Phenotypic and genotypic characterization of canine pyoderma isolates of Staphylococcus pseudintermedius for biofilm formation

Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.     

嗜水气单胞菌双组分系统FlrBC的功能探究

为明确双组分系统FlrBC在嗜水气单胞菌极生鞭毛合成、生物被膜形成及致病机制中的作用,通过同源重组法,以嗜水气单胞菌ZYAH72为野生菌株构建双组分系统FlrBC缺失株ΔflrB和ΔflrC,比较不同菌株鞭毛合成及游动性、生物被膜形成、胞外多糖分泌、细胞黏附以及抗全血杀伤能力差异。结果显示:与野生株相比,ΔflrB和ΔflrC仍能形成极生鞭毛,细菌游动能力无显著差异;而结晶紫染色发现ΔflrB和ΔflrC相较于野生株的生物被膜形成能力分别下降了27.2%和22.3%;刚果红检测结果显示,与野生株相比,ΔflrB和ΔflrC的胞外多糖分泌分别下降了18.4%和14.2%;实时荧光定量PCR的结果显示,flrB和flrC的缺失不同程度抑制了鞭毛合成、生物被膜相关通路基因的表达;与草鱼肾细胞共孵育后,ΔflrB和ΔflrC的细胞黏附率与野生株相比分别下降了23.2%和18.2%;全血杀伤实验结果显示,ΔflrB和ΔflrC抗全血杀伤能力均显著减弱。以上结果表明,双组分系统FlrBC不是嗜水气单胞菌极生鞭毛形成所必需,但在细菌鞭毛形成组装和生物被膜形成中发挥调控作用,并且影响嗜水气单胞菌致病性...     

口服嗜水气单胞菌生物被膜疫苗的动物免疫试验

将热灭活的嗜水气单胞菌(Aeromonashydrophila)生物被膜(biofilm,BF)菌苗和浮游(freecell,FC)菌苗口服免疫小鼠和剑尾鱼,通过测定抗体效价、相对保护率、小鼠的淋巴细胞转化和肠道sIgA浓度,评价免疫效果。免疫20d后,BF菌苗组50尾剑尾鱼的相对保护率为68%;FC菌苗组为42%。BF菌苗组12只小鼠的相对保护率为70%;FC菌苗组为50%;对照组为17%。BF菌苗组小鼠外周血淋巴细胞转化率是7.28、FC菌苗组为3.87、对照组为2.36。BF菌苗组小鼠肠道sIgA浓度是1.22μg/mL;FC菌苗组为0.73μg/mL;对照组为0.465μg/mL。免疫30d后,小鼠的血清凝集抗体达到最高,BF组抗体达128:1,FC组为32:1,对照组为2:1。各组间差异显著(P<0.01)。结果表明,BF菌苗较FC菌苗能更有效地刺激机体的免疫系统,尤其是肠道淋巴组织的免疫应答,对剑尾鱼和小鼠具有较好的保护力。     

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.     

两种生物膜工艺对污染物去除效果的比较研究

在室温下,用竹丝、塑料两种材料作为生物膜载体,使用SBR工艺对比研究这两种生物膜处理工艺去除污染物的实验结果及作用机理。实验结果表明:竹丝填料反应器对高锰酸盐指数、NH3-N(氨氮)、硝酸盐氮、亚硝酸盐氮、TP(总磷)的去除率分别为31.8%~51.9%、53.3%~71.9%、69.2%~84.0%、21.1%~33.3%、51.1%~74.5%,与塑料填料反应器相比具有明显的优势。因为竹丝填料是天然载体,其具有亲水基团和良好的生物亲和性,并且生物量多,这就使得微生物容易在竹丝填料上生长并快速形成生物膜,并且其生物膜的总体性能要比塑料填料反应器内生物膜的总体性能要好。总的来看,塑料填料反应器的处理效果不如竹丝填料反应器。     

The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.     

Effect of submerged time of collector ropes on the settlement capacity of Mytilus galloprovincialis L.

Submersion time of collector ropes influences settlement and larval metamorphosis of Mytilus galloprovincialis, which might be related to the biofilm development. In the present study, the effect of submersion time of collector ropes on the settlement of mussel seed was studied. The results of two experiments performed in an experimental long‐line in the Ría de Ares‐Betanzos have established a minimum submersion time of 41–46 days.     

金黄色葡萄球菌分离株的菌膜测定及其影响因素

采用96孔细胞培养板法,对10株不同来源的金黄色葡萄球菌分离株的菌膜形成能力进行调查,并研究外部环境因素对金黄色葡萄球菌菌膜形成的影响。首先以金黄色葡萄球菌标准菌株ATCC6538为模式菌株,确定了亲水性细胞培养板、培养48 h为适宜的菌膜体外培养条件。在此条件下测定了10株分离株的菌膜形成量,并研究了不同培养温度（25~46℃）和不同营养条件（葡萄糖和氯化钠）对菌膜形成的影响。结果显示,体外培养条件下,金黄色葡萄球菌普遍可以形成菌膜,10株菌中仅1株为菌膜阴性,不同菌株菌膜形成能力差异较大;较高的培养温度有利于菌膜的形成,有3株菌在46℃时菌膜形成量达到最大;0.5%葡萄糖和1%氯化钠的添加可显著改变各菌株的菌膜形成,葡萄糖倾向于促进临床分离株菌膜形成量的增加,而氯化钠倾向于促进食品分离株菌膜形成量的增加。这一结果,可以为实际环境中菌膜的预防及控制提供指导和方向,同时也说明金黄色葡萄球菌的菌膜形成存在着不同的调控模式。     

金黄色葡萄球菌生物被膜在不锈钢表面的形成及其对二氧化氯的敏感性

通过扫描电镜观察和测定生物被膜生长曲线的研究得出 :在不锈钢表面形成的金黄色葡萄球菌生物被膜约 6h后进入稳定期 ,培养 5d后其细菌密度达到 3× 10 6cfu·cm-2 ;金黄色葡萄球菌以生物被膜的形式比以浮游形式生长对二氧化氯具有更强的抵抗能力 ;胰酶大豆肉汤 (tryptonesoybroth ,TSB)的营养物质能明显减弱二氧化氯的杀菌效果     

鸡源大肠杆菌浮游菌与生物被膜菌超广谱β-内酰胺酶的检测

采用平板培养法建立鸡源大肠杆菌生物被膜的体外模型,银染后经显微镜观察鉴定,并用双纸片协同法检测浮游菌与生物被膜菌超广谱β-内酰胺酶的产生情况,同时用双光束紫外分光光度计测定超广谱β-内酰胺酶的活性,为研究鸡源大肠杆菌生物被膜的耐药机制奠定方法学基础。结果表明,27株鸡源大肠杆菌均形成了生物被膜,肉眼可以看到硅胶片上有黑色膜样物质形成,用银染法处理过的生物被膜菌在显微照相系统下可清楚地看到生物被膜的结构;鸡源大肠杆菌生物被膜中超广谱β-内酰胺酶的检出率(70.4%)高于浮游菌的检出率(55.6%),且生物被膜菌中超广谱β-内酰胺酶的酶活性((0.71±0.23)U.mg-1)高于浮游菌的酶活性((0.42±0.12)U.mg-1),前者是后者的1.73倍。