建立食品中椰毒假单胞菌酵米面亚种实时荧光PCR检测方法。方法根据椰毒假单胞菌酵米面亚种16S～23S rRNA基因片段序列,用Oligo7设计一对特异性引物和一个TaqMan探针,摸索最佳退火温度,最佳引物和探针浓度,建立椰毒假单胞菌酵米面亚种实时荧光PCR检测方法。通过对唐菖蒲伯克霍尔德氏菌以及22种其他标准菌株进行实时荧光PCR检测,验证本法的特异性和抗干扰性。同时从菌液浓度和DNA浓度水平上进行研究,确定该检测方法的灵敏度。结果用该方法检测23种菌,除唐菖蒲伯克霍尔德氏菌外,其他22种细菌均为阴性。抗干扰性试验显示杂菌对检测结果不影响,本法抗干扰能力强。通过灵敏度试验的研究,确定本法检测椰毒假单胞菌酵米面亚种的菌液灵敏度为1×10~2 CFU/mL,DNA灵敏度为250 fg/μL。结论本试验设计的引物和探针具有较强特异性、抗干扰性以及灵敏度,该方法具有很好的研究价值和应用前景。     

茶多酚对肉源腐败菌和致病菌的抑制效果

研究了茶多酚对肉源荧光假单胞菌、鼠伤寒沙门氏菌、大肠杆菌、金黄色葡萄球菌的抑制效果。设计不同浓度茶多酚溶液(0.0%、0.5%、1.0%、2.0%、2.5%),采用滤纸片法评判抑制效果。结果表明,茶多酚溶液(0.5%、1.0%、2.0%、2.5%)对荧光假单胞菌有极显著的抑制效果(P<0.01),常用的防腐剂山梨酸钾(1.0%)对其抑制效果却不显著(P>0.05)。茶多酚溶液(1.0%、2.0%、2.5%)和山梨酸钾溶液(1.0%)对鼠伤寒沙门氏菌、大肠杆菌、金黄色葡萄球菌有极显著的抑制效果(P<0.01),茶多酚溶液(0.5%)对鼠伤寒沙门氏菌、大肠杆菌、金黄色葡萄球菌无显著的抑制效果(P>0.05)。通过茶多酚溶液的浓度与4种菌抑菌圈直径的直线相关图可以看出,茶多酚溶液的浓度与鼠伤寒沙门氏菌抑菌圈直径的相关系数最大,荧光假单胞菌次之,其次是金黄色葡萄球菌、大肠杆菌。