Anti-biofilm Drugs Research Articles

Candida albicans Dispersed Cells Are Developmentally Distinct from Biofilm and Planktonic Cells.

ABSTRACTCandida albicans surface-attached biofilms such as those formed on intravenous catheters with direct access to the bloodstream often serve as a nidus for continuous release of cells capable of initiating new infectious foci. We previously reported that cells dispersed from a biofilm are yeast cells that originate from the top-most hyphal layers of the biofilm. Compared to their planktonic counterparts, these biofilm dispersal yeast cells displayed enhanced virulence-associated characteristics and drug resistance. However, little is known about their molecular properties. To address that issue, in this study we aimed to define the molecular characteristics of these biofilm dispersal cells. We found that the inducer of dispersal, PES1, genetically interacts with the repressor of filamentation, NRG1, in a manner consistent with the definition of dispersed cells as yeast cells. Further, using a flow biofilm model, we performed comprehensive comparative RNA sequencing on freshly dispersed cells in order to identify unique transcriptomic characteristics. Gene expression analysis demonstrated that dispersed cells largely inherit a biofilm-like mRNA profile. Strikingly, however, dispersed cells seemed transcriptionally reprogrammed to acquire nutrients such as zinc and amino acids and to metabolize alternative carbon sources, while their biofilm-associated parent cells did not induce the same high-affinity transporters or express gluconeogenetic genes, despite exposure to the same nutritional signals. Collectively, the findings from this study characterize cell dispersal as an intrinsic step of biofilm development which generates propagules more adept at colonizing distant host sites. This developmental step anticipates the need for virulence-associated gene expression before the cells experience the associated external signals.

A Promising Copper(II) Complex as Antifungal and Antibiofilm Drug against Yeast Infection.

The high mortality rate of candidemia and the limited option for the treatment of Candida spp. infection have been driving the search for new molecules with antifungal property. In this context, coordination complexes of metal ions and ligands appear to be important. Therefore, this study aimed to synthesize two new copper(II) complexes with 2-thiouracil and 6-methyl-2-thiouracil ligands and to evaluate their mutagenic potential and antifungal activity against Candida. The complexes were synthesized and characterized by infrared vibrational spectroscopy, CHN elemental analysis, UV-Vis experiments and ESI-HRMS spectrometry studies. The antifungal activity was evaluated by broth microdilution against 21 clinical isolates of Candida species. The mutagenic potential was evaluated by the Ames test. The complexes were Cu(Bipy)Cl2(thiouracil) (Complex 1) and Cu(Bipy)Cl2(6-methylthiouracil) (Complex 2). Complex 1 showed fungicidal and fungistatic activities against all isolates. Furthermore, the Minimum Inhibitory Concentration (MIC) from 31 to 125 µg/mL and inhibition percentage of 9.9% against the biofilms of C. krusei and C. glabrata were demonstrated. At the concentrations tested, complex 1 exhibited no mutagenic potential. Complex 2 and the free ligands exhibited no antifungal activity at the concentrations evaluated. Since complex 1 presented antifungal activity against all the tested isolates and no mutagenic potential, it could be proposed as a potential new drug for anti-Candida therapy.

Inhibitory effects of Myricetin derivatives on curli-dependent biofilm formation in Escherichia coli

Biofilms are well-organised communities of microbes embedded in a self-produced extracellular matrix (e.g., curli amyloid fibers) and are associated with chronic infections. Therefore, development of anti-biofilm drugs is important to combat with these infections. Previously, we found that flavonol Myricetin inhibits curli-dependent biofilm formation by Escherichia coli (IC50 = 46.2 μM). In this study, we tested activities of seven Myricetin-derivatives to inhibit biofilm formation by E. coli K-12 in liquid culture. Among them, only Epigallocatechin gallate (EGCG), a major catechin in green tea, inhibited biofilm formation of K-12 (IC50 = 5.9 μM) more efficiently than Myricetin. Transmission electron microscopy and immunoblotting analyses demonstrated that EGCG prevented curli production by suppressing the expression of curli-related proteins. Quantitative RT-PCR analysis revealed that the transcripts of csgA, csgB, and csgD were significantly reduced in the presence of EGCG. Interestingly, the cellular level of RpoS, a stationary-phase specific alternative sigma factor, was reduced in the presence of EGCG, whereas the rpoS transcript was not affected. Antibiotic-chase experiments and genetic analyses revealed that EGCG accelerated RpoS degradation by ATP-dependent protease ClpXP in combination with its adaptor RssB. Collectively, these results provide significant insights into the development of drugs to treat chronic biofilm-associated infections.

Staphylococcus Aureus Surface Protein G is An Immunodominant Protein and a Possible Target in An Anti-Biofilm Drug Development.

Background: Staphylococcus aureus is a Gram-positive bacterium that causes severe illnesses in the human population. The capacity of S. aureus strains to form biofilms on biotic and abiotic surfaces creates serious problems for treatment of hospital infections and has stimulated efforts to develop new means of specific protection or immunotherapy.Material and Methods:We found that rabbit serum raised against crude concentrated S. aureus liquid culture significantly decreased the development of staphylococcal biofilm in vitro. To discover the corresponding staphylococcal antigen, we used mass-spectrometry and molecular cloning and identified three major immunodominant proteins. They included α-haemolysin, serine proteinase SplB and S. aureus surface protein G, known as adhesin SasG.Results:Although according to literature data, all these proteins represent virulence factors of S. aureus and play diverse and important roles in the pathogenesis of staphylococcal diseases, only SasG can be directly implicated into the biofilm formation because of its surface location on a staphylococcal cell. Indeed, rabbit serum directed against purified recombinant SasG, similar to serum against crude staphylococcal liquid culture, prevented the formation of a biofilm.Conclusion:SasG can be considered as a target in an anti-biofilm drug development and a component of the vaccine or immunotherapeutic preparations directed against staphylococcal infections in humans.

Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein.

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.

Changing facades of Vibrio cholerae: An enigma in the epidemiology of cholera.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged humanity from time immemorial. Although the disease can be treated using antibiotics along with administration of oral rehydration salts and controlled by good sanitation, cholera is known to have produced mayhems in ancient times when little was known about the pathogen. By the 21st century, ample information about the pathogen, its epidemiology, genetics, treatment and control strategies was revealed. However, there is still fear of cholera outbreaks in developing countries, especially in the wake of natural calamities. Studies have proved that the bacterium is mutating and evolving, out-competing all our efforts to treat the disease with previously used antibiotics and control with existing vaccines. In this review, the major scientific insights of cholera research are discussed. Considering the important role of biofilm formation in the V. cholerae life cycle, the vast availability of next-generation sequencing data of the pathogen and multi-omic approach, the review thrusts on the identification of suitable biofilm-inhibiting targets and the discovery of anti-biofilm drugs from nature to control the disease.

Combination Strategies to Enhance the Efficacy of Antimicrobial Peptides against Bacterial Biofilms.

The great clinical significance of biofilm-associated infections and their inherent recalcitrance to antibiotic treatment urgently demand the development of novel antibiofilm strategies. In this regard, antimicrobial peptides (AMPs) are increasingly recognized as a promising template for the development of antibiofilm drugs. Indeed, owing to their main mechanism of action, which relies on the permeabilization of bacterial membranes, AMPs exhibit a strong antimicrobial activity also against multidrug-resistant bacteria and slow-growing or dormant biofilm-forming cells and are less prone to induce resistance compared to current antibiotics. Furthermore, the antimicrobial potency of AMPs can be highly increased by combining them with conventional (antibiotics) as well as unconventional bioactive molecules. Combination treatments appear particularly attractive in the case of biofilms since the heterogeneous nature of these microbial communities requires to target cells in different metabolic states (e.g., actively growing cells, dormant cells) and environmental conditions (e.g., acidic pH, lack of oxygen or nutrients). Therefore, the combination of different bioactive molecules acting against distinct biofilm components has the potential to facilitate biofilm control and/or eradication. The aim of this review is to highlight the most promising combination strategies developed so far to enhance the therapeutic potential of AMPs against bacterial biofilms. The rationale behind and beneficial outcomes of using AMPs in combination with conventional antibiotics, compounds capable of disaggregating the extracellular matrix, inhibitors of signaling pathways involved in biofilm formation (i.e., quorum sensing), and other peptide-based molecules will be presented and discussed.

Kaempferol Inhibits the Primary Attachment Phase of Biofilm Formation in Staphylococcus aureus.

The ability to form biofilms on surfaces makes Staphylococcus aureus the main pathogenic factor in implanted medical device infections. The aim of this study was to discover a biofilm inhibitor distinct from the antibiotics used to prevent infections resulting from S. aureus biofilms. Here, we describe kaempferol, a small molecule with anti-biofilm activity that specifically inhibited the formation of S. aureus biofilms. Crystal violet (CV) staining and fluorescence microscopy clearly showed that 64 μg/ml kaempferol inhibited biofilm formation by 80%. Meanwhile, the minimum inhibitory concentration (MIC) and growth curve results indicated that kaempferol had no antibacterial activity against the tested bacterial strain. Kaempferol inhibited the primary attachment phase of biofilm formation, as determined by a fibrinogen-binding assay. Moreover, a fluorescence resonance energy transfer (FRET) assay and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that kaempferol reduced the activity of S. aureus sortaseA (SrtA) and the expression of adhesion-related genes. Based on these results, kaempferol provides a starting point for the development of novel anti-biofilm drugs, which may decrease the risk of bacterial drug resistance, to prevent S. aureus biofilm-related infections.

Natural Antibiofilm Agents and the Need for Antibiofilm Drug Leads

Plant with medicinal property contains different types of bioactive compounds and these compounds are effective against certain diseases. Biofilm infection is a disease caused by biofilm which is associated with the respiratory and gastrointestinal infections. Biofilm-producing bacteria as such Pseudomonas aeruginosa strain PA14 is a serious treat in hospitals. The bacterium possesses many virulent factors that contributed to its pathogenesis and resistance thus caused the inhibition and removal of its biofilms a difficult task. This study therefore attempts to identify natural antibiofilm agents against the P. aeruginosa PA14 biofilms. The activity of plant extracts and subsequently purified compounds was evaluated using a stepwise separation process called bioassay-guided fractionation. The fractionation process involved the use of chromatographic techniques and compounds were identified using NMR, GC-MS and LC-MS. A total of four bioactive fractions namely (E333F1S1), (E341), (HA6) and (M338B) have been discovered with antibiofilm activity and were selected for further investigation. The active fraction from Ribes nigrum leaf (E333F1S1) contained mixtures of alkanes while active extract of Sambucus nigra flower (E341) contained a mixture of ursolic acid and oleanolic acid. The LC-MS analysis on Coriandrum sativum seeds (HA6) revealed the presence mixture of oxygenated monoterpenes, while another active fraction of C. sativum seeds (M338B) showed the presence of carboxylic acid, carboxylate, tetraone, glycerol, carbohydrate and fatty acids. Thus, the discovery of potential bioactive compounds from this study could be further investigated as antibiofilm drug leads to battle the infections by P. aeruginosa PA14 biofilm in the near future.

Riboswitches as Potential Targets for the Development of Anti-Biofilm Drugs.

In nature, bacteria can exist as single motile cells or as sessile cellular community, known as microbial biofilms. Bacteria within biofilms are embedded in a self-produced extracellular matrix that makes them more resistant to antibiotic treatment and responses of the host immune system. Microbial biofilms are very important in medicine since they are associated with several human diseases such as dental caries, periodontitis, otitis media, infective endocarditis, infectious kidney stones, osteomyelitis or prostatitis. In addition, biofilms formed on the surface of clinical devices such as pacemakers, implants and catheters are difficult to treat, which underlines the clinical relevance of biofilm formation. At the molecular level, the switch from the planktonic state to biofilm formation is regulated primarily by bis- (3'-5)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP performs its function by binding to a wide variety of proteins, but also to riboswitches. C-di-GMP riboswitches are RNA regulatory elements located in the 5'-untranslated regions (5'-UTRs) of RNA messengers (mRNA) from genes involved in virulence, motility and biofilm formation, which are regulated by changes in the intracellular concentration of c-di-GMP. This review discusses the role of c-di-GMP responsive riboswitches as potential targets for the design of anti-biofilm agents.

Development of gold nanoparticles coated with silica containing the antibiofilm drug cinnamaldehyde and their effects on pathogenic bacteria.

Emerging resistance to antibiotics is a mounting worldwide health concern and increases the need for nonantibiotic strategies to combat infectious diseases caused by bacterial pathogens. In this study, the authors used the antibiofilm activity of the naturally occurring antimicrobial cinnamaldehyde (CNMA) conjugated to the surface of gold nanoparticles (GNPs) to deliver CNMA efficiently and eradicate biofilms of Gram-negative organisms (enterohemorrhagic Escherichia coli O157:H7, and Pseudomonas aeruginosa), Gram positive (methicillin-sensitive Staphylococcus aureus organisms, and methicillin-resistant Staphylococcus aureus) bacteria. CNMA-GNPs containing 0.005% (v/v) of CNMA were found to inhibit biofilm formation efficiently. The distributions of nanoparticles in biofilm cells and their biofilm disruption activities, including distorted cell morphology, were determined by transmission electron microscopy. In addition to their antibiofilm activities, CNMA-GNPs attenuated S. aureus virulence and protected Caenorhabditis elegans (C. elegans) worms. Here, the authors report the antibiofilm effects of CNMA-GNPs and suggest that they could be used to treat pathogenic bacterial infections in vivo.

Genetic Tools to Study c-di-GMP-Dependent Signaling in Pseudomonas aeruginosa.

Pseudomonas aeruginosa infections are often difficult or impossible to treat, mainly due to its ability to form antibiotic-resistant biofilms. Since c-di-GMP signaling strongly influences P. aeruginosa biofilm development and sensitivity to antibiotics, it is considered a promising target for the development of anti-biofilm drugs and it is under intensive investigation. However, studying c-di-GMP signaling in P. aeruginosa is challenging, mainly due to (1) the multiplicity of enzymes involved in c-di-GMP metabolism, (2) the difficulty to extract and measure c-di-GMP intracellular levels by chemical methods, and (3) the lack of genetic tools specifically dedicated to this purpose.Here, a bioluminescence-based reporter system convenient for studying cellular processes or compounds expected to cause an increase or a decrease in intracellular c-di-GMP levels produced by P. aeruginosa cultures is described. Bioluminescence is particularly appropriate in P. aeruginosa research, due to the high intensity of the signal and total lack of background noise. In addition, the use of genetic cassettes allowing the fine control of P. aeruginosa c-di-GMP intracellular levels via arabinose induction is described.Overall, the genetic tools described here could facilitate investigations tackling the c-di-GMP signaling process on different fields, from cellular physiology to drug-discovery research.

High-Throughput Screening for Compounds that Modulate the Cellular c-di-GMP Level in Bacteria.

Bacteria in the biofilm mode of growth cause numerous problematic infections due to their resistance to antimicrobials and the immune system. Because conventional antimicrobial compounds cannot efficiently eradicate biofilm infections, we urgently need new efficient anti-biofilm drugs. The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. We describe a protocol for high-throughput screening of chemical libraries for compounds that lower the c-di-GMP level in bacteria, and potentially can serve as lead compounds in the development of novel biofilm dismantling drugs.

Antibiofilm Activity of Plant Polyphenols.

In the history of human medicine, antibiotics represent epochal examples of medical progress. However, with an approaching antibiotic crisis due to the emergence and extensive spread of antimicrobial resistance among bacterial agents, as well as to increasing number of patients with chronic and recalcitrant bacterial biofilm-associated infections, the naturally occurring molecules may become new sources of antibacterial and antibiofilm drugs for clinical usage. Polyphenols represent a class of plant natural products which are important in plant defense against microbial pathogens. The main focus of the review is on the antibiofilm activities of phenolic compounds against bacteria which play an essential role in medical device biofilm-associated infections. The other, not negligible part of the review is devoted to polyphenols’ activity against bacterial agents that cause dental caries and periodontal disease.

English

Biofilm formation is a well organised, genetically-driven process, well characterized for numerous bacterial species which plays important role in urinary tract infections (UTIs). Adherence is a key event initiating each step in UTI pathogenesis. Such UTIs are difficult to treat owing to increased drug resistance within the biofilm cells. The review is mainly focused on biofilm-growing microorganisms because this form of growth poses a threat to chronically infected or immunocompromised patients and is difficult to eradicate from medical devices. Biofim formation process and mechanisms to its increased resistance to various antimicrobials is also discussed together with newer prophylactic and therapeutic approaches like catheters coated with hydrogels or antibiotics, nanoparticles, ionotrophoresis, biofilm enzyme inhibitors, liposomes, bacterial interference, bacteriophages, quorum sensing inhibitor, combining antimicrobial photodynamic therapy and antiadhesion agents. The review justifies the need for new antibiofilm drug.   Key words: Biofilm, uropathogens, catheter, urinary tract infection, catheter-associated UTI (CAUTI).

The dlt genes play a role in antimicrobial tolerance of Streptococcus mutans biofilms

Microbial biofilms are tolerant to antibiotic treatment and therefore cause problematic infections. Knowledge about the molecular mechanisms underlying biofilm-associated antimicrobial tolerance will aid the development of antibiofilm drugs. Screening of a Streptococcus mutans transposon mutant library for genes that are important for biofilm-associated antimicrobial tolerance provided evidence that the dlt genes play a role in the tolerance of S. mutans biofilms towards gentamicin. The minimum bactericidal concentration for biofilm cells (MBC-B) for a dltA transposon mutant was eight-fold lower than that of the wild-type. The minimum bactericidal concentration for planktonic cells (MBC-P) was only slightly reduced, indicating that the mechanism involved in the observed antimicrobial tolerance has a predominant role specifically in biofilms. Experiments with a knockout dltA mutant and complemented strain confirmed that the dlt genes in S. mutans play a role in biofilm-associated tolerance to gentamicin. Confocal laser scanning microscopy analyses of biofilms grown on glass slides showed that the dltA mutant produced roughly the same amount of biofilm as the wild-type, indicating that the reduced antimicrobial tolerance of the dltA mutant is not due to a defect in biofilm formation. The products of the dlt genes have been shown to mediate alanylation of teichoic acids, and in accordance the dltA mutant showed a more negatively charged surface than the wild-type, which likely is an important factor in the reduced tolerance of the dltA mutant biofilms towards the positively charged gentamicin.

Polymeric prodrug⿿functionalized polypropylene films for sustained release of salicylic acid

Medical devices decorated with salicylic acid-based polymer chains (polymeric prodrug) that slowly release this anti-inflammatory and anti-biofilm drug at the implantation site were designed. A ⿿grafting from⿿ method was implemented to directly grow chains of a polymerizable derivative of salicylic acid (2-methacryloyloxy-benzoic acid, 2MBA) onto polypropylene (PP). PP was modified both at bulk and on the surface with poly(2MBA) by means of an oxidative pre-irradiation method (60Co source), in order to obtain a grafted polymer in which salicylic acid units were linked by means of labile ester bonds. The grafting percent depended on absorbed dose, reaction time, temperature and monomer concentration. The functionalized films were analyzed regarding structure (FTIR-ATR, SEM-EDX, fluorescence microscopy), temperature stability (TGA), interaction with aqueous medium (water contact angle and swelling), pH-responsive release and cytocompatibility (fibroblasts). In the obtained poly(2MBA)-grafted biomaterial, poly(2MBA) behaved as a polymeric prodrug that regulates salicylic acid release once in contact with aqueous medium, showing pH-dependent release rate.

Establishment of a multi-species biofilm model and metatranscriptomic analysis of biofilm and planktonic cell communities.

We collected several biofilm samples from Japanese rivers and established a reproducible multi-species biofilm model that can be analyzed in laboratories. Bacterial abundance at the generic level was highly similar between the planktonic and biofilm communities, whereas comparative metatranscriptomic analysis revealed many upregulated and downregulated genes in the biofilm. Many genes involved in iron-sulfur metabolism, stress response, and cell envelope function were upregulated; biofilm formation is mediated by an iron-dependent signaling mechanism and the signal is relayed to stress-responsive and cell envelope function genes. Flagella-related gene expression was regulated depending upon the growth phase, indicating different roles of flagella during the adherence, maturation, and dispersal steps of biofilm formation. Downregulation of DNA repair genes was observed, indicating that spontaneous mutation frequency would be elevated within the biofilm and that the biofilm is a cradle for generating novel genetic traits. Although the significance remains unclear, genes for rRNA methyltransferase, chromosome partitioning, aminoacyl-tRNA synthase, and cysteine, methionine, leucine, thiamine, nucleotide, and fatty acid metabolism were found to be differentially regulated. These results indicate that planktonic and biofilm communities are in different dynamic states. Studies on biofilm and sessile cells, which have received less attention, are important for understanding microbial ecology and for designing tailor-made anti-biofilm drugs.

New frontiers for anti-biofilm drug development.

Pathogenic microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered a worldwide challenge due to the inherent antibiotic resistance conferred by its lifestyle. Living, as it does, in a community of microbial organisms in a clinical situation, makes it responsible for severe and dangerous cases of infection. Combating this organisation of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence. In addition to therapeutic limitations, biofilms can be a source of infections when they grow in medical devices. The challenge imposed by biofilms has mobilised researchers in the entire world to prospect or develop alternatives to control biofilms. In this context, this review summarises the new frontiers that could be used in clinical circumstances in order to prevent or eliminate pathogenic biofilms.

Natural Sources as Innovative Solutions Against Fungal Biofilms.

Fungal cells are capable of adhering to biotic and abiotic surfaces and form biofilms containing one or more microbial species that are microbial reservoirs. These biofilms may cause chronic and acute infections. Fungal biofilms related to medical devices are particularly responsible for serious infections such as candidemia. Nowadays, only a few therapeutic agents have demonstrated activities against fungal biofilms in vitro and/or in vivo. So the discovery of new anti-biofilm molecules is definitely needed. In this context, biodiversity is a large source of original active compounds including some that have already proven effective in therapies such as antimicrobial compounds (antibacterial or antifungal agents). Bioactive metabolites from natural sources, useful for developing new anti-biofilm drugs, are of interest. In this chapter, the role of molecules isolated from plants, lichens, algae, microorganisms, or from animal or human origin in inhibition and/or dispersion of fungal biofilms (especially Candida and Aspergillus biofilms) is discussed. Some essential oils, phenolic compounds, saponins, peptides and proteins and alkaloids could be of particular interest in fighting fungal biofilms.