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.     

大蒜素对铜绿假单胞菌生物膜群体密度感应系统调控毒力因子表达的影响

目的通过体外测定铜绿假单胞菌（Pseudomonas aeruginosa）群体密度感应（Quorum Sensing,QS）系统调控的毒力因子表达,比较大蒜素干预前后毒性因子表达的差异以及对铜绿假单胞菌PAO1成熟生物膜（biofilm,BF）的影响。方法应用ELISA法比较处理前后外毒素A的含量差异;利用弹性蛋白一刚果红染色的方法,测定处理前后弹性蛋白酶活性;用蒽酮一硫酸法测定鼠李糖脂;利用荧光酶标仪检测青脓素含量变化;利用激光共聚焦显微镜观察干预前后大蒜素对铜绿假单胞菌成熟BF的影响。结果大蒜素干预后与生理盐水对照组比较,外毒素A、弹性蛋白酶、鼠李糖脂和青脓素表达分别由（19．630±0．573）pg/μl、（0．467±0．003）、（2．009±0．063）g／L、（9325．833±367．675）下降到（6．529±0．289）pg／μl、（0．032±0．001）、（0．269±0．009）g／L、（7819．167±111．800）。激光共聚焦显微镜观察可见生理盐水对照组细菌菌落云集呈蘑菇状分布,干预组黏附的细菌稀疏散在平坦分布。结论大蒜素可抑制铜绿假单胞菌群体密度感应系统调控的毒力因子表达,减弱其毒力,干扰BF分化成熟。     

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

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.     

Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa

In cystic fibrosis individuals, chronic lung infections and hospital-acquired pneumonia are caused by Pseudomonas aeruginosa. P. aeruginosa generates siderophores such as pyoverdine (PVD) as iron uptake systems to cover its needs of iron ions for growth and infection. lasR quorum sensing (QS) gene has a crucial function in PVD production and biofilm generation in P. aeruginosa. Fifty isolates of P. aeruginosa were obtained from clinical specimens of sputum (collected from individuals suffering from pulmonary infections). Antibiotic sensitivity test was performed for 50P. aeruginosa isolates by using 10 different types of antibiotics. All isolates of P. aeruginosa showed resistance for all 10 using antibiotics in this study. Ten multidrug resistant isoloates of P. aeruginosa were selected for next tests. Virulence factors of ten multidrug resistant isolates of P. aeruginosa, such as biofilm generation, PVD production, and lasR gene were detected. From results, all 10P. aeruginosa isolates can produce biofilm, PVD, and contain lasR gene. The produced amplicon for the lasR gene was 725 bp. After mice injection by fresh and heated PVD produced by P. aeruginosa PS10 LC619328.2, the fresh PVD caused 100 % mortality within five days using 0.3 ml of its concentration (37.4 µM), while (15.3 µM) of heated PVD (toxoid) caused 50 % mortality.     

Aluminium oxide nanoparticles inhibit EPS production,adhesion and biofilm formation by multidrug resistant Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a biofilm forming multidrug resistant (MDR) pathogen responsible for respiratory tract infections. In this study, aluminium oxide nanoparticles (Al₂O₃ NPs) were synthesized and characterized by TEM and EDX and shown to be spherical shaped nanoparticles with a diameter < 10?nm. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for the Al₂O₃ NPs ranged between 125 and 1,000?µg ml^?1. Exposure to NPs caused cellular membrane disruption, indicated by an increase in cellular leakage of the contents. Biofilm inhibition was 11.64 to 70.2%, whereas attachment of bacteria to polystyrene surfaces was reduced to 48.8 to 51.9% in the presence of NPs. Nanoparticles also reduced extracellular polymeric substance production and the biomass of established biofilms. The data revealed the non-toxic nature of Al₂O₃ NPs up to a concentrations of 120?µg ml^?1 in HeLa cell lines. These results demonstrate an effective and safer use of Al₂O₃ NPs against the MDR A. baumannii by targeting biofilm formation, adhesion and EPS production.     

Efficacy of silver-releasing rubber for the prevention of Pseudomonas aeruginosa biofilm formation in water

Abstract

The aim of the present study was to evaluate the efficacy of Elastoguard? silver-releasing rubber in preventing Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under various conditions in an in vitro model system was compared for silver-releasing and conventional rubber. Under most conditions tested, the numbers of sessile cells attached to silver-releasing rubber were considerably lower with reference to conventional rubber, although the effect diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic cells. By exposing both materials simultaneously to conditions for biofilm growth, it became obvious that the antibiofilm effect was due to a reduction in the number of planktonic cells, rather than to contact-dependent killing of sessile cells. The data demonstrate that the use of silver-releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells present in the surrounding solution.     

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.     

依地酸钠对黏液型铜绿假单胞菌生物膜的作用

目的探讨金属螯合剂依地酸钠（EDTA）对黏液型铜绿假单胞菌（PA）成熟生物膜的杀菌作用和对其结构的影响。方法平板法培养成熟铜绿假单胞菌生物膜,微量肉汤稀释法测量EDTA、环丙沙星的最低抑菌浓度,平板计数法计算EDTA、环丙沙星单独及联合对生物膜菌落数的影响,荧光探针FITC-ConA染细菌胞外多糖、荧光显微镜下观察EDTA作用前后多糖差别,荧光探针SYT09／H标记生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（ISA）对EDTA作用前后的生物膜结构参数进行定量分析。结果当EDTA浓度为5MIC时达到对PA生物膜的最大杀菌效应,可使菌落数由10^7CFU／ml降至10^4CFU／ml,0．1MIC、5 MIC的EDTA均可增强环丙沙星对生物膜的杀菌作用,高浓度组效果更明显、使菌落数降至10^2CFU／ml。EDTA作用后荧光显微镜下可见多糖被破坏,明显减少。激光共聚焦显微镜下可见EDTA作用后生物膜死茵比例增加,菌落变稀疏。ISA软件分析结果显示：5MIC的EDTA作用后生物膜厚度（d）由（22．59±4．13）μm降至（8．97±2．45）μm,t=8．515,P〈0．05;AP（区域孔率）由0．89±0．07增加至0．97±0．02,t=-2．653,P〈0．05;ADD（平均扩散距离）由3．08±0．96降至1．59±0．24,t=4．510,P〈0．05;TE（结构熵）由6．25±0．79降至3．02±0．67,t=9．375,P〈0．05;0．1MIC的EDTA效果没有5MIC明显。结论EDTA可以破坏铜绿假单胞菌生物膜的结构,增强抗生素对生物膜杀菌活性。     

纳米银水凝胶涂膜干预气管导管表面铜绿假单胞菌黏附及生物膜形成的实验研究

目的研究纳米银水凝胶涂膜对气管导管（endotracheal tube,ETT）表面铜绿假单胞菌粘附及细菌生物膜（biofilm,BF）形成的干预作用。方法实验共设6组,分别为空白对照组,涂纳米银3．5、7．0、10．5、14．0和17．5μg／cm^2组。参考Brown平板法,制备ETT、表面铜绿假单胞菌BF模型。通过超声振荡-平板菌落计数法检测体外培养6、12、18h时各组ETT表面BF中的活细菌粘附数量。借助激光共聚焦显微镜观察BF中的活死菌分布情况,并测量BF厚度。结果（1）与空白对照组相比,最小量涂膜组（3．5μg／cm^2）体外培养6h时,导管表面活细菌的粘附量显著减少（P〈0．05）,12h时差异无显著性（P〉0．05）;最大量涂膜组（17．5μg／cm^2）体外培养6h时,ETT表面几乎未见细菌粘附（P〈0．05）,18h时BF中的活菌数量及BF厚度均显著减少（P〈0．05）。（2）激光共聚焦显微镜观察可见,培养6h时,空白对照组ETT表面粘附的死活菌呈不规则散点样分布,未见明显的细菌菌落形成,而各实验组ETT表面仅有细菌零星分布,其数量少于空白组。18h时空白对照组表面可见大量活死菌堆积粘连,有小菌落形成并相互交通成地图状,可见典型BF结构,而此时最大量涂膜组（17．5μg／cm^2）表面仅见数量不等的菌落形成,菌落周围可见数量不等的细菌分布。结论纳米银水凝胶涂膜可有效减少ETT表面铜绿假单胞菌的粘附数量,延缓导管表面细菌BF形成,其作用强弱随培养时间及单位面积中的纳米银剂量的变化而变化。     

Pseudomonas aeruginosa biofilm: Potential therapeutic targets

Pseudomonas aeruginosa is a gram-negative pathogen that has become an important cause of infection, especially in patients with compromised host defense mechanisms. It is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteremia. The biofilm formed by the bacteria allows it to adhere to any surface, living or non-living and thus Pseudomonal infections can involve any part of the body. Further, the adaptive and genetic changes of the micro-organisms within the biofilm make them resistant to all known antimicrobial agents making the Pseudomonal infections complicated and life threatening. Pel, Psl and Alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell–cell and cell–surface interactions during biofilm formation. Understanding the bacterial virulence which depends on a large number of cell-associated and extracellular factors is essential to know the potential drug targets for future studies. Current novel methods like small molecule based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides, monoclonal antibodies and nanoparticles to curtail the biofilm formed by P. aeruginosa are being discussed in this review.     

铜绿假单胞菌生物被膜与宿主免疫的关系

铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)是一种常见的革兰阴性条件致病菌,能引起严重的院内感染,可从支气管扩张、肺囊性纤维化(CF)等患者体内分离。机体免疫系统可以通过识别不同的病原体相关分子模式(PAMPs)来抵御P.aeruginosa的感染,但P.aeruginosa生物被膜(BF)的形成可以导致这些成分被遮蔽从而引起免疫逃逸,导致疾病的迁延难愈。BF是一种与游离细菌相对立的生活方式,能帮助细菌有效适应外部环境,其可以通过藻酸盐的屏障作用,抵抗吞噬细胞的吞噬,干扰多核白细胞(PMNs)的激活,从而逃避宿主免疫。研究P.aeruginosa-BF的免疫逃逸机制,发现有效清除P.aeruginosa-BF的方法,从而为临床治疗P.aeruginosa引起的感染性疾病提供科学依据。现以P.aeruginosa为例对近年来国内外BF的免疫逃逸机制的研究进展进行综述。     

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。     

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.     

镁离子对黏液型铜绿假单胞菌生物膜形成过程的影响

目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FTTC-ConA染细菌胞外多糖（Extracellular Polymeric Substances,EPS）、荧光显微镜下观察各组多糖差别;SYTO9／PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（Image Structor Analyzer,ISA）对各组生物膜结构参数进行定量分析。结果2d时,空白组和1mmol／L镁组的黏附细菌的荧光强度分别为1845．67±45．3和2254．78±42．45,t=-9．96,P〈0．05;0．1mmol／L的镁浓度下荧光强度也有增加,其余各时间组趋势与2d组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物膜活菌增加、菌落变密集;ISA软件分析结果示：空白组和1mmol／L镁组的6d生物膜厚度分别为（25．80±1．16）μm和（34．87±1．59）μm,t=-13．85,P〈0．05;区域孔率分别为0．96±0．05和0．90±0．04,t=2．48,P〈0．05;平均扩散距离分别为1．54±0．15和1．92±0．16,t=5．23,P〈0．05;结构熵分别为3．64±0．57和4．70±1．09,t=-2．6,P〈0．05,3d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物膜的形成及结构。     

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.