A perspective on the safety of parabens as preservatives in wound care products.

Antimicrobial and/or preservative ingredients incorporated in wound care products are subjected to certain safety restrictions. However, several of those agents, and paraben preservatives in particular, have been criticised. Conflicting reports on the potential of parabens to induce allergic contact dermatitis, and their assumed oestrogen-like activity, raised public health concerns about their overall safety. Here, we seek to provide a balanced perspective on the most significant purported adverse health effects, and thereby allay the many misconceptions regarding the safety of parabens. Extensive and long-term monitoring of paraben allergy frequencies illustrate that allergic reactions are quite uncommon, especially when compared with other antimicrobial and preservative agents. The estrogenic potential of parabens was illustrated to be far less potent than that of natural oestrogen receptor ligands, and the etiological significance of their presence in human tissue has not been established. The general consensus based on investigations by both the scientific community and regulatory agencies indicates that, with current safety regulations regarding their use in place, this effective and well-documented group of preservatives should not warrant drastic measures to replace them. As such, despite the ongoing concern, it is indicated that, when used at typical concentrations, parabens are unlikely to affect human health.

Novel injectable gellan gum hydrogel composites incorporating Zn- and Sr-enriched bioactive glass microparticles: High-resolution X-ray microcomputed tomography, antibacterial and in vitro testing.

Mineralization of hydrogel biomaterials is desirable to improve their suitability as materials for bone regeneration. In this study, gellan gum (GG) hydrogels were formed by simple mixing of GG solution with bioactive glass microparticles of 45S5 composition, leading to hydrogel formation by ion release from the amorphous bioactive glass microparticles. This resulted in novel injectable, self-gelling composites of GG hydrogels containing 20% bioactive glass. Gelation occurred within 20 min. Composites containing the standard 45S5 bioactive glass preparation were markedly less stiff. X-ray microcomputed tomography proved to be a highly sensitive technique capable of detecting microparticles of diameter approximately 8 µm, that is, individual microparticles, and accurately visualizing the size distribution of bioactive glass microparticles and their aggregates, and their distribution in GG hydrogels. The widely used melt-derived 45S5 preparation served as a standard and was compared with a calcium-rich, sol-gel derived preparation (A2), as well as A2 enriched with zinc (A2Zn5) and strontium (A2Sr5). A2, A2Zn, and A2Sr bioactive glass particles were more homogeneously dispersed in GG hydrogels than 45S5. Composites containing all four bioactive glass preparations exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. Composites containing A2Zn5 and A2Sr5 bioactive glasses supported the adhesion and growth of osteoblast-like cells and were considerably more cytocompatible than 45S5. All composites underwent mineralization with calcium-deficient hydroxyapatite upon incubation in simulated body fluid. The extent of mineralization appeared to be greatest for composites containing A2Zn5 and 45S5. The results underline the importance of the choice of bioactive glass when preparing injectable, self-gelling composites.

Elucidation of the mechanism behind the potentiating activity of baicalin against Burkholderia cenocepacia biofilms.

Reduced antimicrobial susceptibility due to resistance and tolerance has become a serious threat to human health. An approach to overcome this reduced susceptibility is the use of antibiotic adjuvants, also known as potentiators. These are compounds that have little or no antibacterial effect on their own but increase the susceptibility of bacterial cells towards antimicrobial agents. Baicalin hydrate, previously described as a quorum sensing inhibitor, is such a potentiator that increases the susceptibility of Burkholderia cenocepacia J2315 biofilms towards tobramycin. The goal of the present study is to elucidate the molecular mechanisms behind the potentiating activity of baicalin hydrate and related flavonoids. We first determined the effect of multiple flavonoids on susceptibility of B. cenocepacia J2315 towards tobramycin. Increased antibiotic susceptibility was most pronounced in combination with apigenin 7-O-glucoside and baicalin hydrate. For baicalin hydrate, also other B. cepacia complex strains and other antibiotics were tested. The potentiating effect was only observed for aminoglycosides and was both strain- and aminoglycoside-dependent. Subsequently, gene expression was compared between baicalin hydrate treated and untreated cells, in the presence and absence of tobramycin. This revealed that baicalin hydrate affected cellular respiration, resulting in increased reactive oxygen species production in the presence of tobramycin. We subsequently showed that baicalin hydrate has an impact on oxidative stress via several pathways including oxidative phosphorylation, glucarate metabolism and by modulating biosynthesis of putrescine. Furthermore, our data strongly suggest that the influence of baicalin hydrate on oxidative stress is unrelated to quorum sensing. Our data indicate that the potentiating effect of baicalin hydrate is due to modulating the oxidative stress response, which in turn leads to increased tobramycin-mediated killing.

Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein ß-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20-48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Pitfalls associated with evaluating enzymatic quorum quenching activity: the case of MomL and its effect on Pseudomonas aeruginosa and Acinetobacter baumannii biofilms.

BACKGROUND: The enzymatic degradation of quorums sensing (QS) molecules (called quorum quenching, QQ) has been considered as a promising anti-virulence therapy to treat biofilm-related infections and combat antibiotic resistance. The recently-discovered QQ enzyme MomL has been reported to efficiently degrade different N-acyl homoserine lactones (AHLs) of various Gram-negative pathogens. Here we investigated the effect of MomL on biofilms formed by two important nosocomial pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. METHODS: MomL was expressed in E.coli BL21 and purified. The activity of MomL on AHLs with hydroxyl substituent was tested. Biofilms of P. aeruginosa PAO1 and Acinetobacter strains were formed in 96-well microtiter plates. Biofilm formation was evaluated by crystal violet staining, plating and fluorescence microscopy. The effect of MomL on biofilm susceptibility to antibiotics was also tested. We further evaluated MomL in dual-species biofilms formed by P. aeruginosa and A. baumannii, and in biofilms formed in a wound model. The effect of MomL on virulence of A. baumannii was also tested in the Caenorhabditis elegans model. RESULTS: MomL reduced biofilm formation and increased biofilm susceptibility to different antibiotics in biofilms of P. aeruginosa PAO1 and A. baumannii LMG 10531 formed in microtiter plates in vitro. However, no significant differences were detected in the dual-species biofilm and in wound model biofilms. In addition, MomL did not affect virulence of A. baumannii in the C. elegans model. Finally, the effect of MomL on biofilm of Acinetobacter strains seems to be strain-dependent. DISCUSSION: Our results indicate that although MomL showed a promising anti-biofilm effect against P. aeruginosa and A. baumanii biofilms formed in microtiter plates, the effect on biofilm formation under conditions more likely to mimic the real-life situation was much less pronounced or even absent. Our data indicate that in order to obtain a better picture of potential applicability of QQ enzymes for the treatment of biofilm-related infections, more elaborate model systems need to be used.

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models.

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

Screening a repurposing library for potentiators of antibiotics against Staphylococcus aureus biofilms.

Staphylococcus aureus biofilms are involved in a wide range of infections that are extremely difficult to treat with conventional antibiotic therapy. We aimed to identify potentiators of antibiotics against mature biofilms of S. aureus Mu50, a methicillin-resistant and vancomycin-intermediate-resistant strain. Over 700 off-patent drugs from a repurposing library were screened in combination with vancomycin in a microtitre plate (MTP)-based biofilm model system. This led to the identification of 25 hit compounds, including four phenothiazines among which thioridazine was the most potent. Their activity was evaluated in combination with other antibiotics both against planktonic and biofilm-grown S. aureus cells. The most promising combinations were subsequently tested in an in vitro chronic wound biofilm infection model. Although no synergistic activity was observed against planktonic cells, thioridazine potentiated the activity of tobramycin, linezolid and flucloxacillin against S. aureus biofilm cells. However, this effect was only observed in a general biofilm model and not in a chronic wound model of biofilm infection. Several drug compounds were identified that potentiated the activity of vancomycin against biofilms formed in a MTP-based biofilm model. A selected hit compound lost its potentiating activity in a model that mimics specific aspects of wound biofilms. This study provides a platform for discovering and evaluating potentiators against bacterial biofilms and highlights the necessity of using relevant in vitro biofilm model systems.

Ca:Mg:Zn:CO3 and Ca:Mg:CO3-tri- and bi-elemental carbonate microparticles for novel injectable self-gelling hydrogel-microparticle composites for tissue regeneration.

Injectable composites for tissue regeneration can be developed by dispersion of inorganic microparticles and cells in a hydrogel phase. In this study, multifunctional carbonate microparticles containing different amounts of calcium, magnesium and zinc were mixed with solutions of gellan gum (GG), an anionic polysaccharide, to form injectable hydrogel-microparticle composites, containing Zn, Ca and Mg. Zn and Ca were incorporated into microparticle preparations to a greater extent than Mg. Microparticle groups were heterogeneous and contained microparticles of differing shape and elemental composition. Zn-rich microparticles were 'star shaped' and appeared to consist of small crystallites, while Zn-poor, Ca- and Mg-rich microparticles were irregular in shape and appeared to contain lager crystallites. Zn-free microparticle groups exhibited the best cytocompatibility and, unexpectedly, Zn-free composites showed the highest antibacterial activity towards methicilin-resistant Staphylococcus aureus. Composites containing Zn-free microparticles were cytocompatible and therefore appear most suitable for applications as an injectable biomaterial. This study proves the principle of creating bi- and tri-elemental microparticles to induce the gelation of GG to create injectable hydrogel-microparticle composites.

Novel Potentiators for Vancomycin in the Treatment of Biofilm-Related MRSA Infections via a Mix and Match Approach.

A library of 52 hamamelitannin analogues was synthesized and investigated for its ability to potentiate the effect of vancomycin toward Staphylococcus aureus biofilms. Several compounds were found to effectively increase the susceptibility of staphylococcal biofilms toward this glycopeptide. The most active analogue identified in this study showed an EC50 value of 0.26 µM.

Composites of gellan gum hydrogel enzymatically mineralized with calcium-zinc phosphate for bone regeneration with antibacterial activity.

Gellan gum hydrogels functionalized with alkaline phosphatase were enzymatically mineralized with phosphates in mineralization medium containing calcium (Ca) and zinc (Zn) to improve their suitability as biomaterials for bone regeneration. The aims of the study were to endow mineralized hydrogels with antibacterial activity by incorporation of Zn in the inorganic phase, and to investigate the effect of Zn incorporation on the amount and type of mineral formed, the compressive modulus of the mineralized hydrogels and on their ability to support adhesion and growth of MC3T3-E1 osteoblast-like cells. Mineralization medium contained glycerophosphate (0.05 m) and three different molar Ca:Zn ratios, 0.05:0, 0.04:0.01 and 0.025:0.025 (all mol/dm3 ), hereafter referred to as A, B and C, respectively. FTIR, SAED and TEM analysis revealed that incubation for 14 days caused the formation of predominantly amorphous mineral phases in sample groups A, B and C. The presence of Zn in sample groups B and C was associated with a drop in the amount of mineral formed and a smaller mineral deposit morphology, as observed by SEM. ICP-OES revealed that Zn was preferentially incorporated into mineral compared to Ca. Mechanical testing revealed a decrease in compressive modulus in sample group C. Sample groups B and C, but not A, showed antibacterial activity against biofilm-forming, methicillin-resistant Staphylococcus aureus. All sample groups supported cell growth. Zn incorporation increased the viable cell number. The highest values were seen on sample group C. In conclusion, the sample group containing the most Zn, i.e. group C, appears to be the most promising. Copyright © 2015 John Wiley & Sons, Ltd.

Novel hamamelitannin analogues for the treatment of biofilm related MRSA infections-A scaffold hopping approach.

Antimicrobial research is increasingly being focused on the problem of resistance and biofilm formation. Hamamelitannin (HAM) was recently identified as an antimicrobial potentiator for conventional antibiotics towards Staphylococcus aureus. This paper describes the synthesis and biological evaluation of novel hamamelitannin analogues with alternative central scaffolds. Via a ligand-based approach, several interesting compounds with improved synthetic accessibility were identified as potentiators for vancomycin in the treatment of MRSA infections.

Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo.

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia.

Enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich Ecklonia cava extract Seanol(®) to endow antibacterial properties and promote mineralization.

Hydrogels offer several advantages as biomaterials for bone regeneration, including ease of incorporation of soluble substances such as mineralization-promoting enzymes and antibacterial agents. Mineralization with calcium phosphate (CaP) increases bioactivity, while antibacterial activity reduces the risk of infection. Here, gellan gum (GG) hydrogels were enriched with alkaline phosphatase (ALP) and/or Seanol(®), a seaweed extract rich in phlorotannins (brown algae-derived polyphenols), to induce mineralization with CaP and increase antibacterial activity, respectively. The sample groups were unmineralized hydrogels, denoted as GG, GG/ALP, GG/Seanol and GG/Seanol/ALP, and hydrogels incubated in mineralization medium (0.1 M calcium glycerophosphate), denoted as GG/ALP_min, GG/Seanol_min and GG/Seanol/ALP_min. Seanol(®) enhanced mineralization with CaP and also increased compressive modulus. Seanol(®) and ALP interacted in a non-covalent manner. Release of Seanol(®) occurred in a burst phase and was impeded by ALP-mediated mineralization. Groups GG/Seanol and GG/ALP/Seanol exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. GG/Seanol/ALP_min, but not GG/Seanol_min, retained some antibacterial activity. Eluates taken from groups GG/ALP_min, GG/Seanol_min and GG/ALP/Seanol_min displayed comparable cytotoxicity towards MG-63 osteoblast-like cells. These results suggest that enrichment of hydrogel biomaterials with phlorotannin-rich extracts is a promising strategy to increase mineralizability and antibacterial activity.

Design, synthesis and biological evaluation of novel hamamelitannin analogues as potentiators for vancomycin in the treatment of biofilm related Staphylococcus aureus infections.

Staphylococcus aureus is a frequent cause of biofilm-related infections. Bacterial cells within a biofilm are protected from attack by the immune system and conventional antibiotics often fail to penetrate the biofilm matrix. The discovery of hamamelitannin as a potentiator for antibiotics, recently led to the design of a more drug-like lead. In the present study, we want to gain further insight into the structure-activity relationship (S.A.R.) of the 5-position of the molecule, by preparing a library of 21 hamamelitannin analogues.

Hamamelitannin Analogues that Modulate Quorum Sensing as Potentiators of Antibiotics against Staphylococcus aureus.

The modulation of bacterial communication to potentiate the effect of existing antimicrobial drugs is a promising alternative to the development of novel antibiotics. In the present study, we synthesized 58 analogues of hamamelitannin (HAM), a quorum sensing inhibitor and antimicrobial potentiator. These efforts resulted in the identification of an analogue that increases the susceptibility of Staphylococcus aureus towards antibiotics in vitro, in Caenorhabditis elegans, and in a mouse mammary gland infection model, without showing cytotoxicity.

The Quorum Sensing Inhibitor Hamamelitannin Increases Antibiotic Susceptibility of Staphylococcus aureus Biofilms by Affecting Peptidoglycan Biosynthesis and eDNA Release.

Treatment of Staphylococcus aureus infections has become increasingly challenging due to the rapid emergence and dissemination of methicillin-resistant strains. In addition, S. aureus reside within biofilms at the site of infection. Few novel antibacterial agents have been developed in recent years and their bacteriostatic or bactericidal activity results in selective pressure, inevitably inducing antimicrobial resistance. Consequently, innovative antimicrobials with other modes of action are urgently needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. Hamamelitannin (2',5-di-O-galloyl-d-hamamelose; HAM) was previously suggested to block QS through the TraP QS system and was shown to increase S. aureus biofilm susceptibility towards vancomycin (VAN) although mechanistic insights are still lacking. In the present study we provide evidence that HAM specifically affects S. aureus biofilm susceptibility through the TraP receptor by affecting cell wall synthesis and extracellular DNA release of S. aureus. We further provide evidence that HAM can increase the susceptibility of S. aureus biofilms towards different classes of antibiotics in vitro. Finally, we show that HAM increases the susceptibility of S. aureus to antibiotic treatment in in vivo Caenorhabditis elegans and mouse mammary gland infection models.

Dressings Loaded with Cyclodextrin-Hamamelitannin Complexes Increase Staphylococcus aureus Susceptibility Toward Antibiotics Both in Single as well as in Mixed Biofilm Communities.

Bacteria reside within biofilms at the infection site, making them extremely difficult to eradicate with conventional wound care products. Bacteria use quorum sensing (QS) systems to regulate biofilm formation, and QS inhibitors (QSIs) have been proposed as promising antibiofilm agents. Despite this, few antimicrobial therapies that interfere with QS exist. Nontoxic hydroxypropyl-ß-cyclodextrin-functionalized cellulose gauzes releasing a burst of the antibiotic vancomycin and the QSI hamamelitannin are developed, followed by a sustained release of both. The gauzes affect QS and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro model of chronic wound infection and can be considered as candidates to be used to prevent wound infection as well as treat infected wounds.

In Vitro and In Vivo Biofilm Wound Models and Their Application.

Chronic wounds are wounds which are detained in one or more phases of normal wound healing. It is estimated that 1-2 % of the population of developed countries will experience a chronic wound during their lifetime and this number is expected to increase given the growing world population, increase in age, body mass index and associated diseases such as diabetes and cardiovascular diseases. Although several factors contribute to wound healing, presence of bacterial biofilms significantly affects healing and success of wound treatment. This indicates that wound-care therapies should be directed towards targeting biofilms within chronic wounds. Despite this, the role of biofilms in chronic wound pathogenesis and the effect of wound-care therapies against biofilms within wounds are not well understood. In order to address these issues, appropriate biofilm models are necessary. To this end, several model systems mimicking the conditions observed in a biofilm infected chronic wound have been developed. In this review we present an overview of these different in vitro and in vivo biofilm wound model systems and discuss their advantages and disadvantages.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Inhibition of Quorum Sensing in Staphylococcus spp.

The Gram-positive, facultative anaerobic coccus-shaped bacteria of the genus Staphylococcus are among the most important causative agents of acute and chronic bacterial infections in humans as well as in animals. Treatment of Staphylococcus infections has become increasingly challenging due to the growing problem of antibiotic resistance. For this reason innovative antimicrobials with novel targets and modes of action are needed. Since the discovery that QS is used by Staphylococcus spp. to coordinate the expression of several genes involved in virulence, biofilm formation and pathogenicity, QS inhibition has gained increasing attention as an alternative anti-pathogenic strategy. A major advantage compared with antibiotic therapy is that QSIs are used in concentrations that do not affect bacterial growth. For this reason, it is expected that these compounds would exert less pressure towards the development of resistance. However, some important points still need to be addressed. Although several inhibitors have proven to be active antipathogenic agents in vitro and in several in vivo models, it is still unknown whether these compounds will also be useful in humans. Furthermore, several fundamental mechanisms by which the different QS systems in Staphylococcus spp. exert their regulatory functions and how they are inhibited by QSIs are still poorly understood. In order to achieve real-life applications with QSIs, these challenges should be addressed and more research will be needed. In this article, we will discuss the different QS systems present in Staphylococcus spp., how they are used to control virulence and biofilm formation and how they can be blocked.