Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.     

Common plant flavonoids prevent the assembly of amyloid curli fibres and can interfere with bacterial biofilm formation

Like all macroorganisms, plants have to control bacterial biofilm formation on their surfaces. On the other hand, biofilms are highly tolerant against antimicrobial agents and other stresses. Consequently, biofilms are also involved in human chronic infectious diseases, which generates a strong demand for anti-biofilm agents. Therefore, we systematically explored major plant flavonoids as putative anti-biofilm agents using different types of biofilms produced by Gram-negative and Gram-positive bacteria. In Escherichia coli macrocolony biofilms, the flavone luteolin and the flavonols myricetin, morin and quercetin were found to strongly reduce the extracellular matrix. These agents directly inhibit the assembly of amyloid curli fibres by driving CsgA subunits into an off-pathway leading to SDS-insoluble oligomers. In addition, they can interfere with cellulose production by still unknown mechanisms. Submerged biofilm formation, however, is hardly affected. Moreover, the same flavonoids tend to stimulate macrocolony and submerged biofilm formation by Pseudomonas aeruginosa. For Bacillus subtilis, the flavonone naringenin and the chalcone phloretin were found to inhibit growth. Thus, plant flavonoids are not general anti-biofilm compounds but show species-specific effects. However, based on their strong and direct anti-amyloidogenic activities, distinct plant flavonoids may provide an attractive strategy to specifically combat amyloid-based biofilms of some relevant pathogens.     

The activity of a small lytic peptide PTP-7 on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

One of the most important features of bacterial biofilms is their resistance to antibiotics and to the host immune system. In this study, we have found that a small lytic peptide, PTP-7, is very potent to Gram-positive bacteria and is able to kill antibiotic sensitive and resistant Staphylococcus aureus indiscriminately. Further studies have revealed that despite being a cationic peptide, the antibacterial activity of PTP-7 was not affected by the negatively charged extracellular polymeric substance (EPS) of biofilms. PTP-7 could diffuse into the deep layer of S. aureus biofilms to kill bacteria inside biofilms efficiently and effectively. Neither the high concentrations of metal ions nor the acidic pH in biofilms affected the activity of peptide PTP-7. It seems that the unique sequence/structure together with the resistant bacteria killing ability of peptide PTP-7 confers its anti-biofilm activity. This study sheds new light on the treatment of bacterial biofilms, especially various biofilm related infections.     

Control of bacterial biofilms with marine alkaloid derivatives

Bacterial biofilms are defined as a community of surface-attached bacteria that are protected by an extracellular matrix of biomolecules. We have recently reported the synthesis of a small molecule, denoted TAGE, based on the natural product bromoageliferin and demonstrated that TAGE has anti-biofilm activity against Pseudomonas aeruginosa. Herein we demonstrate that TAGE: (1) does not have selective toxicity against cells within the biofilm state, (2) will inhibit biofilm development under flow conditions, indicating that the CV staining protocol correlates with the ability to be active under biomimetic conditions, and (3) will disperse preformed P. aeruginosa biofilms. We also present preliminary toxicity work that indicates that TAGE is devoid of cytotoxicity in rat and mice cell lines. Advanced derivatives of TAGE have generated compounds shown to be exceedingly effective as biofilm inhibitors against the gamma-proteobacteria in this study (P. aeruginosa strains PAO1, PA14, PDO300, and Acinetobacter baumannii). TAGE derivatives also possessed anti-biofilm activity against the beta-proteobacterium Bordetella bronchiseptica (Rb50) and the Gram-positive bacterium Staphylococcus aureus;TAGE derivatives inhibited the formation of biofilms, however, some of this activity is attributed to microbicidal activity. The TAGE derivatives presented in this study, however, do not disperse pre-formed biofilms with the same efficiency as TAGE.     

Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (～10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.     

抗生物膜肽研究进展

细菌生物膜导致的细菌耐药性增加受到了广泛关注。抗生物膜肽是一类具有抑制和杀灭细菌生物膜独特优势的抗微生物肽,有望成为理想的抗细菌生物膜的新型抗菌药物。就抗生物膜肽与生物膜各组分间的相互作用、抗生物膜肽对生物膜形成的干预作用及其调控、抗生物膜肽目前存在的问题及其解决思路以及抗生物膜肽未来的应用领域等展开综述。     

Influence of aptamer-targeted antibiofilm agents for treatment of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> biofilms

Biofilms are bacterial communities consisting of numerous extracellular polymeric substances. Infections caused by biofilm-forming bacteria are considered to be a major threat to health security and so novel approaches to control biofilm are of importance. Aptamers are single-strand nucleic acid molecules that have high selectivity to their targets. Single-walled carbon nanotubes (SWNTs) are common nanomaterials and have been shown to be toxic to bacterial biofilms. The aim of this study was to test whether an aptamer could play a role as targeting agents to enhance the efficiency of anti-biofilm agents. Hence, two complexes (aptamer–SWNTs and aptamer–ciprofloxacin–SWNTs) based on an aptamer which targets Pseudomonas aeruginosa and SWNTs were constructed. Both complexes were assessed against P. aeruginosa biofilms. In vitro tests demonstrated that the aptamer–SWNTs could inhibit ~36% more biofilm formation than SWNTs alone. Similarly, the aptamer–ciprofloxacin–SWNTs had a higher anti-biofilm efficiency than either component or simple mixtures of two components. Our study underscores the potential of aptamers as targeting agents for anti-biofilm compounds, as well as providing a new strategy to control biofilms.     

抗菌肽的抗生物膜机理研究进展

生物膜,也称为生物被膜,是指附着于有生命或无生命物体表面被细菌胞外大分子包裹的有组织的细菌群体。与浮游菌相比,生物膜内的细菌对抗生素的耐受性提高了10–1000倍,是造成目前细菌耐药的主要原因之一。作为一种新型抗菌制剂,抗菌肽的使用为生物膜感染的治疗提供了一种新的思路和手段。抗菌肽在抑制生物膜形成、杀灭生物膜内细菌以及消除成熟生物膜的过程中发挥了独特的优势。文中分析了近30年的数据,从细菌生物膜的结构入手,对抗菌肽可能的抗生物膜机理进行了综述,以期为抗菌肽临床治疗生物膜感染提供一定参考。     

Enzymatic removal and disinfection of bacterial biofilms.

Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata. Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite. The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured. Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata. A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity. Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.     

Nanocarriers for combating biofilms: Advantages and challenges

Bacterial biofilms are highly resistant to antibiotics and pose a great threat to human and animal health. The control and removal of bacterial biofilms have become an important topic in the field of bacterial infectious diseases. Nanocarriers show great anti-biofilm potential because of their small particle size and strong permeability. In this review, the advantages of nanocarriers for combating biofilms are analysed. Nanocarriers can act on all stages of bacterial biofilm formation and diffusion. They can improve the scavenging effect of biofilm by targeting biofilm, destroying extracellular polymeric substances and enhancing the biofilm permeability of antimicrobial substances. Nanocarriers can also improve the antibacterial ability of antimicrobial drugs against bacteria in biofilm by protecting the loaded drugs and controlling the release of antimicrobial substances. Additionally, we emphasize the challenges faced in using nanocarrier formulations and translating them from a preclinical level to a clinical setting.     

A surface-active agent from Saccharomyces cerevisiae influences staphylococcal adhesion and biofilm development

Bacterial biofilms which are responsible for a number of diseases are very difficult to control effectively because of their high resistance to antibiotics and the host defence system. The use of natural products decreasing or preventing initial adhesion of bacteria and biofilm formation is one of the alternative therapeutic strategies taken into consideration. We ask the question, whether a crude extract from the cell wall of Saccharomyces cerevisiae (mannoprotein), which possesses surfactant activity, may be used as inhibitor of Staphylococcus aureus and S. epidermidis biofilm development. By using the "bactericidal spot assay" it was demonstrated that mannoprotein had no direct antibiotic activity against the tested strains. The influence of this extract on initial adhesion, biofilm formation and dispersal of preformed biofilms was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. In this assay, live bacteria with an active electron transport system reduce the tetrazolium salt to a water-soluble purple formazan product, and optical density reading (A550) values are directly dependent on their cell numbers. Yeast-derived surfactant, when adsorbed in the microplate wells or present in the medium, was effective both in decreasing the initial deposition of staphylococci and in reducing the amount of growing biofilm, quantitated after 24 h of co-incubation with the bacteria. It also changed the parameters of biofilm morphology analyzed by PHLIP - the confocal laser scanning microscopy image quantification package. Mannoprotein also accelerated the detachment of mature staphylococcal biofilms, preformed in optimal conditions. It was concluded that mannoprotein anti-biofilm action reflects its influence on cell surface hydrophobicity.     

Insights into discharge argon-mediated biofilm inactivation

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in biomedical sciences. Conventional sterilization and decontamination methods are not suitable for new and more sophisticated biomaterials. In this paper, the efficiency and effectiveness of gas discharges in the inactivation and removal of biofilms on biomaterials were studied. It was found that although discharge oxygen, nitrogen and argon all demonstrated excellent antibacterial and antibiofilm activity, gases with distinct chemical/physical properties underwent different mechanisms of action. Discharge oxygen- and nitrogen-mediated decontamination was associated with strong etching effects, which can cause live bacteria to relocate thus spreading contamination. On the contrary, although discharge argon at low powers maintained excellent antibacterial ability, it had negligible etching effects. Based on these results, an effective decontamination approach using discharge argon was established in which bacteria and biofilms were killed in situ and then removed from the contaminated biomaterials. This novel procedure is applicable for a wide range of biomaterials and biomedical devices in an in vivo and clinical setting.     

Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo

Biofilms are complex communities of bacteria encased in a matrix composed primarily of polysaccharides, extracellular DNA, and protein. Staphylococcus aureus can form biofilm infections, which are often debilitating due to their chronicity and recalcitrance to antibiotic therapy. Currently, the immune mechanisms elicited during biofilm growth and their impact on bacterial clearance remain to be defined. We used a mouse model of catheter-associated biofilm infection to assess the functional importance of TLR2 and TLR9 in the host immune response during biofilm formation, because ligands for both receptors are present within the biofilm. Interestingly, neither TLR2 nor TLR9 impacted bacterial density or inflammatory mediator secretion during biofilm growth in vivo, suggesting that S. aureus biofilms circumvent these traditional bacterial recognition pathways. Several potential mechanisms were identified to account for biofilm evasion of innate immunity, including significant reductions in IL-1β, TNF-α, CXCL2, and CCL2 expression during biofilm infection compared with the wound healing response elicited by sterile catheters, limited macrophage invasion into biofilms in vivo, and a skewing of the immune response away from a microbicidal phenotype as evidenced by decreases in inducible NO synthase expression concomitant with robust arginase-1 induction. Coculture studies of macrophages with S. aureus biofilms in vitro revealed that macrophages successful at biofilm invasion displayed limited phagocytosis and gene expression patterns reminiscent of alternatively activated M2 macrophages. Collectively, these findings demonstrate that S. aureus biofilms are capable of attenuating traditional host proinflammatory responses, which may explain why biofilm infections persist in an immunocompetent host.     

A novel compound from the marine bacterium Bacillus pumilus S6-15 inhibits biofilm formation in gram-positive and gram-negative species

Biofilm formation is a critical problem in nosocomial infections and in the aquaculture industries and biofilms show high resistance to antibiotics. The aim of the present study was to reveal a novel anti-biofilm compound from marine bacteria against antibiotic resistant gram-positive and gram-negative biofilms. The bacterial extract (50 μg ml(-1)) of S6-01 (Bacillus indicus = MTCC 5559) showed 80-90% biofilm inhibition against Escherichia coli, Shigella flexneri, Proteus mirabilis and S6-15 (Bacillus pumilus = MTCC 5560) showed 80-95% biofilm inhibition against all the 10 tested organisms. Furthermore, they also reduced the hydrophobicity index and extracellular polymeric substances (EPS) production. Structural elucidation of the active principle in S6-15 using GC-MS, (1)H NMR, and (13)C NMR spectral data revealed it to be 4-phenylbutanoic acid. This is the first report of 4-phenylbutanoic acid as a natural product. The purified compound (10-15 μg ml(-1)) showed potential activity against a wide range of biofilms. This study for the first time, reports a novel anti-biofilm compound from a marine bacterium with wide application in medicine and the aquaculture industry.     

Antibiofilm activity of selected plant essential oils and their major components

The aim of the study was to examine the antibiofilm activity of selected essential oils (EO): Lavandula angustifblia (LEO), Melaleuca alternifolia (TTO), Melissa officinalis (MEO) and some of their major constituents: linalool, linalyl acetate, alpha-terpineol, terpinen-4-ol. Biofilms were formed by Staphylococcus aureus ATCC 29213 and Escherichia coli NCTC 8196 on the surface of medical biomaterials (urinary catheter, infusion tube and surgical mesh). TTC reduction assay was used for the evaluation of mature biofilm eradication from these surfaces. Moreover, time-dependent eradication ofbiofilms preformed in polystyrene 96-well culture microplates was examined and expressed as minimal biofilm eradication concentration (evaluated by MTT reduction assay). TTO, alpha-terpineol and terpinen-4-ol as well as MEO, showed stronger anti-biofilm activity than LEO and linalool or linalyl acetate. Among the biomaterials tested, surgical mesh was the surface most prone to persistent colonization since biofilms formed on it, both by S. aureus and E. coli, were difficult to destroy. The killing rate studies of S. aureus biofilm treated with TTO, LEO, MEO and some of their constituents revealed that partial (50%) destruction of 24-h-old biofilms (MBEC50) was achieved by the concentration 4-8 x MIC after 1 h, whereas 2-4 x MIC was enough to obtain 90% reduction in biomass metabolic activity (MBEC90) after just 4 h of treatment. A similar dose-dependent effect was observed for E. coli biofilm which, however, was more susceptible to the action of phytochemicals than the biofilms of S. aureus. It is noteworthy that an evident decrease in biofilm cells metabolic activity does not always lead to their total destruction and eradication.     

Synthesis,Biological Evaluation and in Silico Study of N-(2- and 3-Pyridinyl)benzamide Derivatives as Quorum Sensing Inhibitors against Pseudomonas aeruginosa

The effectiveness of treating bacterial infections is seriously threatened by the emergence of bacterial resistance to chemical treatment. Growth of microbes in biofilm is one of the main causes of resistance to antimicrobial drugs. Quorum sensing (QS) inhibition, which targets the QS signalling system by obstructing cell-cell communication, was developed as an alternative treatment by creating innovative anti-biofilm drugs. Therefore, the goal of this study is to develop novel antimicrobial drugs that are effective against Pseudomonas aeruginosa by inhibiting QS and acting as anti-biofilm agents. In this study, N-(2- and 3-pyridinyl)benzamide derivatives were selected to design and syntheses. Antibiofilm activity was revealed by all the synthesized compounds and the biofilm was visibly impaired, and the OD_595nm readings of solubilized biofilm cells presented a momentous difference between the treated and untreated biofilms. The best anti-QS zone was observed for compound 5d and found to be 4.96 mm. Through in silico research, the physicochemical characteristics and binding manner of these produced compounds were examined. For the purpose of understanding the stability of the protein and ligand complex, molecular dynamic simulation was also carried out. The overall findings showed that N-(2- and 3-pyridinyl)benzamide derivatives could be the key to creating effective newer anti-quorum sensing drugs that are effective against different bacteria.     

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.     

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 μg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 μg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 μg ml(-1) (FA) and 5000 μg ml(-1) (GA), for L. monocytogenes it was >5000 μg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 μg ml(-1) (FA) and >5000 μg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.     

Biofilm formation by Pseudomonas aeruginosa in solid murine tumors - a novel model system

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds.