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Covering: 2009 to 2021Antimicrobial resistance is now rising to dangerously high levels in all parts of the world, threatening the treatment of an ever-increasing range of infectious diseases. This has becoming a serious public health problem, especially due to the emergence of multidrug-resistance among clinically important bacterial species and their ability to form biofilms. In addition, current anti-infective therapies have low efficacy in the treatment of biofilm-related infections, leading to recurrence, chronicity, and increased morbidity and mortality. Therefore, it is necessary to search for innovative strategies/antibacterial agents capable of overcoming the limitations of conventional antibiotics. Natural compounds, in particular those obtained from plants, have been exhibiting promising properties in this field. Plant secondary metabolites (phytochemicals) can act as antibiofilm agents through different mechanisms of action from the available antibiotics (inhibition of quorum-sensing, motility, adhesion, and reactive oxygen species production, among others). The combination of different phytochemicals and antibiotics have revealed synergistic or additive effects in biofilm control. This review aims to bring together the most relevant reports on the antibiofilm properties of phytochemicals, as well as insights into their structure and mechanistic action against bacterial pathogens, spanning December 2008 to December 2021.

Diabetic foot ulcers (DFUs) are a common result of a complex secondary complication of diabetes mellitus. More than half of DFUs become infected due to frequent colonization with Staphylococcus aureus. The use of topical antibiotics is proposed, especially in combination with natural adjuvants, to minimize the negative impacts caused by generalized use of systemic antibiotics. In this study, 13 different phytochemicals—namely chalcone, juglone, cinnamic acid, trigonelline, Furvina—and four nitrovinylfuran derivatives—guaiazulene, α-bisabolol, farnesol and nerolidol—were selected to be tested as antibiotic enhancers. After minimum inhibitory and bactericidal concentration (MIC and MBC) determination of each molecule against different strains of S. aureus, including clinical isolates from diabetic foot wounds (CECT 976, Xu212, SA 1199B, RN4220, MJMC102, MJMC109, MJMC110 and MJMC111), their potentiation effects on the antibiotics fusidic acid, mupirocin, gentamicin, oxacillin and methicillin were evaluated through the disc diffusion method. Farnesol at sub-MIC was able to restore the activity of methicillin and oxacillin on the MJMC102 and MJMC111 strains, as well as two MRSA clinical isolates, and potentiated the effect of the remaining antibiotics. The results obtained demonstrate the great potential for the topical application of phytochemicals and derivatives as antibiotic resistance modifier agents to combat multidrug resistance in bacterial wound infections.

The effect that the hydrodynamic conditions under which biofilms are formed has on their persistence is still unknown. This study assessed the behaviour of Pseudomonas fluorescens biofilms, formed on stainless steel under different shear stress (sw) conditions (1, 2 and 4 Pa), to chemical (benzalkonium chloride – BAC, glutaraldehyde – GLUT and sodium hypochlorite – SHC) and mechanical (20 Pa) treatments (alone and combined). The biofilms formed under different sw showed different structural characteristics. Those formed under a higher sw were invariably more tolerant to chemical and mechanical stresses. SHC was the biocide which caused the highest biofilm killing and removal, followed by BAC. The sequential exposure to biocides and mechanical stress was found to be insufficient for effective biofilm control. A basal layer containing biofilm cells mostly in a viable state remained on the surface of the cylinders, particularly for the 2 and 4 Pa-generated biofilms., This work was financially supported by: Base Funding – UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE – funded by national funds through the FCT/ MCTES (PIDDAC); Projects Biocide_for_Biofilm – PTDC/ BII-BTI/30219/2017 – POCI-01-0145-FEDER-030219; Germirrad – POCI-01-0247-FEDER-072237, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalizac¸~ao (POCI) and by national funds (PIDDAC) through FCT/MCTES. PRESAGE – Aquatic/0007/2020 – from the programme ERA-NET – European Research Area networks. This study was further supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the stra tegic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. Anabela Borges and Henrique Sousa thank the Portuguese Foundation for Science and Technology (FCT) for financial support for their work through the Scientific Employment Stimulus – Individual Call – [CEECIND/01261/2017] and through the research grant for a doctoral degree (2020.09538.BD), respectively., info:eu-repo/semantics/publishedVersion

Diabetic foot ulcers (DFUs) are a serious complication from diabetes mellitus, with a huge economic, social and psychological impact on the patients life. One of the main reasons why DFUs are so difficult to heal is related to the presence of biofilms. Biofilms promote wound inflammation and a remarkable lack of response to host defences/treatment options, which can lead to disease progression and chronicity. In fact, appropriate treatment for the elimination of these microbial communities can prevent the disease evolution and, in some cases, even avoid more serious outcomes, such as amputation or death. However, the detection of biofilm-associated DFUs is difficult due to the lack of methods for diagnostics in clinical settings. In this review, the current knowledge on the involvement of biofilms in DFUs is discussed, as well as how the surrounding environment influences biofilm formation and regulation, along with its clinical implications. A special focus is also given to biofilm-associated DFU diagnosis and therapeutic strategies. An overview on promising alternative therapeutics is provided and an algorithm considering biofilm detection and treatment is proposed., This work was supported by: Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE—funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm-PTDC/BII-BTI/30219/2017- POCI-01-0145-FEDER-030219, ABFISH–PTDC/ASP-PES/28397/2017-POCI-01-0145-FEDER- 028397 and Germirrad-POCI-01-0247-FEDER-072237, funded by FEDER funds through COMPETE2020— Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PID DAC) through FCT/MCTES. The authors also thank the CITAB (Centre for the Research and Technol ogy of Agro-Environmental and Biological Sciences) under the scope of the FCT funds with reference UIDB/AGR/04033/2020. Ana Afonso (2020.04773.BD) and Diana Oliveira (SFRH/BD/138217/2018) acknowledge the FCT grants. Anabela Borges thanks the FCT for the financial support of their work contract through the Scientific Employment Stimulus—Individual Call—[CEECIND/01261/2017]., info:eu-repo/semantics/publishedVersion

This study sought to assess the prevalence and impact of biofilms on two commonly biofilm-related infections, bloodstream and urinary tract infections (BSI and UTI). Separated systematic reviews and meta-analyses of observational studies were carried out in PubMed and Web of Sciences databases from January 2005 to May 2020, following PRISMA protocols. Studies were selected according to specific and defined inclusion/exclusion criteria. The obtained outcomes were grouped into biofilm production (BFP) prevalence, BFP in resistant vs. susceptible strains, persistent vs. non-persistent BSI, survivor vs. non-survivor patients with BSI, and catheter-associated UTI (CAUTI) vs. non-CAUTI. Single-arm and two-arm analyses were conducted for data analysis. In vitro BFP in BSI was highly related to resistant strains (odds ratio-OR: 2.68, 95% confidence intervals-CI: 1.60–4.47, p &lt, 0.01), especially for methicillin-resistant Staphylococci. BFP was also highly linked to BSI persistence (OR: 2.65, 95% CI: 1.28–5.48, 0.01) and even to mortality (OR: 2.05, 95% CI: 1.53–2.74, 0.01). Candida spp. was the microorganism group where the highest associations were observed. Biofilms seem to impact Candida BSI independently from clinical differences, including treatment interventions. Regarding UTI, multi-drug resistant and extended-spectrum β-lactamase-producing strains of Escherichia coli, were linked to a great BFP prevalence (OR: 2.92, 95% CI: 1.30–6.54, 0.01 and OR: 2.80, 95% CI: 1.33–5.86, 0.01). More in vitro BFP was shown in CAUTI compared to non-CAUTI, but with less statistical confidence (OR: 2.61, 95% CI: 0.67–10.17, 0.17). This study highlights that biofilms must be recognized as a BSI and UTI resistance factor as well as a BSI virulence factor.

Biofilm control is mainly based on chemical disinfection, without a clear understanding of the role of the biocides and process conditions on biofilm removal. This study aims to understand the effects of a biocide (benzyldimethyldodecyl ammonium chlorideBDMDAC) and mechanical treatment (an increase of shear stress -w) on single- and dual-species biofilms formed by Bacillus cereus and Pseudomonas fluorescens on high-density polyethene (HDPE). BDMDAC effects were initially assessed on bacterial physicochemical properties and initial adhesion ability. Then, mature biofilms were formed on a rotating cylinder reactor (RCR) for 7 days to assess the effects of chemical and mechanical treatments, and the combination of both on biofilm removal. The results demonstrated that the initial adhesion does not predict the formation of mature biofilms. It was observed that the dual-species biofilms were the most susceptible to BDMDAC exposure. The exposure to increasing w emphasised the mechanical stability of biofilms, as lower values of w (1.66 Pa) caused high biofilm erosion and higher w values (17.7 Pa) seem to compress the remaining biofilm. In general, the combination of BDMDAC and the mechanical treatment was synergic in increasing biofilm removal. However, these were insufficient to cause total biofilm removal (100%; an average standard deviation of 11% for the method accuracy should be considered) from HDPE., This work was financially supported by: Base Funding—UIDB/00511/2020 of LEP-ABE funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm—TDC/BII-BTI/30219/2017—POCI-01-0145-FEDER-030219; Germirrad—POCI-01-0247-FEDER-072237; pBio4.0—POCI-01-0247-FEDER-033298, funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES. This study was further supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Persistent infections, usually associated with biofilm-producing bacteria, are challenging for both medical and scientific communities. The potential interest in drug repurposing for biofilm control is growing due to both disinvestment in antibiotic R&amp, D and reduced efficacy of the available panel of antibiotics. In the present study, the antibacterial and antibiofilm activities of four non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam (PXC), diclofenac sodium (DCF), acetylsalicylic acid (ASA) and naproxen sodium (NPX) were evaluated against Escherichia coli and Staphylococcus aureus. The minimum inhibitory/bactericidal concentrations (MICs and MBCs) and the dose&ndash, response curves from exposure to the selected NSAIDs were determined. MICs were found for PXC (800 &mu, g/mL) and ASA (1750 &mu, g/mL) against E. coli, and for DCF (2000 &mu, g/mL) and ASA (2000 &mu, g/mL) against S. aureus. No MBCs were found (&gt, 2000 &mu, g/mL). The potential of NSAIDs to eradicate preformed biofilms was characterized in terms of biofilm mass, metabolic activity and cell culturability. Additionally, the NSAIDs were tested in combination with kanamycin (KAN) and tetracycline (TET). ASA, DCF and PXC promoted significant reductions in metabolic activity and culturability. However, only PXC promoted biofilm mass removal. Additive interactions were obtained for most of the combinations between NSAIDs and KAN or TET. In general, NSAIDs appear to be a promising strategy to control biofilms as they demonstrated to be more effective than conventional antibiotics.

Research on the discovery of new drugs to treat bacterial biofilm infections has been a priority for biomedical and scientific communities. Actually, the sessile mode of growth accounts for more than 80% of human bacterial infections and displays increased resistance to antibiotics. To date, there are no drugs with total efficacy against biofilms and the treatment failure is a recurrent clinical situation. This lack of alternatives boosted up again the exploration of the pharmacological properties of health-promoting agents from natural origin. In this connection, the potential of phytotherapeutic agents for the treatment/prevention of complex infectious diseases, such as those that involve biofilm formation has been intensified[1]. In view of that, the main aim of the present study was to evaluate the activity of four phytochemicals bellowing to terpenoids class [cis-6-nonen-1-ol (CIS), citronellic acid (CITACID), citronellol (CITRO) and 3-7-dimethyl-1-octanol (3,7DOC)] against Staphylococcus aureus, both in planktonic and sessile state. Firstly, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined by the broth microdilution method and culturability on plate count agar, respectively. Then, the potential of each terpenoid as resistance modifying agent was assessed by the disc diffusion method, using antibiotics from different classes. Besides, its potential to eradicate pre-formed S. aureus biofilms (24-h old) was performed using a microtiter plate assay and characterized in terms of biofilm mass removal (crystal violet staining), metabolic activity reduction (alamar blue staining) and culturability (colony forming units - CFU - counts). Considering that the selected terpenoids are chemical structurally related, i.e. present a similar backbone and differ only on the functional groups location, a structure activity relationship (SAR) analysis was also established. Both CITO and 3,7DOC presented the lowest MIC value (200 μg/mL) followed by CIS (400 μg/mL) and CITACID (1000 μg/mL). The MBC was found to be 1000 µg/mL with CIS, 2000 µg/mL with CITACID and > 2000 µg/mL (the maximum concentration tested) with CITO/3,7DOC. Apparently, the hydrophobicity of the molecules appear to affect positively its inhibitory proprieties – molecules with higher hydrophobicity presented lower MIC values. Moreover, it seems that the hydroxyl and methyl functional groups play the major influence on the antimicrobial properties. Indeed, CITO and 3,7DOC presented the higher hydrophobicity values and both had hydroxyl and methyl functional groups, possessing the lowest MIC value. Independently of the terpenoid tested, all combinations (terpenoid-antibiotic) resulted in a potentiation effect. Regarding biofilm eradication, although no biomass removal was observed, metabolic activity reductions from 25% (CIS at 5×MIC) up to 44% (CITACID at 10×MIC) and total loss of culturability (CITACID at 10×MIC; 6-log CFU/cm2 reduction) was found. These effects were found to be dose dependent. Overall, the results obtained suggest that all the tested terpenoids might be interesting antibiotic adjuvants and emphasize the use of CITACID for biofilm cells inactivation. The results obtained are promising since the terpenoids studied are natural occurring flavoring ingredients, generally recognized as safe by the FDA, which are usually applied as food additives for human consumption. References: [1] A.Borges, A.Abreu, C.Dias, M.Saavedra, F.Borges, M.Simões, Molecules 21(2016)877.

Chronic skin wounds are disruptions of the skin that remain in the inflammatory state for more than one month. They affect approximately 3% of the people with more than 60 years and are mostly associated with diabetic foot, pressure ulcers, blood vessels or trauma1. Besides that, it is expected that 578 million people worldwide will suffer from the disease by 2030, that will subsequently increase the risk of developing infection and potentially result in amputation or osteomyelitis2. One of the main difficulties in the wound healing process is the fact that bacteria persist due to biofilm formation. Biofilms delay not only healing, but they also make bacteria more resistant to the antimicrobial therapy. Among the bacteria present at the wound site, Staphylococcus aureus was found to be one of the most prevalent biofilm producers3. In this sense, other alternatives to fight biofilm infections should be considered due to the resistance to the current antibiotics. One possible strategy consists of using antibiotic adjuvants to enhance the activity of current drugs and to minimize or even block resistance. For that, plants are used as a resource of such adjuvant compounds. The use of natural compounds from plants have been applied in skin wound care for millennials, generating a lot of interest from the scientific community. Indeed, not only are phytochemicals great antibiotic potentiators but they also possess numerous therapeutic properties4. Therefore, in this study, the phenolic compound juglone (5-hydroxy-1,4-naphtoquinone) was investigated, alone and combined with fusidic acid antibiotic, for its potential to eradicate pre-formed S. aureus biofilms. Although no biomass removal was observed, there was a total loss of cell culturability (about 6-log CFU/cm2 reduction) and a considerable metabolic activity reduction. Juglone reduced metabolic activity by 83% both alone and in combination with fusidic acid, which is an improvement over the 70% reduction obtained by the antibiotic. Therefore, an additive interaction of juglone when combined with fusidic acid was attained in the control of S. aureus biofilms. In addition, according with Lipinsksi’s rule of five, it was assessed that juglone possesses important molecular properties with respect to pharmacokinetics in the human body. Overall, this study reveals the great potential for the topical application of juglone as an adjuvant to the widely used fusidic acid to combat multi-drug resistant wound infections. References: (1) Manu, C., et al. J. Wound Care 27.3 (2018): 186–192. (2) International Diabetes Federation. https://www.diabetesatlas.org (accessed May 27, 2020). (3) Kadam, S., et al. Biomedicines 7.2 (2019): 35. (4) Borges, A.; et al. Molecules 21.7 (2016): 877. Acknowledgements: This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC); - PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219; POCI-01-0247-FEDER-035234; POCI-01-145-FEDER-006939, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through CEECIND/01261/2017 (Anabela Borges), SFRH/BD/138217/2018 (Diana Oliveira) and SFRH/BSAB/150379/2019 (Manuel Simões).

Ethnopharmacological use of plant natural extracts has been known since ancient times. The optimization of plant molecule extraction is fundamental in obtaining relevant extraction yields. The main purpose of this study was to understand the role of different extraction techniques (solid-liquid, ultrasound, Soxhlet, and microwave) and solvents (water, methanol, ethanol, acetone, dichloromethane, and hexane) on the antimicrobial and antioxidant activities of extracts from Olea europaea (olive) and Acacia dealbata (mimosa). Crude plant extracts were evaluated for their antimicrobial activity against Staphylococcus aureus and Escherichia coli by the disk diffusion method. The antioxidant capacity of the extracts was determined by ABTS (2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) methods. In terms of extraction yield, ultrasound extraction and the solvents methanol, acetone (O. europaea) or water (A. dealbata) were found to be the best options. However, ethanol and acetone proved to be the best solvents to extract compounds with antimicrobial activity and antioxidant capacity, respectively (regardless of the extraction method employed). Soxhlet and microwave were the best techniques to extract compounds with antimicrobial activity, whereas any of the tested techniques showed the ability to extract compounds with antioxidant capacity. In most of the cases, both plant extracts (mimosa and olive) were more efficient against S. aureus than E. coli. In the present study, both mimosa and olive leaf crude extracts proved to have antimicrobial and antioxidant activities, increasing the demand of these natural products as a source of compounds with health benefits.

The emergence of multidrug-resistant microorganisms in persistent biofilm infections represents a public health threat. The limited efficacy of currently available antimicrobials has been imposing the search for new approaches to overcome bacterial and fungal biofilm infections. Drug repurposing arises as an interesting strategy that can reduce time, cost, and risk associated with drug innovation. This can be achieved by finding new applications beyond the original medical indication for approved or investigational drugs. This chapter collects the antimicrobial effects known so far against bacteria and fungi caused by different classes of drugs, including antineoplastic, antipsychotic, antidepressant, and antiinflammatory, among others. Besides the action on microbial growth in vitro and in vivo, some of these drugs are also able to synergize with traditional antibiotics and antifungals. Finally, a special focus on antibiofilm activity was included, reveling the promising drugs for biofilm prevention and control.

Microorganisms have the ability to multiply, attach to wet surfaces, and produce a matrix of extracellular polymeric substances (EPS), forming biofilms. The development of biofilms occurs on almost all surfaces (inanimate and living) and can cause serious problems in medical field, food industry, and systems. The multiple layers of cells and EPS confer to biofilms a complex and compact structure that hinders penetration of antimicrobial agents, decreasing their capacity to reach internal layers, and rendering them ineffective. Thus, when microorganisms form biofilms, the resistance to conventional antimicrobial agents increases significantly, and the control of those biofilms becomes hard to achieve. Besides, due to the complexity/heterogeneity of sessile communities, the application of a single strategy is considered insufficient. In this context, there is a need for new strategies, new antimicrobials, and biofilm prevention/control techniques. An ideal antibiofilm strategy should involve approaches to prevent biofilm formation, disintegrate biofilm structure, or remove preformed biofilms. Promising approaches can be based on the application of “green” and biological-based products, such as enzymes. Enzymes are applied in many fields of investigation and are known to be capable of degrading key components of the biofilm matrix. Moreover, it is recognized that the combination of enzymes with other biofilm control agents can enhance their activity. However, enzymes are specific, and to degrade the complex matrix that involves the biofilm, a mixture of enzymes is often necessary. In this chapter, the factors that play a crucial role in the resistance of biofilm-associated bacteria will be addressed. In addition, emergent strategies for biofilm control and eradication will be highlighted. As the use of biofilm-disrupting enzymes appears to be a very promising option to effectively control and eradicate microbial biofilms, a literature review for this strategy will be presented.

Foodborne microbial diseases are still considered a growing public health problem worldwide despite the global continuous efforts to ensure food safety. The traditional chemical and thermal-based procedures applied for microbial growth control in the food industry can change the food matrix and lead to antimicrobial resistance. Moreover, currently applied disinfectants have limited efficiency against biofilms. Therefore, antimicrobial photodynamic therapy (aPDT) has become a novel alternative for controlling foodborne pathogenic bacteria in both planktonic and sessile states. The use of aPDT in the food sector is attractive as it is less likely to cause antimicrobial resistance and it does not promote undesirable nutritional and sensory changes in the food matrix. In this review, aspects on the antimicrobial photodynamic technology applied against foodborne pathogenic bacteria and studied in recent years are presented. The application of photodynamic inactivation as an antibiofilm strategy is also reviewed.

Antibiotic resistance has been increasingly reported for a wide variety of bacteria of clinical significance. This widespread problem constitutes one of the greatest challenges of the twenty-first century. Faced with this issue, clinicians and researchers have been persuaded to design novel strategies in order to try to control pathogenic bacteria. Therefore, the discovery and elucidation of the mechanisms underlying bacterial pathogenesis and intercellular communication have opened new perspectives for the development of alternative approaches. Antipathogenic and/or antivirulence therapies based on the interruption of quorum sensing pathways are one of several such promising strategies aimed at disarming rather than at eradicating bacterial pathogens during the course of colonization and infection. This review describes mechanisms of bacterial communication involved in biofilm formation. An overview of the potential of marine bacteria and their bioactive components as QS inhibitors is further provided.

Drug repurposing arises as an interesting alternative to overcome the limited efficacy of current available antibiotics by reducing time, cost and risk associated with drug innovation. In this study, the activity of ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), was evaluated on the control of pre-established adhered cells (2 h) and 24 h old biofilms of Staphylococcus aureus. Bacterial adhesion was performed by S. aureus CECT 976, while biofilm formation was also performed by the antibiotic-resistant strains: SA1199B, RN4220 and XU212. The treatment with ibuprofen promoted metabolic reductions up to 80% and total loss of culturability of adhered cells and 24 h old biofilms. Ibuprofen demonstrated moderate efficacy to remove biofilms of S. aureus CECT 976 (removal ≤ 40%), but did not display removal action against the antibiotic-resistant strains. Antibacterial activity (inhibitory and bactericidal) and mode of action of ibuprofen were also assessed using different bacterial physiological indices. Minimum inhibitory and bactericidal concentrations of the four S. aureus strains ranged between 500 and 2000 mg L−1 and 1400 - >2000 mg L−1, respectively. After S. aureus CECT 976 incubation with ibuprofen, cell permeation to propidium iodide, release of intracellular potassium and changes on cell surface hydrophobicity were observed. The overall results, demonstrate that ibuprofen can control S. aureus planktonic and sessile growth, with strong destabilizing and disrupting action on the cytoplasmic membrane.

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1&ndash, 4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.

Biofilms are encountered on nearly all wet surfaces, with their development being often unwanted due to the serious problems they can cause in different fields, including in the food sector. They are recognized as the preferential microbial lifestyle due to the numerous advantages for the embedded cells. Biofilm cells are highly resistant to stress conditions, particularly to antimicrobials, as their complex and compact structure hampers the penetration of antimicrobials and the access to the deep positioned cells. The increased resistance to the currently employed control strategies emphasizes the urgent need of new alternative and/or complementary eradication approaches. To this direction, the use of enzymes is an interesting alternative anti-biofilm approach due to their capability to degrade crucial components of the biofilm matrix, cause cell lysis, promote biofilm disruption and interrupt the cell-to-cell signaling events governing biofilm formation and maintenance. This review provides an overview of the enzymes used for biofilm control, their targets and examples of effective applications.

Pseudomonas aeruginosa is one of the most dreaded human pathogens, because of its intrinsic resistance to a number of commonly used antibiotics and ability to form sessile communities (biofilms). Innovative treatment strategies are required and that can rely on the attenuation of the pathogenicity and virulence traits. The interruption of the mechanisms of intercellular communication in bacteria (quorum sensing) is one of such promising strategies. A cobalt coordination compound (Co(HL)(2)) synthesized from (E)-2-(2-(pyridin-2-ylmethylene) hydrazinyl)-4-(p-tolyl) thiazole (HL) is reported herein for the first time to inhibit P. aeruginosa 3-oxo-C12-HSL-dependent QS system (LasI/LasR system) and underling phenotypes (biofilm formation and virulence factors). Its interactions with a possible target, the transcriptional activator protein complex LasR-3-oxo-C12-HSL, was studied by molecular modeling with the coordination compound ligand having stronger predicted interactions than those of co-crystallized ligand 3-oxo-C12-HSL, as well as known-binder furvina. Transition metal group 9 coordination compounds may be explored in antipathogenic/antibacterial drug design. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3141]

Staphylococcus aureus is a microorganism resident in the skin and nasal membranes with a dreadful pathogenic potential to cause a variety of community and hospital-acquired infections. The frequency of these infections is increasing and their treatment is becoming more difficult. The ability of S. aureus to form biofilms and the emergence of multidrug-resistant strains are the main reasons determining the challenge in dealing with these infections. S. aureus' infectious capacity and its success as a pathogen is related to the expression of virulence factors, among which the production of a wide variety of toxins is highlighted. For this reason, a better understanding of S. aureus toxins is needed to enable the development of new strategies to reduce their production and consequently improve therapeutic approaches. This review focuses on understanding the toxin-based pathogenesis of S. aureus and their role on infectious diseases.

Objectives The “One Health” concept recognizes that the health of humans, animals and the environment are interconnected. Therefore, the knowledge on the behavior of microorganisms from the most diverse environmental niches is important to prevent the emergence and dissemination of antimicrobial resistance. Wild animals are known to carry antimicrobial resistant microorganisms with potential public health impact. However, no data is available on the sessile behavior of bacteria from wild animals, even if antimicrobial resistance is amplified in biofilms. This study characterizes the antimicrobial susceptibility to ciprofloxacin, and the adhesion and biofilm formation abilities of 14 distinct Aeromonas spp. (eight A. salmonicida , three A. eucrenophila , two A. bestiarum and one A. veronii ) isolated from wild animals and already characterized as resistant to β-lactam antibiotics. Methods Minimal inhibitory concentration (MIC) of ciprofloxacin was determined according to Clinical and Laboratory Standards Institute guidelines. The biofilm formation assay was done according to a modified microtiter plate method. Bacterial surface hydrophobicity was assessed by sessile drop contact angles measurement. Results All Aeromonas spp. strains were resistant to ciprofloxacin (minimum inhibitory concentrations between 6 and 60 μg/ml) and had hydrophilic surfaces (ranging from 2 and 37 mJ/m 2 ). These strains were able to adhere and form biofilms with distinct magnitudes. Biofilm exposure to 10 times minimum inhibitory concentration of ciprofloxacin only caused low to moderate removal. Conclusion This study shows that the strains tested are of potential public health concern and emphasize that wild animals are potential reservoirs of multidrug resistant strains. In fact, Aeromonas species are consistently considered opportunistic pathogens. Moreover, bacterial ability to form biofilms increases antimicrobial resistance and the propensity to cause persistent infections.

Aims The aim of this study was to investigate the antibiofilm potential of five essential oil (EO) components with cyclic (sabinene—SAB, carveol—C1, carvone—C2) and acyclic (citronellol—C3 and citronellal—C4) structures against Escherichia coli and Staphylococcus aureus. Methods and Results The selected EO components prevented biofilm set-up, with C3 and C4 causing remarkable effects. When applied against pre-established biofilms, they promoted high biomass removal and inactivation of biofilm cells. Moreover, no viable E. coli biofilm cells were detected after exposure to SAB at 5 × MIC and 10 × MIC, and a significant viability decrease was observed for both bacteria with the other EO components. SAB, C3 and C4 caused the most prominent effects apparently due to their octanol–water partition coefficient (Po/w), the number of rotatable bonds (n-ROTB) and the free hydroxyl groups. Conclusions The overall results demonstrated that the selected EO components, particularly SAB, C3 and C4 are of interest as new lead molecules to both prevent biofilm set-up and to control pre-established biofilms of E. coli and S. aureus. Significance and Impact of the Study The tested EO components exhibited prominent antibiofilm properties against E. coli and S. aureus providing a novel and effective alternative/complementary approach to counteract chronic infections and the transmission of diseases in clinical settings.

Disruption of cell–cell communication or quorum sensing (QS) is considered a stimulating approach for reducing bacterial pathogenicity and resistance. Although several QS inhibitors (QSIs) have been discovered so far their clinical use remains distant. This problem can be circumvented by searching for QSI among drugs already approved for the treatment of different diseases. In this context, antibiotics have earned special attention. Whereas at high concentrations antibiotics exert a killing effect, at lower concentrations they may act as signaling molecules and as such can modulate gene expression. In this study, the antibiotic furvina was shown to be able to cause inhibition of the 3-oxo-C12-HSL-dependent QS system of Pseudomonas aeruginosa. Furvina interacts with the LasI/LasR system. The data were validated by modeling studies. Furvina can also reduce biofilm formation and decrease the production of QS-controlled virulence factors.

Biofilms are a problem to food industries, causing equipment damage, increased energy costs, and food spoilage, and they are a potential harbour of pathogenic microorganisms. Their extreme antimicrobial resistance means that novel control strategies are necessary. Plant secondary metabolites (phyto-chemicals) have demonstrated promising antimicrobial properties when applied against planktonic cells and biofilms. The aim of this study was to test the effectiveness of two phenolic acids: ferulic (FA) and sali-cylic (SA), alone and in combination (FSA) on the prevention and control of Bacillus cereus and Pseudomonas fluorescens biofilms. Additional tests were performed to ascertain the effects of FA and SA on bacterial motility, surface properties (physicochemical properties and surface charge), and quorum sensing inhibition (QSI). The effects of a concentration two times the minimum inhibitory concentration (500 μg mL −1 ) were assessed on single- and dual-species biofilms. The results demonstrated that only swimming was affected by FA and SA and no clear relationship was obtained between the effects of phenolic acids on motility and biofilm prevention. The bacterial physicochemical surface properties and charge were affected by the phenolic acids. Salicylic acid demonstrated capacity for QSI. However, both bacteria were able to form single- and dual-species biofilms in the presence of the phenolic acids. The application of FA and SA (single and combined) to biofilms caused low to moderate inactivation and removal. However, dual-species biofilms formed in the presence of phenolic acids were highly susceptible to a second exposure to the chemicals. The continuous exposure of dual-species biofilms to the phenolic acids decreased their resilience and resistance to inactivation and removal. This study clarifies the role of FA and SA in the prevention and control of biofilms formed by two important food spoilage bacteria.

Quorum sensing (QS) is an important regulatory mechanism in biofilm formation and differentiation. Interference with QS can affect biofilm development and antimicrobial susceptibility. This study evaluates the potential of selected phytochemical products to inhibit QS. Three isothiocyanates (allylisothiocyanate - AITC, benzylisothiocyanate - BITC and 2-phenylethylisothiocyanate - PEITC) and six phenolic products (gallic acid - GA, ferulic acid - FA, caffeic acid - CA, phloridzin - PHL, (-) epicatechin - EPI and oleuropein glucoside - OG) were tested. A disc diffusion assay based on pigment inhibition in Chromobacterium violaceum CV12472 was performed. In addition, the mechanisms of QS inhibition (QSI) based on the modulation of N-acyl homoserine lactone (AHLs) activity and synthesis by the phytochemicals were investigated. The cytotoxicity of each product was tested on a cell line of mouse lung fibroblasts. AITC, BITC and PEITC demonstrated a capacity for QSI by modulation of AHL activity and synthesis, interfering the with QS systems of C. violaceum CviI/CviR homologs of LuxI/LuxR systems. The cytotoxic assays demonstrated low effects on the metabolic viability of the fibroblast cell line only for FA, PHL and EPI., This work was supported by Operational Programme for Competitiveness Factors - COMPETE, FCT/MEC (PIDDAC) and FEDER through Projects Bioresist - PTDC/EBB-EBI/105085/2008; Phytodisinfectants - PTDC/DTP-SAP/1078/2012 (COMPETE: FCOMP-01-0124-FEDER-028765) and the PhD grants awarded to Ana Abreu (SFRH/BD/84393/2012) and Anabela Borges (SFRH/BD/63398/2009). The authors are also very grateful to Professor Robert McLean (Texas University, USA) for providing the bacterial strains.

The increased resistance of pathogenic microorganisms is frequently attributed to the extreme and inadequate use of antibiotics and transmission of resistance within and between individuals. To counter the emergence of resistant microorganisms, considerable resources have been invested in the search for new antimicrobials. Plants synthesize a diverse array of secondary metabolites (phytochemicals) known to be involved in defense mechanisms, and in the last few years it is recognized that some of these molecules have health beneficial effects, including antimicrobial properties. In this study, the mechanism of action of gallic (GA) and ferulic (FA) acids, a hydroxybenzoic acid and a hydroxycinnamic acid, was assessed on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes. The targets of antimicrobial action were studied using different bacterial physiological indices: minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), membrane permeabilization, intracellular potassium release, physicochemical surface properties, and surface charge. It was found that FA and GA had antimicrobial activity against the bacteria tested with MIC of 500 mu g/mL for P. aeruginosa, 1500 mu g/mL for E. coli, 1750 mu g/mL for S. aureus, and 2000 mu g/mL for L. monocytogenes with GA; 100 mu g/mL for E. coli and P. aeruginosa, 1100 mu g/mL and 1250 mu g/mL for S. aureus and L. monocytogenes, respectively, with FA. The MBC for E. coli was 2500 mu g/mL (FA) and 5000 (GA), for S. aureus was 5000 mu g/mL (FA) and 5250 mu g/mL (GA), for L. monocytogenes was 5300 mu g/mL (FA) and 5500 mu g/mL (GA), and 500 mu g/mL for P. aeruginosa, with both phytochemicals. GA and FA led to irreversible changes in membrane properties (charge, intra and extracellular permeability, and physicochemical properties) through hydrophobicity changes, decrease of negative surface charge, and occurrence of local rupture or pore formation in the cell membranes with consequent leakage of essential intracellular constituents. The overall study emphasizes the potential of plant-derived molecules as a green and sustainable source of new broad spectrum antimicrobial products.