细菌群体感应

Antimicrobial resistance,respiratory tract infections and role of bio films in lung infections in cystic fibrosis patients ☆Oana Ciofu a ,Tim Tolker-Nielsen a ,Peter Østrup Jensen a ,Hengzhuang Wang a ,b ,Niels Høiby a ,b ,⁎a Institute of International Health,Immunology and Microbiology,Costerton Bio film Centre,University of Copenhagen,Copenhagen,Denmark bDepartment of Clinical Microbiology,Rigshospitalet,Copenhagen,Denmarka b s t r a c ta r t i c l e i n f o Available online 2December 2014Keywords:Pseudomonas aeruginosa Bio filmCystic fibrosisLung infection is the main cause of morbidity and mortality in patients with cystic fibrosis and is mainly dominat-ed by Pseudomonas aeruginosa .The bio film mode of growth makes eradication of the infection impossible,and it causes a chronic in flammation in the airways.The general mechanisms of bio film formation and antimicrobial tolerance and resistance are reviewed.Potential anti-bio film therapeutic targets such as weakening of bio films by quorum-sensing inhibitors or antibiotic killing guided by pharmacokinetics and pharmacodynamics of antibiotics are presented.The vicious circle of adaptive evolution of the persisting bacteria imposes important therapeutic challenges and requires development of new drug delivery systems able to reach the different niches occupied by the bacteria in the lung of cystic fibrosis patients.©2014Elsevier B.V.All rights reserved.Contents1.Cystic fibrosis and chronic lung infection ..................................................82.The occurrence and architecture of bacterial bio films –general aspects ....................................83.High cell density and quorum-sensing in bio films ..............................................104.Tolerance of bio films to antimicrobials and immune system .........................................114.1.Restricted penetration .......................................................114.2.Differential physiological activity ..................................................114.3.Differential expression of speci fic genes in bio film ..........................................124.4.Persister cells ...........................................................125.Hydroxyl radical formation during antibiotic treatment of bio films ......................................126.Antibiotic resistance and mutability in bio films ...............................................137.Bio film treatment strategies ........................................................137.1.Quorum-sensing inhibitors .....................................................137.2.Antibiotic killing in bio films:pharmacokinetic and pharmacodynamic issues in Pseudomonas aeruginosa bio film lung infections.......147.2.1.PK/PD of colistin on P .aeruginosa bio films ..........................................147.2.2.PK/PD of β-lactam antibiotics on P .aeruginosa bio films ....................................158.Actual treatment of the P .aeruginosa infection in CF patients .........................................159.Challenges of the treatment of P .aeruginosa airways infection in CF ......................................179.1.P.aeruginosa in the airways of CF patients:sites,niches and bio film ..................................179.2.P .aeruginosa adaptation to the CF lung:bacterial heterogeneity ....................................1810.Therapeutic strategies to circumvent these challenges ............................................1811.Conclusions ...............................................................19Acknowledgement ..............................................................19References (19)Advanced Drug Delivery Reviews 85(2015)7–23☆This review is part of the Advanced Drug Delivery Reviews theme issue on "Inhaled antimicrobial chemotherapy for respiratory tract infections:Successes,challenges and the road ahead".⁎Corresponding author at:Department of Clinical Microbiology,9301,University Hospital Rigshospitalet,Juliane Mariesvej 28,2100Copenhagen Ø,Denmark.Tel.:+4535457778;fax:+4535456412.E-mail address:hoiby@hoibyniels.dk (N.Høiby)./10.1016/j.addr.2014.11.0170169-409X/©2014Elsevier B.V.All rightsreserved.Contents lists available at ScienceDirectAdvanced Drug Delivery Reviewsj o u r na l h om e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a d d r1.Cysticfibrosis and chronic lung infectionCysticfibrosis(CF)is a congenital,recessively inherited disorder which affects one out of2000newborns,in Caucasian populations and affects around70,000people around the world.There are considerable variations through Europe from as high as1in1400live births in Ireland,to1in25,000in Finland[1].The genetic background is one of N2000mutations in the cysticfibrosis transmembrane conductance regulator gene(CFTR)in each of the two chromosomes7which gives malfunction of the chloride channel in CF patients().If intensively treated,the mean expected lifetime of CF patients is N35 years and in some centres N50years(http://www.cysticfibrosis.ca/). Any progress in treatment is therefore important for CF patients.CF is a multiorgans disease affecting the airways,pancreas,small intestine,liver,reproductive tract and the sweat glands.The clinical symptoms from the lungs are viscid mucus and respiratory infections leading to chronic bronchial disease and bronchiectasis.The malfunction of the chloride channel in CF patients leads to decreased volume of the paraciliaryfluid in the lower respiratory tract and to impaired mucus detachment from the submucosal gland ducts in the conducting airways[2]and that in turn leads to impaired mucociliary clearance of inhaled microbes[3].This impairment of the non-inflammatory defense mechanism of the respiratory tract leads to early recruitment of the inflammatory defense mechanisms notably polymorphonuclear leukocytes(PMN)and antibodies[4–6].CF patients,therefore,from early childhood suffer from recurrent and chronic respiratory tract infections characterized by PMN inflammation. In spite of the inflammatory response and intensive antibiotic therapy, however,infections caused by P.aeruginosa,the Burkholderia cepacia complex(mostly B.multivorans and B.cenocepacia)and Achromobacter xylosoxidans,persist and lead to respiratory failure and lung transplanta-tion or death of the patients[7].Several other species including Staphylo-coccus aureus,Haemophilus influenzae,Stenotrophomonas maltophilia and Mycobacteria other than tuberculosis and Aspergillus fumigatus also contribute to the morbidity and mortality[7].Chronic P.aeruginosa lung infection is the cause of much of the morbidity and most of the mortality in CF patients.About80%of CF adults suffer from chronic P.aeruginosa infection.Previously50%of CF patients would succumb within5years after onset of the chronic P.aeruginosa infection.This prognosis has been changed completely by the introduction of intensive early eradication therapy and many patients will therefore not contract the chronic infection during childhood anymore[8].Adaptive mechanisms of P.aeruginosa exists which explain why this pathogen is able to survive and persist for several decades in the respi-ratory tract of CF patients in spite of the defense mechanisms of the host and intensive antibiotic therapy.P.aeruginosa is able to survive by switching to the biofilm mode of growth which provides tolerance to the inflammatory defense mechanism,to the aerobic respiratory zone and to the conductive zone of the lungs which contain anaerobic spu-tum,and to the antibiotic therapy[9–12].The conductive and respirato-ry zones of the lung are presented in Fig.1.During the adaptation mucoid(biofilm mode of growth)and non-mucoid phenotypes are split off due to mutations(Table1).Microscopy pictures of P.aeruginosa biofilms from the lung of CF patients are presented in Fig.2A,B,C.The biofilm strategy is also used by Burkholderia,sp A.xylosoxidans and Stenotrophomonas species[13](Fig.2D.E,F)although this has been questioned by some authors suggesting that Burkholderia species may exist predominantly as single cells in CF lungs[14].2.The occurrence and architecture of bacterialbiofilms–general aspectsMicrobial biofilms are ubiquitous in nature and of importance for a number of environmental processes.However,biofilms are also the un-derlying cause of persistent infections in various parts of the human body,including the teeth[15],urinary tract[16],heart valves[17],CF lungs[11],middle ears[18],nasal passages and sinus cavities[19], bones[20],prostate[21],and chronic wounds[22].In addition,biofilm formation on implants and catheters gives rise to problematic infections in connection with the use of medical devices including urinary cathe-ters,central venous catheters,fracturefixation devices,dental implants, joint prostheses,vascular grafts,cardiac pacemakers,breast implants, mechanical heart valves,penile implants,and heart assist devices[23].The formation and maintenance of biofilms depends critically on the presence of bacteria-to-bacteria interconnecting extracellular substances that serve as a biofilm matrix[24].Many kinds of exopolymers, e.g.polysaccharide,protein,and DNA,may be part of the matrix of biofilms.In addition,outer membrane proteins and a variety of bacterial cell appendages such asfimbriae,pili,andflagella may also have bacteria-to-bacteria interconnecting functions,and can therefore be con-sidered part of the biofilm matrix.The components of the biofilm matrix are usually,but not always,produced by the bacteria themselves.A single bacterial species can produce several different biofilm matrix com-ponents,and it appears that not all of these components are expressed during biofilm formation in a particular environment.It is anticipated that the capacity of bacteria to produce different biofilmmatrix Fig.1.The conductive and respiratory zones of the lungs.Inhalation antibiotic therapy is mainly targeting the conductive zone where sputum is located,whereas systemic antibiotic therapy is mainly targeting the respiratory zones with no sputum[12,75]. Table1Important properties of mucoid and non-mucoid phenotypes of Pseudomonas aeruginosa in the respiratory tract of cysticfibrosis[9,10].Property Mucoid phenotype Nonmucoidphenotype Location in the lungs Respiratory zone andconductive zone in sputumConductivezone in sputum Biofilm formation in vitro Yes YesBiofilm formation in vivo Yes NoMultiply antibiotic resistance dueto conventional mechanismsSeldom FrequentResistance due to biofilm properties Yes No Responsible for lung tissue damage Yes NoInduces pronounced antibody response Yes No8O.Ciofu et al./Advanced Drug Delivery Reviews85(2015)7–23components allows colonization of different niches through different bio film formation pathways.The extracellular bio film matrix is believed to offer protection against various adverse factors including immune responses and antibiotic treatment [25–28].Studies using confocal laser scanning microscopy of bio films formed in laboratory experimental systems by fluorescence-tagged bacteria have provided detailed knowledge about structural bio film develop-ment (e.g.[29–33]).Bio film formation can initiate when bacteria attach to a surface or to each other and form aggregates.The bio film developmental cycle is believed to include the processes:i)transport of solitary microbes to a surface or each other,ii)initial attachment of the microbes to the surface or each other,iii)formation of microcolonies,iv)maturation of the bio film,and v)dispersal of the bio-film.In many bacteria,the initiation of bio film formation and the pro-duction of bio film matrix components occur in response to high intrabacterial levels of the second messenger molecule c-di-GMP,whereas dispersal of the bio film occurs in response to low levels of c-di-GMP [34].Synthesis and degradation of c-di-GMP in the bacteriaisFig.2.Bio films of CF-related pathogensin the lungs of patients with cystic fibrosis.A.Mucoid bio film of P .aeruginosa in an alveolar sac (lower arrow)surrounded by severely in flammed tissue (PMNs,pneumonia)(upper arrow).Autopsy of a CF girl who died due to chronic P .aeruginosa lung infection.HE stain ×100(photo by N.Høiby).B.Pseudomonas aeruginosa bio film in a detached lung alveole in sputum from a CF patient suffering from chronic lung infection.Gram stained smear,×1000magni fication (photo by N.Høiby).C.Pseudomonas aeruginosa bio film in sputum form a cystic fibrosis patient suffering from chronic lung infection.Gram stained smear,×1000magni fication (photo by N.Høiby).D.Achromobacter xylodoxidans bio film in sputum from a CF patient suffering from chronic lung infection.Gram stained smear,×400magni fication (photo by N.Høiby).E.Burkholderia multivorans bio film in sputum from a CF patient suffering from chronic lung infection.Gram stained smear,×1000magni fication (photo by N.Høiby).F.Stenotrophomonas maltophilia bio film in sputum from a CF patient suffering from chronic lung infection.Gram stained smear,×1000magni fication (photo by N.Høiby).9O.Ciofu et al./Advanced Drug Delivery Reviews 85(2015)7–23accomplished by diguanylate cyclases and phosphodiesterases that con-tain sensory domains,enabling translation of diverse environmental cues into synthesis or degradation of c-di-GMP,which bind to down-stream effector molecules and modulates their function,resulting in regulation of the production of different adhesins and bio film matrix products.In between initiation and termination of bio film formation we have de fined speci fic bio film stages,but the currently available evi-dence suggests that these transitions are mainly governed by adaptive responses,and not by speci fic genetic programs.In the case of P .aeruginosa a number of microarray analyses have been performed in order to monitor genes that are expressed during bio film formation [35–38].However,the transcriptomic analyses performed by the differ-ent research groups have failed to consistently identify speci fic bio film regulons,in support of the suggestion that bio film formation is mainly governed by adaptive responses.Bio film formation can occur through multiple pathways,and the spatial structure of the bio films is species dependent as well as dependent on the environmental conditions.For example,P .aeruginosa forms bio films with mushroom shaped struc-tures in flow-chambers perfused with glucose-medium,whereas it forms flat bio films in flow-chambers perfused with citrate medium [29](Fig.3).The bacteria are still susceptible to antibiotics at the earliest stage of bio film formation,and this is in accordance with the success of peroperative antibiotic prophylaxis for e.g.alloplastic surgery,however more developed bio films display recalcitrance to antibiotic treatment (as addressed below).When dispersal occurs at focal areas of bio films the liberated bacteria can spread to other locations where new bio films can be formed.The difference in the properties of bio film-growing and planktonic bacteria has signi ficant diagnostic and therapeutic consequences.In clinical specimens (e.g.biopsies,pus,sputum)bio films can often be rec-ognized by light microscopy,but precise identi fication of the bacteria within a bio film can only be done by the use of nucleic acid hybridiza-tion techniques,and identi fication of the components of the bio film ma-trix requires specialized staining techniques [11].Traditional sampling techniques may not be suf ficient to culture bacteria from bio films unless the bacteria are released from the bio film by ultasonic pre-treatment [39].The ordinary culture techniques,however,reveals only the proper-ties of planktonic bacteria,and antibiotic susceptibility testing therefore does not indicate the susceptibility of the bio film-growing bacteria.Methods to test antibiotic susceptibility of bio film-growing bacteria have been developed,but their clinical relevance awaits con firmation [25,40,41].3.High cell density and quorum-sensing in bio filmsMany bacterial species use cell-to-cell signaling,also referred to as quorum-sensing,to coordinate gene expression in relation to population density.Production by the bacteria of extracellular signal molecules gives rise to a high concentration of signal molecule in high-density bacterial populations.The signal molecules are sensed by the bacteria via speci fic quorum-sensing receptors,and the bacteria can therefore induce target genes when the concentration of quorum-sensing signal molecule is high,and restrict expression of speci fic genes to conditions of high cell density.P .aeruginosa utilizes the three quorum-sensing signal molecules 3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL),N-butanoyl homoserine lactone (C4-HSL),and 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal,denoted PQS)[42].The 3-oxo-C12-HSL molecule is synthesized via the LasI synthase and sensed by the LasR re-ceptor,whereas C4-HSL is synthesized via the RhlI synthase and sensed by the RhlR receptor [43],and PQS is synthesized via the pqsABCDE and pqsH gene products and sensed by the PqsR receptor [44].In many P .aeruginosa strains,the quorum-sensing systems are interconnected and work in a hierarchical manner with the Las system affecting expres-sion of the Rhl system [45,43],and the Pqs system intertwined between the Las and Rhl systems [46],but exceptions to this hierarchical organiza-tion have been found [47].Transcriptomic analysis of P .aeruginosa bio films has con firmed that quorum-sensing controls gene expression in these high density populations [35].Quorum-sensing primarily regulates the expression of virulence factors in P .aeruginosa [48],but in addition quorum-sensing appears to regulate the production of several factors that play roles in P .aeruginosa bio film formation.Davies et al.[49]found that a P .aeruginosa lasI quorum-sensing mu-tant formed flat and undifferentiated bio films in flow-chambers,where-as the wild-type strain formed bio films with large mushroom-shaped structures.The flat bio films formed by the lasI mutant strain were vulnerable to treatment with the detergent SDS,while the structured bio films formed by the wild-type strain resisted SDS treatment.Quorum-sensing plays a role in the generation of extracellular DNA in P .aeruginosa bio films [32],and the extracellular DNA functions as a matrix component in the bio films [50].In addition to a small amount of extracellular DNA present in the initial phase in P .aeruginosa bio films,release of a large amount of extracellular DNA occurs at a later stage of P .aeruginosa bio film formation,regulated via the PQS-based quorum-sensing system,and evidently as a consequence of lysis of a small sub-population of the bacteria [32].In agreement with a role ofPQS-basedFig.3.Confocal laser scanning microscopy (CLSM)micrographs acquired in 5day-old P .aeruginosa -Gfp bio films grown in flow-chambers on glucose minimal medium (A)or citrate min-imal medium (B).The central pictures show-top down fluorescence projections and the flanking pictures show vertical sections.Bars,20μm.Adapted from Mol Microbiol .48:1511–1524with permission from Wiley-Blackwell publishing.10O.Ciofu et al./Advanced Drug Delivery Reviews 85(2015)7–23QS in the generation of extracellular DNA that functions as a biofilm ma-trix component,P.aeruginosa pqs mutants were found to have defects in biofilm formation,and theflat biofilms that were formed by the pqs mu-tants contained little extracellular DNA[51,32,33,52].The mechanism involved in PQS-mediated DNA-release is unknown,but evidence has been provided that PQS can act as a cell-sensitizing pro-oxidant[53], which may cause lysis of a subpopulation of P.aeruginosa bacteria.In ad-dition to extracellular DNA,quorum-sensing signaling controls the pro-duction of the biosurfactant rhamnolipid,which was shown to be important for biofilm formation by P.aeruginosa[54–56],and to play a role in the tolerance of P.aeruginosa biofilms towards immune cells [57].Furthermore,the production of the P.aeruginosa LecA and LecB lectins was shown to be regulated by quorum-sensing[58,59],and the lectins were shown to play a role in P.aeruginosa biofilm formation [60,61].Moreover,transcriptome analysis has indicated that genes encoding CupA and CupBfimbriae are regulated by quorum-sensing in P.aeruginosa biofilms[35],and thefimbriae were shown to function as biofilm matrix components[62].Quorum-sensing signaling in P.aeruginosa also controls the production of siderophores such as pyoverdine and pyochelin,which are also of importance for biofilm for-mation[63].Furthermore,evidence has been presented that the Rhl quorum-sensing system is necessary for preventing accumulation of toxic nitric oxide during anaerobic nitrate respiration which may play an important role in P.aeruginosa biofilms in clinical settings[64–66]. In agreement,genes required for respiratory nitrate reduction,e.g. nirCMSQ and napEF,were found to be strongly up-regulated in P.aeruginosa during biofilm growth[35],and genes in the nar operon, encoding the denitrification pathway,were found to be upregulated in some P.aeruginosa CF lung isolates[67].4.Tolerance of biofilms to antimicrobials and immune systemMicrobial biofilms can tolerate activities of the host immune system and treatment with antimicrobial compounds[26–28]The minimal in-hibitory concentration(MIC)and minimal bactericidal concentration (MBC)of antibiotics to biofilm-growing bacteria may be100–1000 fold higher than to planktonic bacteria[25,28,41].The tolerance of biofilms to immune defenses and antimicrobials is connected to the biofilm mode of growth,and if bacteria originating from a biofilm are grown in planktonic culture they will usually display susceptibility to the antimicrobial activities[68].Biofilm-associated antimicrobial toler-ance is therefore fundamentally different from antimicrobial resistance, which can be displayed by bacteria in planktonic culture and is not connected to the biofilm mode of growth per se.4.1.Restricted penetrationRestricted penetration of antimicrobials through the biofilm matrix can in some cases contribute to the antimicrobial tolerance of biofilms. Although biofilm matrices do not inhibit diffusion of antibiotics in gen-eral,restricted penetration of antibiotics through biofilms may occur in cases where the antibiotics bind to components of the biofilm matrix or the bacterial membranes e.g.;[69,70].For example,alginate and extra-cellular DNA in the matrix of P.aeruginosa biofilms may bind aminogly-coside antibiotics and thereby play a role in the antimicrobial tolerance of P.aeruginosa biofilms to antibiotics such as tobramycin[71].demon-strated that biofilms formed by an alginate over-producing P.aeruginosa strain displayed enhanced tolerance to tobramycin in comparison to a biofilm formed by an isogenic nonmucoid P.aeruginosa strain.Much of the extracellular DNA that is present in P.aeruginosa biofilms is released by the bacteria via a quorum-sensing controlled process(as described above),and[70]provided evidence that the fragile biofilms formed by a DNA-release deficient P.aeruginosa quorum-sensing mu-tant are susceptible to tobramycin treatment,but become tobramycin tolerant if they were supplied with exogenous DNA.Furthermore, it was demonstrated that addition of lysed polymorphonuclear leukocytes,which are thought to be a source of extracellular DNA at sites of infection,increased the tolerance of P.aeruginosa biofilms to-wards tobramycin treatment[70].In accordance with these studies [72],demonstrated that co-administration of DNase and alginate lyase led to increased killing activity of tobramycin in P.aeruginosa biofilms.4.2.Differential physiological activityDifferential physiological activity of the bacteria in biofilms can also be an underlying cause of biofilm-associated antimicrobial tolerance. Studies of P.aeruginosa biofilms grown in various in vitro laboratory setups have provided evidence that the metabolic activity of the bacte-ria is high in the outer part of the biofilm whereas it is low in the inner part of the biofilm[69,73,74].The available evidence suggests that the differential physiological activity seen in biofilms is caused by limited oxygen and nutrient penetration through the biofilm due to bacterial consumption(e.g.[69].Because many antibiotics targets processes that occur in growing bacteria(e.g.replication,transcription,transla-tion,and cell wall synthesis),biofilm bacteria with low metabolic activity may display increased antimicrobial tolerance.Studies of P.aeruginosa biofilms grown inflow-chambers have provided evidence that the antibiotics tobramycin,ciprofloxacin,and tetracycline preferen-tially kill the metabolically active bacteria located in the outer part of the biofilm,whereas the non-growing bacteria in the inner part of the bio-film survives treatment with these antibiotics[75–77,33,78,74,79] (Fig.4).However,some antimicrobials such as colistin,SDS,EDTA,and chlorhexidine preferentially kill the non-growing bacteria located in the inner part of the biofilm[74,79,80](see Fig.4).Colistin,SDS,EDTA, and chlorhexidin,on the contrary,do not kill the actively growing biofilm bacteria as they are able to induce the expression of defense mechanisms such as efflux pumps and the pmr genes,the latter encoding enzymes that adds amino arabinose to LPS and thereby abolish binding of the antimicrobials to the bacterial surface[74,80].Fig.4.Visualization of live and dead bacteria inflow-chamber-grown P.aeruginosa-Gfp biofilms treated with antibiotics.P.aeruginosa-Gfp biofilms were grown for4days and then continuously exposed to no antibiotic(A),25μg/ml colistin(B),60μg/ml ciproflox-acin(C),or200μg/ml tetracycline(D)for24hours.The central pictures show horizontal CLSM optical sections,and theflanking pictures show vertical CLSM optical sections.Live cells appear green due to expression of Gfp and dead cells appear red due to staining with the dead-cell indicator propidium iodide.Adapted from Mol.Microbiol.68:223–240with permission from Wiley-Blackwell publishing.11O.Ciofu et al./Advanced Drug Delivery Reviews85(2015)7–234.3.Differential expression of specific genes in biofilmAs indicated above,biofilms can display antimicrobial tolerance mechanisms that can be related to the expression of a few specific genes in the bacteria.Another example of this is the ndvB gene in P.aeruginosa PA14which encodes an enzyme that is involved in the syn-thesis of periplasmic glucans that binds tobramycin and prevents cell death by sequestering the antibiotic[81].Evidence was provided that the ndvB gene is expressed specifically in P.aeruginosa PA14biofilms but not in planktonic cells,and biofilms formed by a P.aeruginosa ndvB mutant were demonstrated to be more sensitive to tobramycin than wild type biofilms,whereas the ndvB mutant and wild type strain showed no difference in tobramycin sensitivity in planktonic culture [81].Another biofilm-specific resistance mechanism was discovered by Zhang and Mah[82]who reported that the PA1874-1877genes in P. aeruginosa encode a putative efflux pump that is expressed only during the biofilm mode of growth,and mediates tolerance to tobramycin,gen-tamicin,and ciprofloxacin.Moreover,Liao and Sauer[83]reported that the transcriptional regulator BrlR is expressed in P.aeruginosa biofilms but not in planktonic cells,and plays a role in the antimicrobial tolerance of P.aeruginosa biofilms.Inactivation of the brlR gene rendered biofilms, but not planktonic cells,significantly more susceptible to hydrogen per-oxide andfive different classes of antibiotics.Subsequently,evidence was provided that BrlR is required for maximal expression in P.aeruginosa biofilms of genes encoding the MexAB-OprM and MexEF-OprN efflux pumps[84].Earlier studies have suggested that efflux pumps such as MexAB-OprM,MexCD-OprJ,MexEF-OprN,and MexXY-oprM do not play a role in the tolerance of P.aeruginosa biofilms to antibiotics[85]. However,more recent studies have provided evidence that these efflux pumps are involved in the tolerance of P.aeruginosa biofilms to azithromycin[86],colistin[74,80],tobramycin,tetracycline,trimetho-prim,and norfloxacin[84].Resistance of biofilms to tobramycin mediat-ed by efflux-pump has also been reported to occur in B.cenocepacia[87]. Efflux pumps might also have a general detoxifying role in P.aeruginosa biofilms.The P.aeruginosa MexEF-OprN and MexXY-oprM efflux sys-tems were shown to be up-regulated in response to reactive oxygen spe-cies,and it was proposed that these efflux systems export cellular constituents damaged by reactive oxygen species[88].Because bacteria in biofilms experience increased oxidative stress due to accumulation of waste products[89],efflux pumps might have a detoxifying role in P.aeruginosa and Burkholderia sp.biofilms[90].In line with this,Kvist et al.[91]reported that efflux pumps involved in removal of toxic sub-stances were highly up-regulated in E.coli biofilms.In addition,it was found that mutant E.coli strains which were unable to produce efflux pumps showed defects in biofilm formation[91].Evidence has been provided that quorum-sensing is involved in the tolerance of P.aeruginosa biofilms to tobramycin,kanamycin, and hydrogen peroxide[92,93,75].There is currently no evidence that quorum-sensing promotes antibiotic tolerance in planktonic P. aeruginosa cells,and the involvement of quorum-sensing in antimicro-bial tolerance therefore appears to be biofilm-specific.The involvement of quorum-sensing in antimicrobial tolerance of P.aeruginosa biofilms may in part be explained by its role in the production of extracellular DNA which inhibits penetration of some antibiotics into the biofilm. The extracellular DNA that is produced in P.aeruginosa biofilms also has a stabilizing effect[32].Because biofilms formed by P.aeruginosa quorum-sensing mutants are more fragile than wild type biofilms they may be more vulnerable to for example shear forces and phagocy-tosis,and because of the high bacterial turnover a large proportion of the bacteria will be growing actively and may therefore show increased sensitivity to some antibiotics.4.4.Persister cellsIn addition to the mechanism described above,slowly-dividing or non-dividing bacteria that show diminished susceptibility to antibiotics also contribute to the antimicrobial tolerance of biofilms[94–96].The fraction of these so-called persister cells is usually low(b0.1%)and they should be distinguished from the bulk subpopulation of metaboli-cally inactive bacteria that are present in biofilms as described above. Persister cells are believed to be the result of bacterial differentiation into a dormant state.The reduced metabolism exhibited by persister cells evidently enable them to escape the activity of antibiotics that tar-get fundamental cellular processes such as replication,translation,and cell wall synthesis,but persister cells also in some cases show tolerance to antibiotics that kill non-growing cells[94,95].Substantial evidence suggests that the formation of persister cells is connected to bacterial toxin/antitoxin systems[97,96,98].Increased expression of toxins was shown to block cell metabolism,indicating that randomfluctuations in the expression of toxin and antitoxin genes can lead to the formation of persister cells.However,mutant screens have suggested that a number of other genes(e.g.rpoS,spoT,relA,dksA,dinG,spuC,algR,pilH,ycgM,pheA) than those encoding toxin/antitoxins are involved in persister formation in P.aeruginosa[99,100,95],suggesting that multiple pathways can lead to the persister cell phenotype.Exogenous addition of quorum-sensing molecules has been shown to increase the formation of planktonic per-sister cells for P.aeruginosa PAO1,but had no such effect for P.aeruginosa PA14[101].However,there is currently no evidence that physiological levels of quorum-sensing molecules promote persister cell formation.Be-sides the persister cells,other phenotypic variants can also contribute to the antimicrobial tolerance of biofilms Drenkard and Ausubel[102],for example,identified a class of antibiotic tolerant P.aeruginosa variants in which the regulatory protein PvrR,was shown to control the conversion between the parental form and the tolerant variant.5.Hydroxyl radical formation during antibiotic treatmentof biofilmsThe involvement of hydroxyl radical(OH·)formation in the bacteri-cidal activity of major classes of antibiotics[103]may be attenuated in biofilm resulting in enhanced tolerance.As comprehensively reviewed [104,105],bactericidal treatment with antibiotics may cause hyper-oxidation of NADH produced in the tricarboxylic acid(TCA)cycle resulting in leakage of electrons from the respiratory electron transport chain.The leaking electrons reduce molecular oxygen(O2)to produce su-peroxide(O2-),which subsequently dismutates into hydrogenperoxide (H2O2)and liberates ferrous iron by disintegrating iron-sulphur clusters thereby providing the necessary substrates for generation of lethal amounts of highly reactive OH·through the Fenton reaction.The actual positions of the electron leak in the respiratory electron transport chain has not yet been determined,however the outflow of electrons is likely to occur upstream of the terminal oxidases,since electrons were mainly leaking from complex I and III in mammalian cells treated with antibiotics [106].Thus,the hyper-oxidation of NADH leads to oxidative stress involv-ing formation of both O2-,H2O2and OH·.But while O2-and H2O2can be enzymatically eradicated by superoxide dismutases,catalases and perox-idases,no known enzyme is able to catalyse the cellular detoxification of OH·.Despite the fact that OH·may impose toxic oxidative lesions on pro-teins,lipids and DNA[107],the main cause of lethality during treatment of E.coli with beta-lactams,quinolones and aminoglycosides was insuffi-cient repair of incorporated oxidized guanine in DNA[108].In addition to being dependent on the activity of the TCA cycle generation of lethal amounts of OH·is reliant on the available electron acceptors as both the bactericidal activity and the formation of OH·was decreased when O2was replaced by nitrate(NO3-)during ciproflox-acin treatment of growing P.aeruginosa[109].Thus,the lethal effect me-diated by formation of OH during bactericidal treatment with antibiotics depends on aerobic respiration and metabolic activity in order to devel-op the required reactive oxygen species(ROS).In biofilms,the contribution of OH·generation to the bactericidal ac-tivity during treatment with antibiotics has so far only been demon-strated for Pseudomonas aeruginosa treated with ciprofloxacin[110].12O.Ciofu et al./Advanced Drug Delivery Reviews85(2015)7–23。