Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.     

Binary culture biofilm formation by Stenotrophomonas maltophilia and Fusarium oxysporum

Binary culture biofilm formation by Stenotrophomonas maltophilia and Fusarium oxysporum was investigated using the recirculating modified Robbins device batch culture system. Sequential attachment studies were carried out in the Robbins device on PVC and glass surfaces, with each species as either the first or the second colonizer. Different surfaces had no significant effect on total numbers of S. maltophilia and F. oxysporum in the binary population biofilm. The attachment of the second colonizer was not influenced significantly by the previous attachment of the first colonizer. These results were confirmed using scanning electron micrographs. Journal of Industrial Microbiology & Biotechnology (2001) 26, 178–183. Received 06 July 1999/ Accepted in revised form 02 November 2000     

Stenotrophomonas maltophilia flagellin is involved in bacterial adhesion and biofilm formation

Stenotrophomonas maltophilia is an emerging drug-resistant pathogen and an important opportunistic pathogen. S. maltophilia flagellin was purified using serial ultracentrifugation. The purity of flagellin was checked by SDS-PAGE. The antibodies were raised in rabbits. The presence of anti-flagellin and the titer of flagellin were detected by immunoblotting and bacterial agglutination techniques. Two methods (viable bacterial count and spectrophotometric methods) were applied to evaluate bacterial adhesion and biofilm formation. Pretreatment of S. maltophilia with dilutions of anti-flagellin (from 1/40 to 1/640) reduced the ability of S. maltophilia to adhere and form biofilms on polystyrene (P < 0.05). In the present study, the inhibition of bacterial adhesion to polystyrene was dose-dependent. The positive correlation was observed between the antibody dilutions and bacterial adhesion (CFU/mL) (r > +0.5, P < 0.05), while, the negative correlation (r < ?0.5, P < 0.05) was observed between the percentage of adhesion inhibition and anti-flagellin dilutions. The current study proved the direct role of S. maltophilia flagellin in bacterial adhesion to and biofilm formation on polystyrene.     

铜绿假单胞菌铁摄取与生物被膜形成研究进展

生物被膜是单细胞微生物通过其分泌的胞外多聚基质粘附于介质表面并将其自身包绕其中而成的膜样微生物细胞聚集物。生物被膜的形成使细菌具有更强的适应外界环境的能力,也是导致微生物产生耐药性及慢性感染性疾病难以治疗的重要原因之一。铜绿假单胞菌在肺部的定殖是肺囊性纤维化病患者发病和死亡主要原因,其造成的感染通常与形成抗生素抗性极强的生物被膜有关。铜绿假单胞菌生物被膜的形成受控于多种复杂的细菌调控体系之下,包括群体感应系统及参与调节胞外多聚基质合成的双组分调控系统等。此外,为了利用低浓度的环境铁来维持生存并完成各种生理功能,铜绿假单胞菌进化出了一系列铁摄取系统,这些系统对其毒力因子的释放和生物被膜的形成又起着重要的调控作用。本文主要对铜绿假单胞菌生物被膜的形成与调控机制及其铁摄取系统进行了综述,为进一步了解及清除铜绿假单胞菌引发的问题提供途径与思路。     

绿原酸通过增强RsmA的表达抑制铜绿假单胞菌生物被膜的形成

铜绿假单胞菌是常见的人类条件致病菌,其生物被膜的形成会增强菌体的耐药性。已有文献报道绿原酸可抑制铜绿假单胞菌生物被膜的形成,本研究在此基础上主要探究了其对全局性次级代谢调控系统Gac-Rsm表达的影响。结果显示,绿原酸可抑制铜绿假单胞菌生物被膜形成的能力,降低胞外总多糖合成量,但关键胞外多糖psl的合成酶基因pslA转录未受影响,还可增强Gac-Rsm系统中关键调控因子RsmA的表达水平,降低细胞内关键信使分子环二鸟苷酸(cyclic dimeric guanosine monophosphate,c-di-GMP)水平。结果表明,绿原酸可通过增强RsmA的表达来抑制铜绿假单胞菌生物被膜的形成。     

Differential biofilm formation and motility associated with lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia WR-C is capable of forming biofilm on polystyrene and glass. The lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes rmlA, rmlC, and xanB are necessary for biofilm formation and twitching motility. Mutants with mutations in rmlAC and xanB display contrasting biofilm phenotypes on polystyrene and glass and differ in swimming motility.     

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

HD-GYP is a protein domain involved in the hydrolysis of the bacterial second messenger cyclic-di-GMP. The genome of the human pathogen Pseudomonas aeruginosa PAO1 encodes two proteins (PA4108, PA4781) with an HD-GYP domain and a third protein, PA2572, which contains a domain with variant key residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa . Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa , consistent with the predicted activity of the encoded proteins as cyclic-di-GMP phosphodiesterases. Mutation of both genes led to reduced swarming motility but had differing effects on production of the virulence factors pyocyanin, pyoverdin and ExoS. Mutation of PA2572 had no effect on cyclic-di-GMP levels and did not influence swarming motility. However, PA2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108 , PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P. aeruginosa to larvae of the Greater Wax moth Galleria mellonella.     

Effects of ginseng on Pseudomonas aeruginosa motility and biofilm formation

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5-2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.     

群体感应对铜绿假单胞菌生物被膜形成的调控

生物被膜是一种与浮游细胞相对应的生长方式,由细菌和自身分泌的包外基质组成。铜绿假单胞菌是研究这一生长方式的模式生物。在过去十年,对铜绿假单胞菌生物被膜的研究已取得显著进展。群体感应(QS)的细胞沟通机制在铜绿假单胞菌生物被膜形成中发挥着重要作用。介绍生物被膜的特点,并重点讨论了QS和生物被膜之间的关系。     

Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two-component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10-fold reduction in biofilm formation capacity resulted relative to wild-type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA(-). Providing gacA in trans on the multicopy vector pUCP-gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA(-) revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA(-) twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar-mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA(-) biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.     

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air–liquid interface.     

Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation

Using flow chamber‐grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that formation of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron‐siderophore pyoverdine in one subpopulation being necessary for development of another subpopulation that does not itself express the pyoverdine synthesis genes. Another example is quorum sensing‐controlled DNA release in one subpopulation being necessary for development of another subpopulation that does not itself express the quorum‐sensing genes.     

Lactoferrin‐derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa

Aims: To investigate the bactericidal activity of lactoferrin‐derived peptides and a new LF‐derived peptides chimera (LFchimera) against P. aeruginosa and the influence on virulence factors of P. aeruginosa. Methods and Results: Lactoferricin (LFcin) and lactoferrampin (LFampin) are highly bioactive peptides isolated from the N‐terminal region of lactoferrin (LF) by pepsin digestion. In this study, we designed LFchimera containing LFcin amino acids 17‐30 and LFampin amino acids 268‐284. Pseudomonas aeruginosa cells were incubated in medium with peptides at different concentrations, and then the assays of viability, pyocyanin, elastase activity and biofilm formation of P. aeruginosa were performed. We found that the concentration‐dependent antibactericidal activity and down‐regulating pyocyanin, elastase and biofilm formation of LFchimera were significantly stronger than those of LF, LFcin, LFampin or LFcin plus LFampin. Conclusions: Our results indicated that LF, LFcin, LFampin and LFchimera were potential candidates to combat P. aeruginosa, and LFchimera was the most effective in them. Significance and Impact of the Study: The new LFchimera has better activity against P. aeruginosa than LF, LFcin and LFampin and may be a promising new compound for treatment of P. aeruginosa infection.