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During the course of our ongoing work to discover new inhibitors of biofilm formation of Staphylococcus aureus from fungal sources, we observed biofilm inhibition by cytochalasans isolated from cultures of the ascomycete Hypoxylon fragiforme for the first time. Two new compounds were purified by a bioassay-guided fractionation procedure; their structures were elucidated subsequently by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This unexpected finding prompted us to test further cytochalasans from other fungi and from commercial sources for comparison. Out of 21 cytochalasans, 13 showed significant inhibition of Staphylococcus aureus biofilm formation at subtoxic levels. These findings indicate the potential of cytochalasans as biofilm inhibitors for the first time, also because the minimum inhibitory concentrations (MIC) are independent of the anti-biofilm activities. However, cytochalasans are known to be inhibitors of actin, making some of them very toxic for eukaryotic cells. Since the chemical structures of the tested compounds were rather diverse, the inclusion of additional derivatives, as well as the evaluation of their selectivity against mammalian cells vs. the bacterium, will be necessary as next step in order to develop structure-activity relationships and identify the optimal candidates for development of an anti-biofilm agent., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The need for effective compounds to combat antimicrobial resistance and biofilms which play important roles in human infections continues to pose a major health challenge. Seven previously undescribed acyclic diterpenes linked to isocitric acid by an ether linkage, microporenic acids A-G (1-7), were isolated from the cultures of a hitherto undescribed species of the genus Microporus (Polyporales, Basidiomycota) originating from Kenya's Kakamega forest. Microporenic acids D and E (4 and 5) showed antimicrobial activity against a panel of Gram positive bacteria and a yeast, Candida tenuis. Moreover, microporenic acids A and B (1 and 2) demonstrated dose-dependent inhibition of biofilm formation by Staphylococcus aureus. Compound 1 further showed significant activity against Candida albicans and Staphylococcus aureus preformed biofilms.

Pathogens embedded in biofilms are involved in many infections and are very difficult to treat with antibiotics because of higher resistance compared with planktonic cells. Therefore, new approaches for their control are urgently needed. One way to search for biofilm dispersing compounds is to look at defense strategies of organisms exposed to wet environments, which makes them prone to biofilm infections. It is reasonable to assume that mushrooms have developed mechanisms to control biofilms on their sporocarps (fruiting bodies). A preliminary screening for biofilms on sporocarps revealed several species with few or no bacteria on their sporocarps. From the edible mushroom Coprinus comatus where no bacteria on the sporocarp could be detected (3R,4S)-2-methylene-3,4-dihydroxypentanoic acid 1,4-lactone, named coprinuslactone, was isolated. Coprinuslactone interfered with quorum-sensing and dispersed biofilms of Pseudomonas aeruginosa, where it also reduced the formation of the pathogenicity factors pyocyanin and rhamnolipid B. Coprinuslactone also damaged Staphylococcus aureus cells in biofilms at subtoxic concentrations. Furthermore, it inhibited UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), essential for bacterial cell wall synthesis. These two modes of action ensure the inhibition of a broad spectrum of pathogens on the fruiting body but may also be useful for future clinical applications., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Microbes attach to surfaces and form biofilms where they are difficult to eradicate. Here they are embedded in a complex matrix of polymers and are much less sensitive against antibiotics or the immune system., Objective: This is a growing problem, especially for implants; hence, novel approaches are urgently needed to control biofilm infections., Methods: Many of these approaches interfere with the communication between the microbial cells required for biofilm formation and maintenance, a process known as quorum sensing. But microbes have also several mechanisms to disperse their own biofilms if conditions become unfavourable., Results: Recently, it has been found that the pathogen Pseudomonas aeruginosa disperses its mature biofilms using cis-2-decenoic acid. This fatty acid belongs to a group of cis-2-alkenoic acids which are known from several bacteria and are also triggering the communication between different species. In this review the biosynthesis of these compounds, their signal transduction and their role in species-species communication are presented. Examples are discussed where cis-2-alkenoic acids have been used to eradicate biofilms and enhance the sensitivity of pathogens against antibiotics, either alone or in combination with antibiotics., Conclusion: This presents an interesting approach for the control of biofilm infections but it is still in its infancy and a much broader characterization of the effects of cis-2-alkenoic acids are needed before moving to any medical application., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Fungi grow under humid conditions and are, therefore, prone to biofilm infections. A 16S rRNA fingerprint analysis was performed on 49 sporocarps of Basidiomycotina in order to determine whether they are able to control these biofilms. Ninety-five bacterial phylotypes, comprising 4 phyla and 10 families, were identified. While ectomycorrhizal fungi harbored the highest bacterial diversity, saprophytic fungi showed little or no association with bacteria. Seven fungal species were screened for antimicrobial and antibiofilm activities. Biofilm formation and bacterial growth was inhibited by extracts obtained from saprophytic fungi, which confirmed the hypothesis that many fungi modulate biofilm colonization on their sporocarps.

Staphylococcus epidermidis, a commensal microorganism at the human skin and mucosae, is nowadays considered an important opportunistic pathogen related to nosocomial infections on indwelling medical devices due biofilm formation. Bacterial biofilms are the worst aspect in the treatment of infections and now efforts have been made in the search for new molecular entities to overcome this situation. In this work, a compound isolated from marine associated fungi was capable to interfere with the adherence and biofilm formation of S. epidermidis. This compound, identified as mevalonolactone, showed significant inhibition of S. epidermidis ATCC 35984 biofilm formation, without antibacterial activity, evaluated by crystal violet assay, turbidimetric assay and scanning electron microscopy. When assayed against 12 clinical isolates of S. epidermidis, this compound exhibited both biofilm inhibition and antimicrobial activity, but no activity against gram-negative bacteria was observed. Therefore, when this constitutive molecule is added in the antibiofilm and antibacterial assays, it might act as an important agent against this pathogen, contributing to the arsenal of antibiofilm compounds.

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization., (© 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.)

Infections associated to microbial biofilms are involved in 80% of human infections and became a challenge concerning public health. Infections related to Staphylococcus epidermidis biofilms are presently commonly associated to medical devices, increasing treatment costs for this type of infection. Alternatives to eliminate this kind of disease have been employed in screening programs using diverse marine-derived fungi source of bioactive compounds capable to combat biofilm formation. In this work was isolated the dipeptide cis-cyclo(Leucyl-Tyrosyl) from a sponge associated Penicillium sp. possessing a remarkable inhibition up to 85% of biofilm formation without interfering with bacterial growth, confirmed by scanning electron microscopy. This is the first demonstration that cis-cyclo(Leucyl-Tyrosyl) is able to specifically inhibit biofilm formation adding another aspect to the broad spectrum of bioactivities of cyclic dipeptides., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Many bacteria grow on surfaces forming biofilms but often high dosages of antibiotics can not clear infectious biofilms. Biofilms are controlled by a process called quorum-sensing triggered by species-specific, small, diffusible autoinducers (N-acyl homoserine lactones (AHL), small peptides or a furanosyl borate diester). Several herbal, animal and microbial extracts possess quorum-quenching activity but only few active compounds and synthetic analogues are known. However, quorum-sensing seems not to be the only way to control biofilms. Bacteria have mechanisms to dissolve their biofilms and return to planktonic lifestyle. Only few compounds responsible for the signalling of these processes are known but may open a completely novel line of biofilm control. Furthermore, controlling the attachment of bacterial by inhibiting the formation of pili is another option and some inhibitors have already been characterized. In this article different approaches to control bacterial biofilms are discussed together with the balance between biofilm formation and virulence.

Biofilms are matrix-enclosed microbial aggregations that adhere to biological or non-biological surfaces. They represent a significant and incompletely understood mode of growth for bacteria and fungi. Biofilm infections cause many deaths and high health costs worldwide. Biofilm infections on indwelling devices or implants are difficult to eradicate because of their much better protection against macrophages and antibiotics, compared to free living cells, leading to severe clinical complications often with lethal outcome. One promising approach to combat biofilm infections independent from the conventional control by antibiotics is the generation of functional surfaces preventing the attachment of bacteria. Another aim is the communication machinery used by bacteria to establish a biofilm, the so called quorum-sensing. Here, small diffusible compounds are produced and sensed by the producing cells to measure their concentration and hence cell density. Natural compounds and synthetic analogues have been used successfully to prevent biofilm formation by quorum-quenching. These compounds are still in the preclinical phase, often struggling with toxicity. A principal problem of quorum-quenchers is their high species specificity, resulting in the control of only some pathogenic strains leaving other pathogens untouched. A field still in its infancy is the control of virulence factors expression not preventing the biofilm but suppressing its virulence. This review will give an overview over the pros and cons of the individual targets and an outlook of future developments.

Many bacteria can form aggregates on interfaces, called biofilms, where they are much more protected against toxic agents such as antibiotics or antibodies. Bacteria organized in biofilms are therefore very difficult to control and often even high dosages of antibiotics cannot clear infectious biofilms. To form biofilms bacteria have to start a complex genetic program to switch from planktonic to sessile lifestyle. This starts with the determination of their cell density, a process called quorum sensing, triggered by small, water soluble molecules, so called autoinducers. From Gram-positive bacteria several small peptides are known, many of them thiolactones. More recently another group of compounds was discovered probably formed from ribose-homocysteine and the first autoinducer-II identified is a furanosyl borate diester. While small peptides are found to trigger the intraspecies communication autoinducer-II is assumed to be used for communication at the interspecies level. Taking the lead structure from these peptides several derivatives have been developed which prevent biofilm formation in many Gram-positive bacteria, including Staphylococcus aureus. Some of these compounds are already in clinical studies. In this review the different approaches to control bacterial biofilms are discussed together with the difficulties arising from the species-specificity of the autoinducers.

Soil contaminated with polychlorinated biphenyls (PCB) was used as an inoculum to grow a complex biofilm community on PCB oil (Aroclor 1242) on a substratum (Permanox). The biofilm was monitored for 31 days by confocal laser scanning microscopy, community fingerprinting using single-strand conformational polymorphism (SSCP), amplicons of the 16S rRNA genes, and chemical analyses of the PCB congeners. SSCP analysis of the young biofilm revealed a rather diverse microbial community with species of the genera Herbaspirillum and Bradyrhizobium as dominant members. The biofilm developing on the PCB droplets displayed pronounced stages of PCB degradation and biofilm development not described before from pure-culture experiments. The first step was the colonization of the substratum while the PCB oil was hardly populated. When a certain density of bacteria was reached on the Permanox, the PCB was colonized, but soon the degradation of the congeners was markedly reduced and many cells were damaged, as seen by LIVE/DEAD staining. Finally, the biofilm formed aggregates and invaded the PCB oil, showing lower numbers of damaged cells than before and a dramatic increase in PCB degradation. This sequence of biofilm formation is understood as a maturation process prior to PCB oil colonization. This is followed by a thin biofilm on the PCB droplet, an aggregation process forming pockets in the PCB, and finally an invasion of the biofilm into the PCB oil. Only the mature biofilm showed degradation of pentachlorinated PCB congeners, which may be reductively dechlorinated and the resulting trichlorobiphenyls then aerobically metabolized.

The two fungal human pathogens, Candida auris and Candida albicans, possess a variety of virulence mechanisms. Among them are the formation of biofilms to protect yeast against harsh conditions through the development of (pseudo)hyphae whilst also facilitating the invasion of host tissues. In recent years, increased rates of antifungal resistance have been associated with C. albicans and C. auris, posing a significant challenge for the effective treatment of fungal infections. In the course of our ongoing search for novel anti-infectives, six selected azaphilones were tested for their cytotoxicity and antimicrobial effects as well as for their inhibitory activity against biofilm and hyphal formation. This study revealed that rubiginosin C, derived from stromata of the ascomycete Hypoxylon rubiginosum, effectively inhibited the formation of biofilms, pseudohyphae, and hyphae in both C. auris and C. albicans without lethal effects. Crystal violet staining assays were utilized to assess the inhibition of biofilm formation, while complementary microscopic techniques, such as confocal laser scanning microscopy, scanning electron microscopy, and optical microscopy, were used to investigate the underlying mechanisms. Rubiginosin C is one of the few substances known to effectively target both biofilm formation and the yeast-to-hyphae transition of C. albicans and C. auris within a concentration range not affecting host cells, making it a promising candidate for therapeutic intervention in the future. [ABSTRACT FROM AUTHOR]

γ-Hexachlorocyclohexane (γ-HCH) persists in the environment and is recalcitrant to microbial degradation. To determine the extent of the microbial potential for the degradation of γ-HCH the diversity of bacteria from 12 soil samples collected around insecticide- and pesticide-producing factories in Egypt were assessed and compared with biofilm communities grown on γ-HCH microcrystals. From all samples, highly diverse microbes were isolated, able to grow on γ-HCH as sole source of carbon. The same soil samples were used to inoculate γ-HCH microcrystals on a substratum in microcosms to grow biofilm communities. All soil samples formed multispecies biofilms on γ-HCH. Biofilms stained with Nile Red showed distinct cell clusters of high hydrophobicity, and it is speculated that these aggregates have a substantial role in the degradation of the hydrophobic substrate. While many Bacillus species were isolated, this group was almost absent in the different biofilm communities. The finding of cells with highly hydrophobic envelopes together with the differences in species composition between isolates and interacting microbial communities points to fundamental differences in the interaction with hydrophobic substrates of single strains and microbial communities. [ABSTRACT FROM AUTHOR]

Most bacteria attach to surfaces where they form a biofilm, cells embedded in a complex matrix of polymers. Cells in biofilms are much better protected against noxious agents than free-living cells. As a consequence it is very difficult to control pathogens with antibiotics in biofilm infections and novel targets are urgently needed. One approach aims at the communication between cells to form and to maintain a biofilm, a process called quorum-sensing. Water soluble small-sized molecules mediate this process and a number of antagonists of these compounds have been found. In this review natural compounds and synthetic drugs which do not interfere with the classical quorum-sensing compounds are discussed. For some of these compounds the targets are still not known, but others interfere with the formation of exopolysaccharides, virulence factors, or cell wall synthesis or they start an internal program of biofilm dispersal. Some of their targets are more conserved among pathogens than the receptors for quorum sensing autoinducers mediating quorum-sensing, enabling a broader application of the drug. The broad spectrum of mechanisms, the diversity of bioactive compounds, their activity against several targets, and the conservation of some targets among bacterial pathogens are promising aspects for several clinical applications of this type of biofilm-controlling compound in the future. [ABSTRACT FROM AUTHOR]

Poor sanitation, poor treatments of waste water, as well as catastrophic floods introduce pathogenic bacteria into rivers, infecting and killing many people. The goal of clean water for everyone has to be achieved with a still growing human population and their rapid concentration in large cities, often megacities. How long introduced pathogens survive in rivers and what their niches are remain poorly known but essential to control water-borne diseases in megacities. Biofilms are often niches for various pathogens because they possess high resistances against environmental stress. They also facilitate gene transfers of antibiotic resistance genes which become an increasing health problem. Beside biofilms, amoebae are carriers of pathogenic bacteria and niches for their survival. An overview about our current understanding of the fate and niches of pathogens in rivers, the multitude of microbial community interactions, and the impact of severe flooding, a prerequisite to control pathogens in polluted rivers, is given. [ABSTRACT FROM AUTHOR]

Abstract: The active bacteria of a biofilm community grown directly on polychlorinated biphenyl (PCB) droplets were analyzed by 16S rRNA fingerprinting, identified by their 16S rRNA gene sequences and fatty acid profiling, and compared with isolates from the biofilm. Although, the multi-species biofilm degraded di- and trichlorinated PCB-congeners these substrates were not attacked by its individual isolated members, which suggests that a metabolic network is responsible for PCB degradation in the biofilm. The community metabolized [U-13C]-2,2′-dichlorobiphenyl incorporating the label into certain phospholipid fatty acids matching those found in Burkholderia species. In contrast, abundant biofilm community members, like Methylobacterium species, did not incorporate the label. These results provide prima faciae evidence for Burkholderia species as the main degraders of PCBs in this type of aerobic soils. [Copyright &y& Elsevier]

A moorland soil site polluted with PCB showed a high diversity of metabolically active bacteria. Beside frequent types of 16S rRNA sequences similar to those of the species of Sphingomonas and the Acidobacterium phylum an unusual high number of sequences from the genus Burkholderia were found. Burkholderia was also the main genus in isolates enriched on biphenyl or various chlorobenzoates. In microcosm experiments sterilized surfaces exposed to PCB polluted soil always showed the presence of clay aggregates formed by bacteria attached to the substratum. The bacteria use the PCB loaded clay colloids as transport medium for the water insoluble substrate to get access to the carbon source. This is a novel mechanism of how bacteria deal with hydrophobic substrates. [ABSTRACT FROM AUTHOR]

Microbial consortia of composite biofilms, grown in surface water of acidic mining lakes near Lauchhammer, Germany, were investigated. The red-brown colored lake water was acidic (pH 2.5), had high concentrations of Fe(III), Al(III), and sulphate and low concentrations of dissolved organic matter. As a result the abundance of bacteria in the lake is with 104 cells mL-1 rather low. One input of organic material into the lake are autumnal leaves from trees, growing in the lakeside area. From aliquots of unfixed birch leave biofilms the 16S rRNA genes were amplified by PCR and community fingerprints were determined by single-strand conformation polymorphism (SSCP) analysis. Specific bands within the fingerprints were extracted from SSCP gels and sequenced for the taxonomical affiliation.These results were compared with those from the second type of biofilms which were grown on sterile substrata, floating submersed in surface waters of the lakes. By excising the bands from the gel and sequencing the individual bands bacterial taxa, common to both types of biofilms, were found but also some, which were only present in one type of biofilm. Ultrathin sectioned biofilms often showed bacteria associated with electron dense particles as main inorganic constituents. Elemental microanalysis by energy dispersive X-ray analysis (EDX) revealed them to contain iron, sulfur and oxygen as main elemental fractions and electron diffraction ring pattern analysis classified them to be schwertmannite. These bacteria and their interactions with each other as well as with the inorganic minerals formed in this lake generally is of great interest, in order to use these results for bioremediation applications. [ABSTRACT FROM AUTHOR]

Infections involving biofilms are difficult to treat due to increased resistances against antibiotics and the immune system. Hence, there is an urgent demand for novel drugs against biofilm infections. During our search for novel biofilm inhibitors from fungi, we isolated linoleic acid from the ascomycete Hypoxylon fragiforme which showed biofilm inhibition of several bacteria at sub-MIC concentrations. Many fatty acids possess antimicrobial activities, but their minimum inhibitory concentrations (MIC) are high and reports on biofilm interferences are scarce. We demonstrated that not only linoleic acid but several unsaturated long-chain fatty acids inhibited biofilms at sub-MIC concentrations. The antibiofilm activity exerted by long-chain fatty acids was mainly against Gram-positive bacteria, especially against Staphylococcus aureus. Micrographs of treated S. aureus biofilms revealed a reduction in the extracellular polymeric substances, pointing to a possible mode of action of fatty acids on S. aureus biofilms. The fatty acids had a strong species specificity. Poly-unsaturated fatty acids had higher activities than saturated ones, but no obvious rule could be found for the optimal length and desaturation for maximal activity. As free fatty acids are non-toxic and ubiquitous in food, they may offer a novel tool, especially in combination with antibiotics, for the control of biofilm infections. [ABSTRACT FROM AUTHOR]