次氯酸钠溶液对不同时期粪肠球菌生物膜的药物作用

背景:有研究指出粪肠球菌能在营养物质缺乏、抗菌药存在的恶劣环境中生存,形成再感染而影响根管再治疗效果。也有实验表明次氯酸钠溶液作为根管冲洗液对残留的粪肠球菌具有很强的清除作用。目的:建立不同时期粪肠球菌生物膜,研究不同浓度次氯酸钠溶液对其的作用效果。方法:建立对数期、稳定期、饥饿期粪肠球菌生物膜模型,以1%,2.5%,5.25%次氯酸钠溶液分别作用于各时期粪肠球菌生物膜表面30 s、5 min、10 min,同时用激光共聚焦显微镜观察用药后生物膜情况。结果与结论:在相同浓度次氯酸钠溶液作用下,饥饿期粪肠球菌生物膜中的活菌降低量低于稳定期与对数期粪肠球菌生物膜中的活菌降低量(P0.05);在1%次氯酸钠溶液作用下,稳定期与对数期粪肠球菌生物膜中的活菌降低量差异有显著性意义(P0.05);在2.5%,5.25%次氯酸钠溶液作用下,稳定期与对数期粪肠球菌生物膜中的活菌降低量差异无显著性意义(P0.05)。在同一时期生物膜模型下,5.25%次氯酸钠溶液作用下的活菌降低量高于2.5%、1%次氯酸钠溶液作用下的活菌降低量(P0.05);2.5%次氯酸钠溶液作用下的活菌降低量高于1%次氯酸钠溶液作用下的活菌降低量,但仅在药物作用30 s时差异有显著性意义(P0.05)。结果表明,在相同药物浓度、相同作用时间内,饥饿期粪肠球菌生物膜较对数期、稳定期粪肠球菌生物膜对次氯酸钠溶液更具耐药性;在相同作用时间内,5.25%次氯酸钠溶液对饥饿期粪肠球菌生物膜的杀菌效果最佳。     

噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的作用研究

目的 检测重组铜绿假单胞菌噬菌体PaP3多糖解聚酶对铜绿假单胞菌生物膜的降解活性.方法 经FTTCConA/PI荧光双染色后,采用激光扫描共聚焦显微镜观察重组多糖解聚酶处理前后PA3生物膜的形态变化,以测定多糖解聚酶对生物膜的降解活性;测定多糖解聚酶作用前、后铜绿假单胞菌对4种敏感抗生素(乳酸环丙沙星、加替沙星、头孢他啶、硫酸庆大霉素)的MIC、MBC变化情况,从而判断多糖解聚酶对抗生素的协同杀菌作用.结果 FTTC-ConA/PI荧光双染色结果显示.经重组多糖解聚酶处理后,铜绿假单胞菌生物膜的胞外多糖组分少而分散,包裹在胞外多糖组分里的细菌数量也大大减少,表明多糖解聚酶确实能够特异性的降解生物膜的胞外多糖组分.多糖解聚酶作用后四种抗生素相应的MIC、MBC都出现了不同程度的降低,其中以头孢他啶降低最为明显,表明多糖解聚酶对抗生素具有协同杀菌活性.结论 重组噬菌体PaP3多糖解聚酶在体外可以特异性的降解铜绿假单胞菌生物膜的胞外多糖,有利于抗生素通透生物膜,作用于菌体,具有协同抗菌作用.     

血液分离肠球菌毒力基因及生物膜形成相关性分析

目的了解血液分离肠球菌毒力基因及其与生物膜形成能力的相关性。方法收集血培养分离粪肠球菌42株和屎肠球菌44株,采用纸片扩散法检测菌株对常规药物的敏感性;PCR法检测肠球菌6种毒力基因cyl、esp、asal、hyl、gelE和agg;采用结晶紫染色法测定肠球菌生物膜形成能力;统计分析肠球菌毒力基因与生物膜形成能力的关系。结果屎肠球菌对青霉素、氨苄西林、左旋氧氟沙星的耐药率高于粪肠球菌;除esp、hyl外,cyl、asal、gelE和agg基因在粪肠球菌检出率均显著高于屎肠球菌(P<0.05);血液分离肠球菌生物膜阳性率为32.6%(28/86),其中粪肠球菌和屎肠球菌生物膜阳性率分别为59.5%(25/42)和6.8%(3/44);肠球菌毒力基因的携带与其生物膜阳性率无相关性(P>0.05)。结论致血流感染的屎肠球菌其耐药率高于粪肠球菌,粪肠球菌生物膜的形成能力高于屎肠球菌,两者的毒力基因与生物膜形成不具相关性。     

解脲脲原体生物膜胞外多糖的检测

目的 研究解脲脲原体(Ureaplasma urealyticum,Uu)4个型别标准株在体外形成的生物膜之胞外多糖的分布及结构成分.方法 将Uu标准株Parvo群中4、8血清型和T960群中3、14血清型进行体外生物膜培养后,扫描电镜下观察生物膜组成及结构,并在FITC-ConA/PI及ECA/PI双荧光染色后进行激光共聚焦显微镜观察及测定平均荧光强度.秩和检验及t检验分别比较两种荧光标记物、两生物群的总体平均荧光强度的差异.结果 4个型别Uu标准株均可在体外形成生物膜,生物膜结构主要呈网格状,胞外物质占大部分比例.在激光共聚焦显微镜下,Uu生物膜胞外多糖均可被FITC-ConA和ECA染色,FTTC-ConA呈网格状分布,ECA小片状聚集分布.FITC-ConA的总体平均荧光强度较ECA高,差异有统计学意义(P＜0.001).结论 Uu体外培养生物膜主要呈网格状结构,胞外多糖中含有葡萄糖、甘露糖、半乳糖、N-乙酰葡聚糖残基,并以葡萄糖、甘露糖残基为主.     

eDNA水平差异对表皮葡萄球菌临床株生物被膜形成能力的影响

目的 检测表皮葡萄球菌(表葡菌)临床株生物被膜的形成能力,研究胞外DNA(eDNA)水平差异对表葡菌临床株生物被膜形成能力的影响.方法 收集227株临床分离表葡菌,黏附试验检测其生物被膜的形成能力,PCR方法扩增icaA基因片段,黏附试验阳性和icaA基因扩增阳性的表葡菌为成膜组,黏附试验阴性和icaA基因扩增阴性的表葡菌为非成膜组,应用浮游培养法和微量板静置培养法检测eDNA水平,激光共聚焦显微镜(CLSM)观察生物被膜中eDNA的分布.结果 227株表葡菌中黏附试验阳性26株,icaA基因阳性32株.选取黏附试验阳性和icaA基因扩增阳性的成膜组表葡菌20株,黏附试验阴性和icaA基因扩增阴性的非成膜组表葡菌19株,浮游培养2、4、6h后,成膜组菌株的eDNA水平分别为(32.2±10.1) μg/ml、(33.6±11.9) μg/ml、(34.3±10.0)μg/ml,非成膜组菌株的eDNA水平分别为(28.7±8.9)μg/ml、(31.5±11.7) μg/ml、(31.8±l2.7)μg/ml,浮游培养成膜组菌株各时相eDNA水平分别高于非成膜组菌株,但差异尚无显著性(P＞0.05).微量板静置培养法成膜组20株eDNA水平为(740.0±264.4) ng/A600,高于非成膜组19株eDNA水平(80.1±31.1) ng,/A600,两组之间比较差异具有显著性(P＜0.05).通过AO-PI荧光染色于CLSM下观察静置培养的成膜株表葡菌Y36,其生物被膜中可见eDNA分布；非成膜株Y26没有生物被膜结构形成,未见eDNA分布.结论 表葡菌临床株具有形成生物被膜的能力,eDNA是表葡菌形成生物被膜的重要基质成分,在判断表葡菌生物被膜形成能力方面微量板静置培养法检测eDNA水平优于浮游培养法.     

聚醚醚酮基纳米复合材料表面口腔微生物黏附与成膜的实验研究

目的制备聚醚醚酮（PEEK）基纳米复合材料,研究材料表面口腔微生物黏附与生物膜形成情况。方法分别制备PEEK基纳米羟磷灰石（n-HA／PEEK）和PEEK基纳米氟磷灰石（n-FA伊EEK）复合材料,以纯钛（CpTi）和PEEK作对照,应用微生物活性剂检测试剂盒和激光共聚焦显微镜研究PEEK基纳米复合材料表面口腔微生物黏附和成膜情况。结果与CpTi相比,黏附初期2小时内PEEK及PEEK基纳米复合材料表面口腔微生物黏附量显著减少;四组材料表面在第14天形成的生物膜形貌和厚度基本一致,但PEEK基纳米复合材料组表面生物膜内死菌／活菌比显著高于CpTi及PEEK组,而且n-FA／PEEK组显著高于n-HA／PEEK组。结论PEEK基纳米复合材料表面细菌黏附量低于CpTi,生物膜死菌量显著高于CpTi,提示材料的成分和表面粗糙度影响口腔微生物的黏附和生物膜的组成结构,PEEK基纳米复合材料具有良好的临床应用前景。     

粪肠球菌类毒力基因sprE功能的动物实验研究

目的构建粪肠球菌丝氨酸蛋白酶基因(sprE)突变株,通过动物实验来研究sprE基因的功能。方法通过小鼠腹膜炎模型和兔心内膜炎模型来评价粪肠球菌sprE基因的突变株(sprE mutant)的毒力下降情况。结果突变株在小鼠腹膜炎模型中的半数致死量(LD50)提高了7倍,小鼠的存活率明显高于野生株感染组,引起的兔心内膜炎的病理改变也较野生株轻微。结论sprE基因在粪肠球菌致病中起着重要的作用,可能是粪肠球菌的毒力因子之一。     

2016年北京市海淀医院细菌耐药性监测

目的 了解北京市海淀医院2016年临床分离细菌对抗菌药物的耐药性.方法 共收集2268株非重复分离菌株,自动化仪器法进行药敏试验,按CLSI 2016年版标准判读药敏结果,采用WHONET 5.6软件进行数据分析.结果 2268株细菌中,革兰阴性菌占75.6％,革兰阳性菌占24.4％.10种最常见的细菌分别为:大肠埃希菌(16.8％),肺炎克雷伯菌(13.4％),铜绿假单胞菌(11.6％),鲍曼不动杆菌(10.1％),凝固酶阴性葡萄球菌(8.0％),屎肠球菌(6.7％),阴沟肠杆菌(5.1％),嗜麦芽窄食单胞菌(3.8％),粪肠球菌(3.4％),金黄色葡萄球菌(金葡菌)(3.2％).耐甲氧西林金葡菌(MRSA)和凝固酶阴性葡萄球菌(MRCNS)的检出率分别为34.2％和70.7％.耐甲氧西林葡萄球菌对β内酰胺类和其他抗菌药物的耐药率明显高于甲氧西林敏感葡萄球菌.未发现对万古霉素、替加环素和利奈唑胺耐药的葡萄球菌.粪肠球菌(除四环素外)对大多数抗菌药物的耐药率明显低于屎肠球菌.发现少数万古霉素耐药肠球菌(VRE),未发现对利奈唑胺耐药的肠球菌.分离到的45株无乳链球菌对青霉素的敏感率为100％.肺炎链球菌对红霉素的耐药率为100％.克雷伯菌属对亚胺培南和美罗培南的耐药率为20.2％和19.6％,其它肠杆菌科细菌对碳青霉烯类仍高度敏感,总耐药率≤6.9％.多重耐药肺炎克雷伯菌的检出率为47.5％.铜绿假单胞菌对亚胺培南的耐药率为26.6％,对阿米卡星的耐药率1.5％.鲍曼不动杆菌亚胺培南的耐药率为42.3％,对阿米卡星的耐药率为2.4％.多重耐药鲍曼不动杆菌和铜绿假单胞菌的检出率分别是42.4％和28.9％.结论 细菌对抗菌药物的耐药率呈下降趋势,特别是多重耐药的鲍曼不动杆菌下降明显,应持续采取有效的医院感染控制措施和加强抗菌药物的合理使用,进一步降低多重耐药菌的比例.     

枸杞多糖对氯化甲基汞诱导海马神经干细胞损伤的保护作用

目的 观察分析枸杞多糖对氯化甲基汞所诱导神经干细胞(NSCs)损伤的影响.方法 取孕16 d SD大鼠胚胎的海马组织,分离纯化得到海马NSCs.将纯化后的海马NSCs增殖、生长10 d后,按随机分组方法 将其分为空白对照组(DMEM/F12培养基);枸杞多糖组(DMEM/F12培养基+枸杞多糖);氯化甲基汞组(DMEM/F12培养基+氯化甲基汞);氯化甲基汞+枸杞多糖组(DMEM/F12培养基+氯化甲基汞+枸杞多糖).分别测定各组生长环境中的海马NSCs分化、生长的情况,观察分析氯化甲基汞对海马NSCs的损伤作用,以及枸杞多糖干预后对海马NSCs的影响.结果 氯化甲基汞组和空白对照组比较,加入氯化甲基汞后的海马NSCs分化后的神经元比例(3.63%±0.62%)和神经元的周长(63.36μm ±5.57 μm)都较空白对照低;枸杞多糖组和其他各组比较,海马NSCs分化后的神经元比例(7.75%±0.59%)和神经元的周长(253.3μm ±11.21μm)都较其他各组高;氯化甲基汞+枸杞多糖组和氯化甲基汞组比较,前者生长环境下的海马NSCs分化后的神经元比例(5.92%±0.98%)和神经元的周长(111.9μm ±6.07μm)较氯化甲基汞组高.结论 氯化甲基汞对海马NSCs的分化、生长具有损伤作用;枸杞多糖可以减轻氯化甲基汞对海马NSCs的损伤作用,并能促进海马NSCs向神经元的分化及神经元的生长.     

肺炎克雷伯菌生物膜相关研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是临床常见的院内感染致病菌,常引起泌尿系统、呼吸系统以及血流感染等。近年来,肺炎克雷伯菌生物膜引起的慢性感染日益得到重视,对肺炎克雷伯菌生物膜的研究也越来越多。为了适应周围环境,肺炎克雷伯菌会形成生物膜(biofilm),这是一种相对于浮游细菌的生存方式,生物膜由细菌及其自身分泌的代谢产物(胞外多糖、蛋白质、胞外DNA、脂质等)组成。生物膜状态下的肺炎克雷伯菌耐药性极强,容易逃避机体的免疫攻击,难以彻底清除,使临床抗感染变得更加棘手。本文就生物膜形成过程、生物膜状态细菌的耐药机制、生物膜测定方法以及生物膜防治作简要综述。     

Aggregation substance-mediated adherence of Enterococcus faecalis to immobilized extracellular matrix proteins

Aggregation substance (AS) of Enterococcus faecalis (E. faecalis), a sex pheromone plasmid encoded cell surface protein, mediates the formation of bacterial aggregates, thereby promoting plasmid transfer. The influence of pAD1-encoded AS, Asa1, on binding to immobilized extracellular matrix proteins was studied. The presence of AS increased enterococcal adherence to fibronectin more than eight-fold, to thrombospondin more than four-fold, to vitronectin more than three-fold, and to collagen type I more than two-fold (P<0.001). In contrast, binding to laminin and collagen type IV occurred independently of AS. Adherence of the constitutively AS expressing E. faecalis OG1X(pAM721) to immobilized fibronectin was found to be approximately five times higher than that of Staphylococcus aureus Cowan and approximately 30 times higher than that of Streptococcus bovis. Investigation of strains with various deletions within the structural gene of asa1 suggests that attachment to immobilized fibronectin is mainly mediated by amino acids within the variable region or by neighbouring residues. Thus, AS may promote adherence to injured epithelium and endothelium, where extracellular matrix proteins are exposed, thereby facilitating colonization and infection.     

Enterococcus spp. produces slime and survives in rat peritoneal macrophages

Enterococcal clinical isolates were investigated for the ability to form biofilm on inert surfaces, as a measure of slime production, in an attempt to find new possible virulence factors for these microorganisms. This property was commonly found among Enterococcus faecalis. Also E. faecium isolates were able to form biofilm, although to a lesser extent; for this species, however, biofilm formation seemed more frequently associated with isolates from infection rather than with environmental strains or isolates from healthy individuals. Biofilm formation was strongly affected by the presence of an additional carbohydrate source in the medium, or by iron deprivation, indicating a role of slime for survival in stressful conditions. Slime-producing E. faecalis were able to survive inside peritoneal macrophages for extended periods compared to slime-negative strains or to slime-positive bacteria grown in conditions depressing slime production. In particular, slime-producing and slime-negative cells showed a decrease of 1 and 2 log units, respectively, at 1 h after infection; slime-negative cells were then rapidly killed, with clearance of bacterial cells at 24 h. Slime-producing bacteria persisted up to 48 h, which was the last time point examined, as after that time viability of both infected and non-infected macrophages started to decline. Scanning electron microscopy observations showed the presence of abundant amorphous extracellular material, of possible polysaccharide nature, embedding bacterial cells to form a multilayered biofilm. Even in conditions not supporting biofilm formation, bacterial cells appeared capsulated, suggesting that capsule and slime might represent different structures. Genes belonging to the epa locus or to a putative icaA homolog did not seem to be involved in synthesis and export of slime.     

Enterococcus faecalis-mediated biomineralized biofilm formation on root canal dentine in vitro

Enterococcus faecalis is the most predominant bacteria in teeth with failed root canal therapy and is found to survive harsh conditions prevailing in the root canals of endodontically treated teeth. This study aims to investigate the interaction between E. faecalis and root canal dentine substrate. Towards this end, tooth specimens were prepared and divided into two groups. The tooth specimens in group 1 were incubated with E. faecalis for periods of 2-, 4-, and 6-week intervals and the chemical composition of the biofilm was determined using X-ray diffraction and Fourier transform infrared (FTIR) spectroscopy. The tooth specimens in group 2 were incubated with E. faecalis for a period of 6 weeks and the topography and ultrastructure of the biofilm were examined using scanning electron microscopy (SEM), light microscopy, and laser confocal scanning microscopy. The sediments formed from the bacterial interaction on the dentine (in group 1) were also examined by SEM and FTIR. These experiments highlighted different stages in the interaction of E. faecalis with root canal dentine. Further, a bacterial-induced apatite reprecipitation on mature biofilm was also observed. This ability of E. faecalis to form such calcified biofilm on root canal dentine may be a factor that contributes to their persistence after endodontic treatment.     

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).     

天然菌斑生物被膜中变形链球菌、远缘链球菌和血链球菌的荧光原位检测

目的 检测菌斑生物被膜初期形成过程中的变形链球菌(Streptpcoccus mutans)、远缘链球菌(Streptpcoccus sobrinus)和血链球菌(Streptpcoccus sanguis).方法 从植入釉质磨片表面获得完整的菌斑生物被膜标本,应用激光共聚焦扫描显微镜和荧光原位杂交技术相结合的方法,对天然菌斑生物被膜形成初期的变形链球菌、远缘链球菌和血链球菌进行原位的、实时的动态观察,通过连续断层扫描及三维重建,观察这3种菌在天然菌斑生物被膜中的空间分布,测量3种细菌的扫描厚度.实验中的扫描厚度结果采用方差分析方法进行统计处理,SPSS11.5统计软件辅助完成,α设在0.05.结果 在激光共聚焦扫描显微镜下观察生物被膜呈三维立体结构,形态多样,生物被膜内层和外层细菌较稀疏,中间层较密集,细菌之间可见许多黑色空隙,贯穿整个生物被膜.菌斑生物被膜变形链球菌、远缘链球菌和血链球菌在菌斑生物被膜形成初期的平均扫描厚度随时间延长而增加,1 h时平均扫描厚度分别为20.43、11.50、14.76 μm,到24 h时平均厚度最大,分别为70.25、75.40、79.98 μm.结论 应用荧光原位杂交技术结合激光共聚焦扫描显微镜,可以快速、灵敏地检测出菌斑生物被膜变形链球菌、远缘链球菌和血链球菌.     

Influence of organic acids present in the oral biofilm on the microtensile bond strength of adhesive systems to human dentin

This study evaluated the influence of organic acids present in the oral biofilm on the microtensile bond strength (μTBS) of adhesive systems to human dentin. Sixty occlusal dentin surfaces were wet ground with 600 grit SiC abrasive paper and divided into four groups according to the adhesive systems: Scotchbond Multipurpose (SMP), Adper Single Bond 2, Adper Scotchbond SE (ASE), and Clearfill SE Bond (CSE). After the adhesive systems were applied, a block of resin composite was built up on the dentin surfaces. After 24 h storage in distilled water at 37°C, the teeth were perpendicularly cut to obtain beams (1 mm(2)). For each adhesive system, the beams were divided into three groups according to storage media: artificial saliva (AS); propionic acid (PA), and lactic acid (LA). After 7 days storage at 37°C, the beams were submitted to μTBS testing. The μTBS ranged from 36.0 ± 1.6 (ASE-PA) to 52.5 ± 1.2 (CSE-AS). For all adhesive systems, the μTBS values after storage in PA were lower than those in AS. Except for the SMP, the values of μTBS after storage in LA were lower than those in AS. The adhesive ASE presented the lowest values of μTBs in the three media. The acids present in the oral biofilm may affect the bond strength of adhesive systems to human dentin.     

Curcumin suppresses Streptococcus mutans adherence to human tooth surfaces and extracellular matrix proteins

Streptococcus mutans is the key causative agent of caries and infective endocarditis. The first step in biofilm development and the consequent initiation of further disease is bacterial adherence to host cell surfaces. Therefore, the aim of this study was to evaluate the inhibitory effect of curcumin on S. mutans adherence to extracellular matrices and tooth surfaces. The effect of curcumin on the ability of S. mutans to adhere to glass surfaces coated with collagen and fibronectin was tested in order to determine whether the decrease of the bacterial adhesion by curcumin is achieved by hindering the bacteria in adhering to collagen and/or fibronectin. Also, human teeth inoculated with S. mutans were treated with curcumin in vitro in order to assess the relevance of the anti-adhesive effect to oral conditions in vivo. The minimum inhibitory concentration (MIC) at which curcumin completely inhibited bacterial growth was 128?μg/mL. The addition of curcumin below the MIC diminished bacterial adherence onto both collagen- and fibronectin-coated glass surfaces and human tooth surfaces. It appears that the anti-adhesive effect of curcumin against S. mutans is mediated through collagen and fibronectin. These results support the widespread use of curcumin as a food-based antimicrobial agent.     

Extracellular carbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcus epidermidis RP62A

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-beta-(1-->6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium.     

Tissue-specific adherent Enterococcus faecalis strains that show highly efficient adhesion to human bladder carcinoma T24 cells also adhere to extracellular matrix proteins

The ability of Enterococcus faecalis clinical isolates to adhere to immobilized extracellular matrixes (ECMs) coating the walls of microtiter plates was examined by microscopy. The ECMs consisted of fibronectin, laminin, collagen types I, II, IV, and V, fibrinogen, and lactoferrin. With the exception of fibrinogen, each isolate showed a different level of adherence to each of the ECMs. No significant level of adherence to fibrinogen was observed for any isolate. The tissue-specific adhesive strains AS11, AS12, AS14, AS15, HT11, and HT12, which showed highly efficient adherence to human bladder carcinoma T24 cells and human bladder epithelial cells, showed strong adherence to fibronectin, laminin, and collagen type I, II, IV, and V ECMs, and the levels were greater than 10(4) cells/mm2 of well surface coated by ECM. None of the isolates that showed little adherence to human bladder carcinoma T24 cells showed efficient adherence to all the ECMs. The levels of adherence of gelatinase-producing isolates to the collagens were lower than the levels of adherence of gelatinase-negative isolates. When tissue-specific adhesive strains that adhered strongly to each ECM were preincubated with fibronectin, the adherence of the strains to fibronectin was inhibited, but the adherence of the strains to collagen type IV was not inhibited. Likewise, preincubation with collagen type IV inhibited adherence to collagen type IV but not adherence to fibronectin. All of the E. faecalis isolates were shown to carry the ace gene by PCR analysis performed with specific primers for collagen binding domain A of ace. The ace gene encodes Ace (adhesin of collagen from enterococci). The prtF gene of group A streptococci, which encodes the fibronectin binding protein of group A streptococci, was not detected in the tissue-specific adhesive strains by Southern analysis performed with the prtF probe of the Streptococcus pyogenes JRS4 strain. Mutants with altered collagen binding were isolated by insertion of Tn916 into the chromosome of tissue-specific adhesive strain AS14. The number of mutant adhesive bacterial cells that adhered to collagen and also to laminin was 1 or 2 orders lower than the number observed for the wild-type strain, but the level of adherence to fibronectin remained the same as that of the wild-type strain.