摘要
背景:
铜绿假单胞菌可以引起各种急慢性感染,并且由该菌引起的院内获得性感染的发病率远高于社区获得性感染,其原因目前并未完全明确。在众多学说中,目前认为产生生物被膜是铜绿假单胞菌耐药和躲避人体免疫系统攻击的重要手段之一。藻酸盐是生物被膜的重要组成成分,在生物被膜病的发病机制中有重要作用。氧疗是临床上常用的治疗方法,目前还没有关于高氧如何影响铜绿假单胞菌产生生物被膜的报道。密度感应系统是细菌协调群体生物学效应的一种有效手段,对于细菌毒力、耐药性等方面有重要影响,但是否在生成生物被膜这种群体行为中起调控作用目前也争议颇多。Ⅲ型分泌系统是铜绿假单胞菌主要的毒力因素之一,在急性感染中发挥重要作用,对于Ⅲ型分泌系统和生物被膜形成这两种生物学行为的关系学术界也有一些分歧。
目的:
观察氧环境变化对铜绿假单胞菌产生生物被膜及藻酸盐的影响,并探讨密度感应系统中Las系统和Rhl系统在生物被膜调控中的作用、以及Ⅲ型分泌系统表达和生物被膜形成的相互关系。
方法:
1.随机扩增DNA多态性基因分型法对23株铜绿假单胞菌临床株进行基因型鉴定,以区别是否有来源于同一克隆的菌株。
2.23株基因型不同的铜绿假单胞菌临床株分别在无氧(0%氧浓度)、低氧(10%)、正常氧(20%)、高氧(30%、40%、50%、60%)七个不同的氧浓度下连续培养3天。用结晶紫染色法检测生物被膜产生量;对-羟基联苯法测定藻酸盐产生量。
3.硝酸银染色法观察标准菌株ATCC27853在不同氧浓度环境下产胞外多糖的时间窗。
4.观察ATCC27853在不同氧浓度环境下生长曲线的差异。
5.采用实时定量PCR测定23株临床菌株在不同氧浓度环境下密度感应系统中信号分子合成酶调控基因LasⅠ和RhlⅠ的表达。
6.Western blot方法观察23株临床菌株在不同氧浓度环境下分泌Ⅲ型分泌系统的代表毒素——胞外酶ExoS的情况。
结果:
1.基因分型显示23株菌株均能产生指纹图谱,分型率为100%。根据所得DNA片段数目及大小对铜绿假单胞菌菌株进行相似度比较,当使用随机引物A时,菌株1、3、6、20、22的DNA扩增条带相似,菌株4和23、8和21分别相似;其余菌株条带均不相同;再采用引物B对菌株1、3、4、6、8、20、21、22、23 DNA进行随机扩增,结果显示扩增条带均不相同。
2.检测23株临床株分别在七种氧浓度环境下培养3天的生物被膜产生量,结果表明在氧浓度为50%时生物被膜产生量达到最高值(4.05 OD/mg/ml),趋势性分析表明随着氧浓度的升高生物被膜产生量逐渐上升,相关性分析显示生物被膜产生量与氧浓度成正相关,R为0.455,P为0.000。
3.测定23株临床株分别在七种氧浓度环境下培养3天的藻酸盐生成量,结果表明藻酸盐产生量是在氧浓度为60%的条件下达到最高值(85.22 ug/mg蛋白),趋势性分析表明随着氧浓度的升高藻酸盐产生量逐渐上升,相关性分析显示藻酸盐产生量与氧浓度成正相关,R为0.367,P为0.000。
4.银染法观察ATCC27853产生胞外多糖的时间窗,结果显示在氧浓度分别为40%、50%、60%时,菌株培养一小时即可出现胞外多糖;氧浓度分别为20%、30%时,菌株需培养2小时以上才出现胞外多糖;氧浓度为10%时,胞外多糖最早出现时间为4小时;无氧条件下,菌株培养8小时出现胞外多糖。
5.ATCC27853生长曲线结果显示当氧浓度分别为0%、10%、20%时,菌株培养3小时开始由延迟期进入对数生长期;氧浓度为30%时,培养2小时进入对数生长期;氧浓度为40%、50%、60%时,菌株培养1小时即进入对数生长期。
6.实时定量PCR结果显示相关性分析未见LasⅠ、RhlⅠ的表达和氧浓度(R分别为0.025和-0.044,P大于0.05)、对应菌株生物被膜产生量(R分别为0.001和0.11,P大于0.05)、藻酸盐产生量(R分别为0.029和0.193,P大于0.05)有统计学意义。
7.Western blot结果表明菌株1、2、3、4、5、7、8、9、10、15、16、19、23共13株菌在氧浓度为20%条件下分泌胞外酶ExoS;当氧浓度为10%时,只有菌株15、16能够分泌ExoS;当氧浓度为30%时,菌株1、2、4、5、9、10、15能够分泌ExoS;氧浓度为0%、40%、50%、60%的条件下均未见菌株分泌ExoS。
结论:
1.铜绿假单胞菌在高氧环境下更容易产生生物被膜,且随着环境氧浓度的升高,生物被膜和藻酸盐产生量增加,表明高氧促进了铜绿假单胞菌产生生物被膜的能力,从而有利于铜绿假单胞菌在人体内的耐药和寄植,不利于肌体对病原菌的清除和抗生素杀菌作用的发挥,所以针对有铜绿假单胞菌感染高危因素的患者应该避免过高浓度的氧疗。
2.在不同氧浓度环境下虽然铜绿假单胞菌产生生物被膜的能力有显著差异,但是生物被膜形成的早期密度感应系统中Las系统和Rhl系统关键基因表达无明显趋势性变化,因此Las系统和Rhl系统可能在生物被膜形成早期阶段因为菌群数量处于低水平而未参与生物被膜的调控过程。
3.随着氧浓度的升高铜绿假单胞菌产生生物被膜增多,但是Ⅲ型分泌系统的代表毒素胞外酶ExoS分泌下调,表明铜绿假单胞菌生物被膜产生增多的同时Ⅲ型分泌系统的表达下调,侵袭力下降。
Backgroud:
The morbidity of hospital acquired infection of Pseudomonas aeruginosa is high.But the real cause is unknown.Producing biofilm barrier is an important way for P.aeruginosa to develop resistance and elude immune attack. Being the important component of biofilm,alginate play a significant role in bacterial biofilm related infection.Oxygen therapy is a usual medical management in hospital,which can offer hyperoxic situation in respiratory tract.Oxygen situation would influence the production of biofilm.Now there are not any reports about the relationship between hyperoxia and biofilm production of P.aeruginosa.On the other hand,Quorum Sensing System is a generic regulatory mechanism employed by P.aeruginosa,which consists of two separate but interrelated system,Las and Rhl.TypeⅢsecretion system is a kind of bacterial device for close combat with cells of their eukaryotic host,which allow bacteria to inject bacterial proteins across the eukaryotic cell membrane to destroy or subvert the target cell.But at present the scholars have not agreed with whether Quorum Sensing System and TypeⅢsecretion system play a part in the process of biofilm production.
Objective:
The aims of this study were to investigate the relationship between oxygen concentration and biofilm production of P.aeruginosa,and whether Las/Rhl system take effects on biofilm.Furthermore,the relationship between TypeⅢsecretion system and biofilm was explored.
Methods:
1.The genotypes of 23 clinical strains of P.aeruginosa were identified by random amplified polymorphic DNA assay.
2.23 genetically different clinical strains of P.aeruginosa were cultured at different levels of environmental oxygen respectively.Then biofilm mass was quantified by crystal violet dye,and alginate by meta-hydroxydiphenyl reaction.
3.Silver nitrate staining to determine the time window of extrapolysaccharide production under different levels of environmental oxygen.
4.Growth curves of ATCC27853 under different levels of environmental oxygen were made.
5.The expression level of LasI and RhlI was detected by real-time polymerase chain reaction.
6.The secretion of exoenzyme S was examined by western blotting assay.
Results:
1.The genotype of 23 clinical strains of P.aeruginosa were assessed to be different by two random primers.
2.For quantifying the amount of biofilm,the highest amount of biofilm (4.05 OD/mg/ml) was produced when the oxygen concentration was at 50%.There was positive correlation between the amount of biofim production and oxygen concentration(R=0.455,P=0.000).
3.For quantifying the amount of alginate,the highest amount of alginate (85.22 ug/mg protein) was produced when the oxygen concentration was 60%. Linear correlation analysis indicated that a significant positive correlation existed between the oxygen concentration and alginate production(r=0.367,P=0.000).
4.The production of extrapolysaccharide by ATCC27853 was not visualized until culture for 1 hour at＞40%environmental oxygen concentration,2 hours for 20%and 30%oxygen concentrations,4 hours for 10%oxygen concentration, and 8 hours for anaerobic conditions.
5.The results of growth curve of ATCC27853 showed that when the concentration of oxygen was 0%,10%,or 20%,the bacteria culture required＞3 hours to shift from the lag period to the logarithmic growth phase,2 hours for 30%oxygen concentration,and 1 hour for＞40%oxygen concentration.
6.The results of real-time polymerase chain reaction did not suggest any correlation between the expression level of LasI and oxygen concentration,or the production of biofilm and alginate.It' s the same for RhlI.
7.The detection of exoenzyme S showed the optimal oxygen concentration range for exoenzyme S secretion of P.aeruginosa was from 10%to 30%.
Conclusion:
1.Hyperoxia promoted the ability of producing biofilm and alginate of P.aeruginosa,which is in favor of resistance and colonization.To avoid and manage P.aeruginosa infection should obviate unnecessary hyperoxia therapy.
2.Las/Rhl System may not take effects on biofilm production at earlier period,maybe for low level of bacteria amount.
3.The results of exoenzyme S showed that although hyperoxia promoted the ability of producing biofilm of P.aeruginosa,TypeⅢsecretion system was down regulated,which may mean that the expression of TypeⅢsecretion system was repressed by the overproduction of biofilm.
引文
[1]Osmon S,Ward S,Fraser VJ,et al.Hospital mortality for patients with bacteremia due to Staphylococcus aureus or Pseudomonas aeruginosa[J].Chest,2004,125(2):607-616.
[2]Driscoll JA,Brody SL,Kollef MH.The Epidemiology,Pathogenesis and Treatment of Pseudomonas aeruginosa Infections[J].Drugs,2007,67(3):351-68.
[3]孙景勇,倪语星。Mohnarin 2006～2007年度报告:华东地区细菌耐药监测[J]。中国抗生素杂志。2008,10:635-639。
[4]汪复。2006年中国CHINET细菌耐药性监测[J]。中国感染与化疗杂志。2008,1:1-9。
[5]Gaynes R,Edwards JR,National Nosocomial Infections Surveillance System.Overview of nosocomial infections caused by gram-negative bacilli[J].Clin Infect Dis.2005,41:848-854.
[6]National Nosocomial Infections Surveillance System Report.National Nosocomial Infections Surveillance(NNIS) System Report,data summary from January 1992 through June 2004,issued October 2004[J].Am J Infect Control 2004;32:470-485.
[7]王辉,陈民钧。1994～2001年中国重症监护病房非发酵糖细菌的耐药变迁[J]。中华医学杂志,2003,83:385-390。
[8]del Pozo JL,Patel R.The Challenge of Treating Biofilm-associated Bacterial Infections[J].Clin Pharmacol Ther.2007,82(2):204-209.
[9]Stoodley P,Sauer K,Davies DG,et al.Biofilms as complex differentiated communities[J].Annu Rev Microbiol.2002,56:187-209.
[10]Sauer K,Camper AK,Ehrlich GD,et al.Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[J].J Bacteriol.2002,184(4):1140-1154.
[11]Hall-Stoodley L,Stoodley P.Biofilm formation and dispersal and the transmission of human pathogens[J].Trends Microbiol.2005,13(1):7-10.
[12]Gacesa P.Bacterial alginate biosynthesis—recent progress and future prospects[J].Microbiology.1998,144:1133-1143.
[13]DeVries CA,Ohman DE.Mucoid-to-nonmucoid conversion in alginate producing Pseudomonas aeruginosa often results from spontaneous mutations in algT,encoding a putative alternate sigma factor,and shows evidence for autoregulation[J].J Bacteriol,1994,176(21):6677-6687.
[14]Ryder C,Byrd M,Wozniak DJ.Role of polysaccharides in Pseudomonas aeruginosa biofilm development[J].Curr Opin Microbiol.2007,10(6):644-648.
[15]Leid JG,Willson CJ,Shirtliff ME,et al.The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing[J].J Immunol.2005 Dec 1;175(11):7512-7518.
[16]Xu KD,McFeters GA,Stewart PS.Biofilm resistance to antimicrobial agents[J].Microbiology.2000,146:547-549.
[17]Davies D.Understanding biofilm resistance to antibacterial agents[J].Nat Rev Drug Discov.2003,2(2):114-122.
[18]Parsek MR,Singh PK.Bacterial biofilms:an emerging link to disease pathogenesis[J].Annu Rev Microbiol.2003,57:677-701.
[19]Xavier JB,Picioreanu C,Rani SA,et al.Biofilm-control strategies based on enzymic disruption of the extracellular polymeric substance matrix--a modelling study[J].Microbiology 151:3817-3832.
[20]Alkawash MA,Soothill JS,Schiller NL.Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms[J].APMIS.2006,114(2):131-138.
[21]Cirioni O,Giacometti A,Ghiselli R,et al.RNAⅢ-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections[J].J Infect Dis.2006,193(2):180-186.
[22]Raad Ⅱ,Fang X,Keutgen XM,et al.The role of chelators in preventing biofilm formation and catheter-related bloodstream infections[J].Curr Opin Infect Dis.2008,21(4):385-392.
[23]Caubet R,Pedarros-Caubet F,Chu M,et al.A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms[J].Antimicrob Agents Chemother.2004,48:4662-4664.
[24]Smith RS,Iglewski BH.P.aeruginosa quorum-sensing systems and virulence[J].Curr Opin Microbiol.2003.6(1):56-60.
[25]Heurlier K,Denervaud V,Haas D.Impact of quorum sensing on fitness of Pseudomonas aeruginosa[J].Int J Med Microbiol.2006.296(2-3):93-102.
[26]Pearson J P,Feldman M,Iglewski BH,et al.Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection[J].Infect Immun.2000.68:4331-4334.
[27]Bjarnsholt T,Jensen PΦ,BurmΦlle M,et al.Pseudomonas aeruginosa tolerance to tobramycin,hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent[J].Microbiology.2005.151:373-383.
[28]Davies DG,Parsek MR,Pearson JP,et al.The involvement of cell-to-cell signals in the development of a bacterial biofilm[J].Science.1998,280:295-298.
[29]Heydorn A,Ersb0ll B,Kato J,et al.Statistical analysis of Pseudomonas aeruginosa biofilm development:impact of mutations in genes involved in twitching motility,cell-to-cell signaling,and stationary-phase sigma factor expression[J].Appl Environ Microbiol.2002.68(4):2008-2017.
[30]Purevdorj B,Costerton JW,Stoodley P.Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms[J].Appl Environ Microbiol.2002.68(9):4457-4464.
[31]Shime N,Sawa T,Fujimoto J,et al.Therapeutic administration of anti-PcrV F(ab)2 in sepsis associated with Pseudomonas aeruginosa[J].J Immunol.2001,167(10):5880-5886.
[32]Roy-Burman A,Savel RH,Racine S.Type Ⅲ secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections[J].J Infect Dis.2001,183:1767-1774.
[33]Sadikot RT,Blackwell TS,Christman JW,et al.Pathogen-Host Interactions in Pseudomonas aeruginosa Pneumonia[J].Am J Respir Crit Care Med.2005,171(11):1209-1223.
[34]Kaufman MR,Jia J,Zeng L,et al.Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS[J].Microbiology.2000,146:2531-2541.
[35]Wu W,Badrane H,Arora S,et al.MucA-Mediated Coordination of Type Ⅲ Secretion and Alginate Synthesis in Pseudomonas aeruginosa[J].J Bacteriol.2004,186(22):7575-7585.
[36]Mikkelsen H,Bond NJ,Skindersoe ME,et al.Biofilms and type Ⅲ secretion are not mutually exclusive in Pseudomonas aeruginosa[J].Microbiology.2009,155:687-698.
[37]Gaynes R,Edwards JR,and the National Nosocomial Infections Surveillance System.Overview of nosocomial infections caused by gram-negative bacilli[J].Clin Infect Dis.2005,41(6):848-854.
[38]Roulenti D,Rello J.Gram-negative bacterial pneumonia:aetiology and management[J].Curr Opin Pulm Med.2006,12:198-204.
[39]Reboli AC,Houston ED,Monteforte JS,et al.Discrimination of epidemic and sporadic isolates of Acinetobacter baumannii by repetitive element PCR-mediated DNA fingerprinting[J].J Clin Microbiol.1994,2(11)2635-2640.
[40]Knezevic P,Petrovic O.A colorimetric microtiter plate method for assessment of phage effect on Pseudomonas aeruginosa biofilm[J].J Microbiol Methods.2008,74:114-118.
[41]谢崧;钱桂生;杨晓静。一种用于研究的经济实用缺氧小容器[J]。第三军医大学学报,1998,1:92。
[42]Bradford,MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Biochem.1976,72:248-254.
[43]Blumenkrantz N,Asboe-Hansen G.New method for quantitative determination of uronic acids[J].Anal Biochem.1973,54:484-489.
[44]Passariello C,Berlutti F,Selan L,et al.A rapid staining procedure to demonstrate glycocalyx production and bacterial biofilms[J].New Microbiol.1994,17:225-230.
[45]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real2time quantitative PCR and the 2 ~(-ΔΔCt)Method[J].Methods,2001,25(4):402-408.
[46]Kobayashi H.Airway biofilms:implications for pathogenesis and therapy of respiratory tract infections[J].Treat Respir Med.2005,4:241-253.
[47]Leid JG,Willson CJ,Shirtliff ME,et al.The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing[J].J Immunol.2005,175:7512-7518.
[48]Williams JGK,Kubelik AR,Livak KJ,et al.DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J].Nuc Aci Res.1990,18:6531-6535.
[49]Bazylinski DA,Soohoo CK,Hollocher TC.Growth of Pseudomonas aeruginosa on nitrous oxide[J].Appl Environ Microbiol.1986,51:1239-1246.
[50]Worlitzsch D,Tarran R,Ulrich M,et al.Effects of reduced mucus oxwen concentration in airway Pseudomonas infections of cystic fibrosis patients[J].J Clin Invest.2002,109:317-325.
[51]Favre-Bonte,K(o|¨)hler T,Delden CV.Biofilm formation by Pseudomonas aeruginosa:role of the C4-HSL cell-to-cell signal and inhibition by azithromycin[J].J Antimicrob Chemother.2003,52:598-604.
[52]Hogardt M,Roeder M,Schreff AM,et al.Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS[J].Microbiology.2004,150(4):843-851.
[53]Wu W,Badrane H,Arora S,et al.MucA-mediated coordination of type Ⅲ secretion and alginate synthesis in Pseudomonas aeruginosa[J].J Bacteriol.2004,186(22):7575-7585.
[54]Kuchma SL,Connolly JP,O'Toole GA.A three-component regulatory system regulates biofilm maturation and type Ⅲ secretion in Pseudomonas aeruginosa[J].J Bacteriol.2005,187(4):1441-1454.
[1]Stoodley P,Sauer K,Davies DG,et al.Biofilms as complex differentiated communities[J].Annu Rev Microbiol.2002,56:187-209
[2]Sauer K,Camper AK,Ehrlich GD,et al.Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm[J].J Bacteriol.2002,184(4):1140-1154.
[3]Hall-Stoodley L,Stoodley P.Biofilm formation and dispersal and the transmission of human pathogens[J].Trends Microbiol.2005,13(1):7-10.
[4]Palmer J,Flint S,Brooks J.Bacterial cell attachment,the beginning of a biofilm[J].J Ind Microbiol Biotechnol.2007 Sep;34(9):577-588.
[5]Ramsey MM,Whiteley M.Pseudomonas aeruginosa attachment and biofilm development in dynamic environments[J].Mol Microbiol.2004 Aug;53(4):1075-1087.
[6]Otto K,Silhavy TJ.Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway[J].Proc Natl Acad Sci USA.2002 Feb 19;99(4):2287-2292.
[7]Price NL,Raivio TL.Characterization of the Cpx regulon in Escherichia coli strain MC4100[J].J Bacteriol.2009 Mar;191(6):1798-1815.
[8]Dorel C,Lejeune P,Rodrigue A.The Cpx system of Escherichia coli,a strategic signaling pathway for confronting adverse conditions and for settling biofilm communities[J]?Res Microbiol.2006 May;157(4):306-314.
[9]Otto K,Silhavy TJ.Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway[J].Proc Natl Acad Sci USA.2002 Feb 19;99(4):2287-2292.
[10]Dorel C,Vidal O,Prigent-Combaret C,et al.Involvement of the Cpx signal transduction pathway of E.coli in biofilm formation[J].FEMS Microbiol Lett.1999 Sep 1;178(1):169-175.
[11]Batchelor E,Goulian M.Imaging OmpR localization in Escherichia coli.Mol Microbiol[J].2006 Mar;59(6):1767-1778.
[12]Jubelin G,Vianney A,Beloin C,et al.CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli[J].J Bacteriol.2005 Mar;187(6):2038-2049.
[13]Cai SJ,Inouye M.EnvZ-OmpR interaction and osmoregulation in Escherichia coli[J].J Biol Chem.2002 Jul 5;277(27):24155-24161.
[14]Donlan RM.Biofilms:microbial life on surfaces[J].Emerg Infect Dis.2002 Sep;8(9):881-890
[15]Reisner A,Haagensen JA,Schembri MA,et al.Development and maturation of Escherichia coli K-12 biofilms[J].Mol Microbiol.2003 May;48(4):933-946.
[16]Watnick PI,Kolter R.Steps in the development of a Vibrio cholerae El Tor biofilm[J].Mol Microbiol.1999 Nov;34(3):586-595.
[17]O'Toole GA,Kolter R.Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development[J].Mol Microbiol.1998 Oct;30(2):295-304.
[18]Heydorn A,ErsbΦll B,Kato J,et al.Statistical analysis of Pseudomonas aeruginosa biofilm development:impact of mutations in genes involved in twitching motility,cell-to-cell signaling,and stationary-phase sigma factor expression[J].Appl Environ Microbiol.2002 Apr;68(4):2008-17.
[19]O'Toole GA,Gibbs KA,Hager PW,et al.The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa[J].J Bacteriol.2000 Jan;182(2):425-431.
[20]Beenken KE,Dunman PM,McAleese F,et al.Global gene expression in Staphylococcus aureus biofilms[J].J Bacteriol.2004 Jul;186(14):4665-4684.
[21]Gotz F.Staphylococcus and biofilms[J].Mol Microbiol.2002 Mar;43(6):1367-1378.
[22]Jager S,Mack D,Rohde H,et al.Disintegration of Staphylococcus epidermidis biofilms under glucose-limiting conditions depends on the activity of the alternative sigma factor sigmaB[J].Appl Environ Microbiol.2005 Sep;71(9):5577-5581.
[23]Cramton SE,Ulrich M,Gotz F,et al.Anaerobic conditions induce expression of polysaccharide intercellular adhesin in Staphylococcus aureus and Staphylococcus epidermidis[J].Infect Immun.2001 Jun;69(6):4079-4085.
[24]Molle V,Fujita M,Jensen ST,et al.The SpoOA regulon of Bacillus subtilis[J].Mol Microbiol.2003 Dec;50(5):1683-1701.
[25]Hamon MA,Lazazzera BA.The sporulation transcription factor SpoOA is required for biofilm development in Bacillus subtilis[J].Mol Microbiol.2001 Dec;42(5):1199-1209.
[26]Srivastava S,Yadav A,Seem K,et al.Effect of high temperature on Pseudomonas putida NBRI0987 biofilm formation and expression of stress sigma factor RpoS[J].Curr Microbiol.2008 May;56(5):453-457.
[27]Stanley NR,BrittonRA,Grossman AD,et al.Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays[J].J Bacteriol.2003 Mar;185(6):1951-1957.
[28]Jackson DW,Simecka JW,Romeo T.Catabolite repression of Escherichia coli biofilm formation[J].J Bacteriol.2002 Jun;184(12):3406-3410.
[29]Waters CM,Lu W,Rabinowitz JD,et al.Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cyclic di-GMP levels and repression of vpsT[J].J Bacteriol.2008 Apr;190(7):2527-2536.
[30]Davies DG,Parsek MR,Pearson JP,et al.The involvement of cell-to-cell signals in the development of a bacterial biofilm[J].Science.1998,280:295-298.
[31]Sauer K,Cullen MC,Rickard AH,et al.Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAOl biofilm[J].J Bacteriol.2004 Nov;186(21):7312-7326.
[32]Barraud N,Hassett DJ,Hwang SH,et al.Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa[J].J Bacteriol.2006 Nov;188(21):7344-7353.
[33]Jackson DW,Suzuki K,Oakford L,et al.Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli[J].J Bacteriol.2002 Jan;184(1):290-301.
[34]Morgan R,Kohn S,Hwang SH,et al.BdlA,a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa[J].J Bacteriol.2006 Nov;188(21):7335-7343.
[35]D'Argenio DA,Miller SI.Cyclic di-GMP as a bacterial second messenger[J].Microbiology.2004 Aug;150(8):2497-2502.
[36]Yan W,Qu T,Zhao H,et al.The effect of c-di-GMP(3'-5'-cyclic diguanylic acid)on the biofilm formation and adherence of Streptococcus mutans[J].Microbiol Res.2009 Jan 30.
[37]Goller CC,Romeo T.Environmental influences on biofilm development[J].Curr Top Microbiol Immunol.2008;322:37-66.
[1]Stoodley P,Sauer K,Davies DG,et al.Biofilms as complex differentiated communities[J].Annu Rev Microbiol.2002,56:187-209.
[2]Xu KD,McFeters GA,Stewart PS.Biofilm resistance to antimicrobial agents[J].Microbiology.2000,146:547-549.
[3]del Pozo JL,Patel R.The Challenge of Treating Biofilm-associated Bacterial Infections[J].Clin Pharmacol Ther.2007,82(2):204-209.
[4]Davies D.Understanding biofilm resistance to antibacterial agents[J].Nat Rev Drug Discov.2003,2(2):114-122.
[5]Parsek MR,Singh PK.Bacterial biofilms:an emerging link to disease pathogenesis[J].Annu Rev Microbiol.2003,57:677-701.
[6]Mack D,Rohde H,Harris LG,et al.Biofilm formation in medical device-related infection[J].lnt J Artif Organs.2006 Apr;29(4):343-359.
[7]Kobayashi H.Airway biofilm disease[J].Int J Antimicrob Agents.2001 May;17(5):351-356.
[8]Kobayashi,H.Biofilm disease:its clinical manifestation and therapeutic possibilities of macrolides[J].Am J Med.1995,99:26S-30S.
[9]Mitsuya Y,Kawai S,Kobayashi H.Influence of macrolides on guanosine diphospho-D-mannose dehydrogenase activity in Pseudomonas biofilm[J].J Infect Chemother.2000 Mar;6(1):45-50.
[10]Kawamura-Sato K,Iinuma Y,Hasegawa T,et al.Effect of subinhibitory concentrations of macrolides on expression of flagellin in Pseudomonas aeruginosa and Proteus mirabilis[J].Antimicrob Agents Chemother.2000 Oct;44(10):2869-2872.
[11]Wozniak DJ,Keyser R.Effects of subinhibitory concentrations of macrolide antibiotics on Pseudomonas aeruginosa[J].Chest.2004 Feb;125(2 Suppl):62S-69S.
[12]Hajdu S,Lassnigg A,Graninger W,et al.Effects of vancomycin,daptomycin,fosfomycin,tigecycline,and ceftriaxone on Staphylococcus epidermidis biofilms[J].J Orthop Res.2009 Apr 24.
[13]Mikuniya T,Kato Y,Ida T,et al.Treatment of Pseudomonas aeruginosa biofilms with a combination of fluoroquinolones and fosfomycin in a rat urinary tract infection model[J].J Infect Chemother.2007 Oct;13 (5):285-290.
[14]Morikawa K,Nonaka M,Yoshikawa Y,et al.Synergistic effect of fosfomycin and arbekacin on a methicillin-resistant Staphylococcus aureus-induced biofilm in a rat model[J].Int J Antimicrob Agents.2005 Jan;25(1):44-50.
[15]Cernohorsk(?)L,Votava M.Antibiotic synergy against biofilm-forming Pseudomonas aeruginosa[J].Folia Microbiol.2008;53(1):57-60.
[16]Tre-Hardy M,Vanderbist F,Traore H,et al.In vitro activity of antibiotic combinations against Pseudomonas aeruginosa biofilm and planktonic cultures[J].Int J Antimicrob Agents.2008 Apr;31(4):329-336.
[17]Smith RS,Iglewski BH.P.aeruginosa quorum-sensing systems and virulence[J].Curr Opin Microbiol.2003.6(1):56-60.
[18]Jayaraman A,Wood TK.Bacterial quorum sensing:signals,circuits,and implications for biofilms and disease[J].Annu Rev Biomed Eng.2008;10:145-167.
[19]Davies DG,Parsek MR,Pearson JP,et al.The involvement of cell-to-cell signals in the development of a bacterial biofilm[J].Science.1998,280:295-298.
[20]Cirioni O,Giacometti A,Ghiselli R,et al.RNAⅢ-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections[J].J Infect Dis.2006 Jan 15;193(2):180-186.
[21]Bjarnsholt T,Givskov M.Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens[J].Philos Trans R Soc Lond B Biol Sci.2007 Jul 29:362(1483):1213-1222.
[22]Rice SA,McDougald D,Kumar N,et al.The use of quorum-sensing blockers as therapeutic agents for the control of biofilm-associated infections[J].Curr Opin Investig Drugs.2005 Feb;6(2):178-184.
[23]de Nys R,Givskov M,Kumar N,et al.Furanones[J].Prog Mol Subcell Biol.2006;42:55-86.
[24]Nalca Y,J(a|¨)nsch L,Bredenbruch F,Geffers R,et al.Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PA01:a global approach[J].Antimicrob Agents Chemother.2006 May;50(5):1680-1688.
[25]Boyd A,Chakrabarty AM.Role of alginate lyase in cell detachment of Pseudomonas aeruginosa[J].Appl Environ Microbiol.1994 Jul;60(7):2355-2359.
[26]Nakamoto DA,Rosenfield ML,Haaga JR,et al.Young Investigator Award.In vivo treatment of infected prosthetic graft material with urokinase:an animal model[J].J Vasc Interv Radiol.1994 Jul-Aug;5(4):549-552.
[27]Selan L,Berlutti F,Passariello C,et al.Proteolytic enzymes:a new treatment strategy for prosthetic infections[J]?Antimicrob Agents Chemother.1993 Dec;37(12):2618-2621.
[28]Nemoto K,Hirota K,Ono T,et al.Effect of Varidase(streptokinase)on biofilm formed by Staphylococcus aureus[J].Chemotherapy.2000 Mar-Apr;46(2):111-115.
[29]Ramasubbu N,Thomas LM,Ragunath C,et al.Structural analysis of dispersin B,a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans[J].J Mol Biol.2005 Jun 10;349(3):475-486.
[30]Donelli G,Francolini I,Romoli D,et al.Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes[J].Antimicrob Agents Chemother.2007 Aug;51(8):2733-2740.
[31]Barraud N,Hassett DJ,Hwang SH,et al.Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa[J].J Bacteriol.2006 Nov;188(21):7344-7353.
[32]Sauer K,Cullen MC,Rickard AH,et al.Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PA01 biofilm[J].J Bacteriol.2004 Nov;186(21):7312-7326.
[33]Wang J,Huang N,Yang P,et al.The effects of amorphous carbon films deposited on polyethylene terephthalate on bacterial adhesion[J].Biomaterials.2004 Jul;25(16):3163-3170.
[34]Stoodley P,deBeer D,Lappin-Scott HM.Influence of electric fields and pH on biofilm structure as related to the bioelectric effect[J].Antimicrob Agents Chemother.1997 Sep;41(9):1876-1879.
[35]Caubet R,Pedarros-Caubet F,Chu M,et al.A radio frequency electric current enhances antibiotic efficacy against bacterial biofilms[J].Antimicrob Agents Chemother.2004 Dec;48(12):4662-4664.
[36]Ensing GT,Neut D,van Horn JR,et al.The combination of ultrasound with antibiotics released from bone cement decreases the viability of planktonic and biofilm bacteria:an in vitro study with clinical strains[J].J Antimicrob Chemother.2006 Dec;58(6):1287-1290.
