Extracellular Polymeric Matrix Research Articles

Lysine aggregates‐based nanostructured antimicrobial peptides for cariogenic biofilm microenvironment‐activated caries treatment

AbstractDental caries is one of the most prevalent and costly biofilm‐induced oral diseases that causes the deterioration of the mineralized tooth tissue. Traditional antimicrobial agents like antibiotics and antimicrobial peptides (AMPs) struggle to effectively eradicate bacteria in biofilms without eliciting resistance. Herein, we demonstrate the construction of FeOOH@Fe‐Lysine@Au nanostructured AMPs (nAMPs) distinguished by their AMP‐like antibacterial activity and self‐producing reactive oxygen species (ROS) capacity for caries treatment. On the one hand, FeOOH@Fe‐Lysine@Au nAMPs can catalyze glucose oxidation to generate ROS within the cariogenic biofilm microenvironment, resulting in the disintegration of the extracellular polymeric substance matrix and the exposure of bacteria. On the other hand, FeOOH@Fe‐Lysine@Au nAMPs can attach to bacterial surfaces via electrostatic attractions, proceeding to damage membranes, disrupt metabolic pathways, and inhibit protein synthesis through the aggregated lysine and the generated ROS. Based on this antibacterial mechanism, FeOOH@Fe‐Lysine@Au nAMPs can effectively eradicate Streptococcus mutans and its associated biofilm, significantly impeding the progression of dental caries. Given the straightforward and cost‐efficient preparation of FeOOH@Fe‐Lysine@Au nAMPs compared to AMPs that require specific sequences, and their minimal adverse impacts on gingival/palatal tissues, major organs, and oral/gut microbiomes, our research may promote the development of novel therapeutic agents in dental health maintenance.

Molecular Aspects of the Functioning of Pathogenic Bacteria Biofilm Based on Quorum Sensing (QS) Signal-Response System and Innovative Non-Antibiotic Strategies for Their Elimination.

One of the key mechanisms enabling bacterial cells to create biofilms and regulate crucial life functions in a global and highly synchronized way is a bacterial communication system called quorum sensing (QS). QS is a bacterial cell-to-cell communication process that depends on the bacterial population density and is mediated by small signalling molecules called autoinducers (AIs). In bacteria, QS controls the biofilm formation through the global regulation of gene expression involved in the extracellular polymeric matrix (EPS) synthesis, virulence factor production, stress tolerance and metabolic adaptation. Forming biofilm is one of the crucial mechanisms of bacterial antimicrobial resistance (AMR). A common feature of human pathogens is the ability to form biofilm, which poses a serious medical issue due to their high susceptibility to traditional antibiotics. Because QS is associated with virulence and biofilm formation, there is a belief that inhibition of QS activity called quorum quenching (QQ) may provide alternative therapeutic methods for treating microbial infections. This review summarises recent progress in biofilm research, focusing on the mechanisms by which biofilms, especially those formed by pathogenic bacteria, become resistant to antibiotic treatment. Subsequently, a potential alternative approach to QS inhibition highlighting innovative non-antibiotic strategies to control AMR and biofilm formation of pathogenic bacteria has been discussed.

Shifting from Ammonium to Phosphonium Salts: A Promising Strategy to Develop Next-Generation Weapons against Biofilms.

Since they are difficult and sometimes impossible to treat, infections sustained by multidrug-resistant (MDR) pathogens, emerging especially in nosocomial environments, are an increasing global public health concern, translating into high mortality and healthcare costs. In addition to having acquired intrinsic abilities to resist available antibiotic treatments, MDR bacteria can transmit genetic material encoding for resistance to non-mutated bacteria, thus strongly decreasing the number of available effective antibiotics. Moreover, several pathogens develop resistance by forming biofilms (BFs), a safe and antibiotic-resistant home for microorganisms. BFs are made of well-organized bacterial communities, encased and protected in a self-produced extracellular polymeric matrix, which impedes antibiotics' ability to reach bacteria, thus causing them to lose efficacy. By adhering to living or abiotic surfaces in healthcare settings, especially in intensive care units where immunocompromised older patients with several comorbidities are hospitalized BFs cause the onset of difficult-to-eradicate infections. In this context, recent studies have demonstrated that quaternary ammonium compounds (QACs), acting as membrane disruptors and initially with a low tendency to develop resistance, have demonstrated anti-BF potentialities. However, a paucity of innovation in this space has driven the emergence of QAC resistance. More recently, quaternary phosphonium salts (QPSs), including tri-phenyl alkyl phosphonium derivatives, achievable by easy one-step reactions and well known as intermediates of the Wittig reaction, have shown promising anti-BF effects in vitro. Here, after an overview of pathogen resistance, BFs, and QACs, we have reviewed the QPSs developed and assayed to this end, so far. Finally, the synthetic strategies used to prepare QPSs have also been provided and discussed to spur the synthesis of novel compounds of this class. We think that the extension of the knowledge about these materials by this review could be a successful approach to finding effective weapons for treating chronic infections and device-associated diseases sustained by BF-producing MDR bacteria.

Nanomedicine against biofilm infections: A roadmap of challenges and limitations.

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Enhanced short-chain fatty acids production from anaerobic fermentation of secondary sludge by lignosulfonate addition: Towards circular economy

This study is the first to unveil the potential of lignosulfonate as an additive in the production of short-chain fatty acids (SCFAs) from anaerobic fermentation of secondary sludge. Batch-scale tests were performed to assess SCFAs production and distribution with lignosulfonate addition of 20–80 mg/g volatile solids (VS), along with potential mechanisms and phytotoxicity. The results showed that lignosulfonate addition improved the SCFAs production and the ratio of acetic acid, while shortening the fermentation time required. The maximum SCFAs production (185.0 ± 1.0 mg COD/g VS) and acetic acid ratio (51.4 ± 0.6%) were achieved with 60 mg lignosulfonate/g VS at 4 d of fermentation time, outperforming the control (93.1 ± 2.7 mg COD/g VS, 44.4 ± 1.8%). Lignosulfonate addition decreased the zeta potential between sludge particles by 19.9%–45.1%, and increased the extractable extracellular polymeric substances matrix by 1.45–2.33 times, compared to the control group. Furthermore, lignosulfonate addition effectively promoted the solubilization, hydrolysis, and acidification processes by up to 116.0%, 58.8%, and 47.6%, respectively. The seed germination index of Chinese cabbage and lettuce using fermented sludge residues increased by up to 486 ± 29% and 158 ± 13%, respectively. These findings suggest that lignosulfonate addition is a sustainable method for SCFAs recovery from sludge, benefiting sludge agricultural reuse, and contributing to the circular economy.

Combination treatment of slightly acidic electrolyzed water with electron beam irradiation to mono- and mixed biofilms of Vibrio parahaemolyticus and Pseudomonas fluorescens

Effective antimicrobial measures are needed to control cross-contamination and rapid spoilage of food caused by Vibrio parahaemolyticus (V. parahaemolyticus) and Pseudomonas fluorescens (P. fluorescens). This study evaluated the combined effect of electron beam irradiation (EBI) combined with slightly acidic electrolytic water (SAEW) treatment on suspended bacteria and single and dual-species biofilms of V. parahaemolyticus and P. fluorescens. Colony counting results showed that EBI combined with SAEW treatment effectively removed biofilm cells. Confocal laser scanning microscopy and ATP leakage analysis showed that EBI combined with SAEW treatment damaged the membrane integrity of biofilm cells and caused ATP leakage. Field emission scanning electron microscopy (SEM) observations showed that EBI with or without SAEW significantly disrupted the three-dimensional reticular structure of mono/co-cultured biofilm (extracellular polymeric substances) EPS, producing reticular perforation damage. Combined treatment with 60 mg/L SAEW and 0.5 kGy (kGy) EBI caused serious damage to the three-dimensional structure of the biofilms. The detection results of biofilm protein and DNA showed that EBI combined with SAEW could effectively destroy the extracellular polymeric matrix of biofilm, but the primary structure of the protein had no significant effect. These results suggest that SAEW(60 mg/L) combined with EBI(0.5 kGy) is effective in reducing microbial and biofilm attachment during the large yellow croaker storage processing.

A novel small-molecule compound S-342-3 effectively inhibits the biofilm formation of Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

Inhibition of hyaluronan synthesis prevents β-cell loss in obesity-associated type 2 diabetes

Pancreatic β-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of β-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on β-cells such that caspase 3 activation did not result in β-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate β-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Review on Anti-Biofilm Potential of CRISPR-Cas System on Various Pathogens

The development of bacterial biofilms entails regulatory as well as signalling mechanisms that regulate the shift from a mobile to astationary state of existence, the production of the extracellular polymeric matrix, and the progression of 3-dimensional biofilm formation withemphasis on how easily they may be created and how crucial they are in biological, ecological, and industrial settings, biofilms are the subject ofextensive research. Biofilms and a variety of pathogenic human disorders are frequently linked. Since bacteria in biofilms can resist antibiotics, theimmune system, and other treatments, biofilm infections are typically long-lasting. Many prokaryotes include CRISPR-associated proteins (Cas), a solidadaptable immunological system that may be programmed to damage the bacterial genomes and induce cell death. Short palindromic repeats that aregrouped and adequately spaced together make up CRISPR-Cas. In light of this, CRISPR-Cas can be seen as an exciting strategy to address andovercome antibiotic resistance. Furthermore, the CRISPR-Cas system can create "precise antimicrobials" that target bacterial infections according tospecific DNA sequences. This CRISPR-Cas technique is susceptible to drug-resistant microorganisms due to its selective targeting of the genesinvolved in biofilm formation, pathogenicity, and antibiotic resistance. However, this method requires potent vectors for the CRISPR-Cas system toaccess the bacterial genomes. As vectors, genetically engineered Phage, liposomes, and lipid-mediated nanoparticles are exciting options. Thistechnique has been used to prevent extracellular and intracellular from forming biofilms. The most current developments in creating innovations andpossible advantages of the various CRISPR-Cas delivery methods for the deliberate eradication of bacterial pathogens will be covered in this review,focussing mainly on the anti-biofilm potential, which is found to be one of the primary causes of the difficulty of irradiation of Multi-drug resistantbacteria. Additionally, each distribution system's positive aspects are highlighted, along with its challenges and potential for advancement in the future.

Bacterial biofilms in the human body: prevalence and impacts on health and disease.

Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states.

Microstructural and Rheological Transitions in Bacterial Biofilms.

Biofilms are aggregated bacterial communities structured within an extracellular matrix (ECM). ECM controls biofilm architecture and confers mechanical resistance against shear forces. From a physical perspective, biofilms can be described as colloidal gels, where bacterial cells are analogous to colloidal particles distributed in the polymeric ECM. However, the influence of the ECM in altering the cellular packing fraction (ϕ) and the resulting viscoelastic behavior of biofilm remains unexplored. Using biofilms of Pantoea sp. (WT) and its mutant (ΔUDP), the correlation between biofilm structure and its viscoelastic response is investigated. Experiments show that the reduction of exopolysaccharide production in ΔUDP biofilms corresponds with a seven-fold increase in ϕ, resulting in a colloidal glass-like structure. Consequently, the rheological signatures become altered, with the WT behaving like a weak gel, whilst the ΔUDP displayed a glass-like rheological signature. By co-culturing the two strains, biofilm ϕ is modulated which allows us to explore the structural changes and capture a change in viscoelastic response from a weak to a strong gel, and to a colloidal glass-like state. The results reveal the role of exopolysaccharide in mediating a structural transition in biofilms and demonstrate a correlation between biofilm structure and viscoelasticresponse.

Dissipation of pesticides by stream biofilms is influenced by hydrological histories.

To evaluate the effects of hydrological variability on pesticide dissipation capacity by stream biofilms, we conducted a microcosm study. We exposed biofilms to short and frequent droughts (daily frequency), long and less frequent droughts (weekly frequency) and permanently immersed controls, prior to test their capacities to dissipate a cocktail of pesticides composed of tebuconazole, terbuthylazine, imidacloprid, glyphosate and its metabolite aminomethylphosphonic acid. A range of structural and functional descriptors of biofilms (algal and bacterial biomass, extracellular polymeric matrix (EPS) concentration, microbial respiration, phosphorus uptake and community-level physiological profiles) were measured to assess drought effects. In addition, various parameters were measured to characterise the dynamics of pesticide dissipation by biofilms in the different hydrological treatments (% dissipation, peak asymmetry, bioconcentration factor, among others). Results showed higher pesticide dissipation rates in biofilms exposed to short and frequent droughts, despite of their lower biomass and EPS concentration, compared to biofilms in immersed controls or exposed to long and less frequent droughts. High accumulation of hydrophobic pesticides (tebuconazole and terbuthylazine) was measured in biofilms despite the short exposure time (few minutes) in our open-flow microcosm approach. This research demonstrated the stream biofilms capacity to adsorb hydrophobic pesticides even in stressed drought environments.

Diffusiophoretic Particle Penetration into Bacterial Biofilms.

Bacterial biofilms are communities of cells adhered to surfaces. These communities represent a predominant form of bacterial life on Earth. A defining feature of a biofilm is the three-dimensional extracellular polymer matrix that protects resident cells by acting as a mechanical barrier to the penetration of chemicals, such as antimicrobials. Beyond being recalcitrant to antibiotic treatment, biofilms are notoriously difficult to remove from surfaces. A promising, but relatively underexplored, approach to biofilm control is to disrupt the extracellular polymer matrix by enabling penetration of particles to increase the susceptibility of biofilms to antimicrobials. In this work, we investigate externally imposed chemical gradients as a mechanism to transport polystyrene particles into bacterial biofilms. We show that preconditioning the biofilm with a prewash step using deionized (DI) water is essential for altering the biofilm so it takes up the micro- and nanoparticles by the application of a further chemical gradient created by an electrolyte. Using different particles and chemicals, we document the transport behavior that leads to particle motion into the biofilm and its further reversal out of the biofilm. Our results demonstrate the importance of chemical gradients in disrupting the biofilm matrix and regulating particle transport in crowded macromolecular environments, and suggest potential applications of particle transport and delivery in other physiological systems.

Combating Bacterial Biofilms: Current and Emerging Antibiofilm Strategies for Treating Persistent Infections

Biofilms are intricate multicellular structures created by microorganisms on living (biotic) or nonliving (abiotic) surfaces. Medically, biofilms often lead to persistent infections, increased antibiotic resistance, and recurrence of infections. In this review, we highlighted the clinical problem associated with biofilm infections and focused on current and emerging antibiofilm strategies. These strategies are often directed at disrupting quorum sensing, which is crucial for biofilm formation, preventing bacterial adhesion to surfaces, impeding bacterial aggregation in viscous mucus layers, degrading the extracellular polymeric matrix, and developing nanoparticle-based antimicrobial drug complexes which target persistent cells within the biofilm core. It is important to acknowledge, however, that the use of antibiofilm agents faces obstacles, such as limited effectiveness in vivo, potential cytotoxicity to host cells, and propensity to elicit resistance in targeted biofilm-forming microbes. Emerging next generation antibiofilm strategies, which rely on multipronged approaches, were highlighted, and these benefit from current advances in nanotechnology, synthetic biology, and antimicrobial drug discovery. The assessment of current antibiofilm mitigation approaches, as presented here, could guide future initiatives toward innovative antibiofilm therapeutic strategies. Enhancing the efficacy and specificity of some emerging antibiofilm strategies via careful investigations, under conditions that closely mimic biofilm characteristics within the human body, could bridge the gap between laboratory research and practical application.

Optimization of Phage-Antibiotic Combinations against Staphylococcus aureus Biofilms.

Phage therapy has gained attention due to the spread of antibiotic-resistant bacteria and narrow pipeline of novel antibiotics. Phage cocktails are hypothesized to slow the overall development of resistance by challenging the bacteria with more than one phage. Here, we have used a combination of plate-, planktonic-, and biofilm-based screening assays to try to identify phage-antibiotic combinations that will eradicate preformed biofilms of Staphylococcus aureus strains that are otherwise difficult to kill. We have focused on methicillin-resistant S aureus (MRSA) strains and their daptomycin-nonsusceptible vancomycin-intermediate (DNS-VISA) derivatives to understand whether the phage-antibiotic interactions are altered by the changes associated with evolution from MRSA to DNS-VISA (which is known to occur in patients receiving antibiotic therapy). We evaluated the host range and cross-resistance patterns of five obligately lytic S. aureus myophages to select a three-phage cocktail. We screened these phages for their activity against 24-h bead biofilms and found that biofilms of two strains, D712 (DNS-VISA) and 8014 (MRSA), were the most resistant to killing by single phages. Specifically, even initial phage concentrations of 107 PFU per well could not prevent visible regrowth of bacteria from the treated biofilms. However, when we treated biofilms of the same two strains with phage-antibiotic combinations, we prevented bacterial regrowth when using up to 4 orders of magnitude less phage and antibiotic concentrations that were lower than our measured minimum biofilm inhibitory concentration. We did not see a consistent association between phage activity and the evolution of DNS-VISA genotypes in this small number of bacterial strains. IMPORTANCE The extracellular polymeric matrix of biofilms presents an impediment to antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. While most phage cocktails are designed for the planktonic state of bacteria, it is important to take the biofilm mode of growth (the predominant mode of bacterial growth in nature) into consideration, as it is unclear how interactions between any specific phage and its bacterial hosts will depend on the physical properties of the growth environment. In addition, the extent of bacterial sensitivity to any given phage may vary from the planktonic to the biofilm state. Therefore, phage-containing treatments targeting biofilm infections such as catheters and prosthetic joint material may not be merely based on host range characteristics. Our results open avenues to new questions regarding phage-antibiotic treatment efficiency in the eradication of topologically structured biofilm settings and the extent of eradication efficacy relative to the single agents in biofilm populations.

Ficin-Cyclodextrin-Based Docking Nanoarchitectonics of Self-Propelled Nanomotors for Bacterial Biofilm Eradication.

Development of bioinspired nanomotors showing effective propulsion and cargo delivery capabilities has attracted much attention in the last few years due to their potential use in biomedical applications. However, implementation of this technology in realistic settings is still a barely explored field. Herein, we report the design and application of a multifunctional gated Janus platinum-mesoporous silica nanomotor constituted of a propelling element (platinum nanodendrites) and a drug-loaded nanocontainer (mesoporous silica nanoparticle) capped with ficin enzyme modified with β-cyclodextrins (β-CD). The engineered nanomotor is designed to effectively disrupt bacterial biofilms via H2O2-induced self-propelled motion, ficin hydrolysis of the extracellular polymeric matrix (EPS) of the biofilm, and controlled pH-triggered cargo (vancomycin) delivery. The effective synergic antimicrobial activity of the nanomotor is demonstrated in the elimination of Staphylococcus aureus biofilms. The nanomotor achieves 82% of EPS biomass disruption and a 96% reduction in cell viability, which contrasts with a remarkably lower reduction in biofilm elimination when the components of the nanomotors are used separately at the same concentrations. Such a large reduction in biofilm biomass in S. aureus has never been achieved previously by any conventional therapy. The strategy proposed suggests that engineered nanomotors have great potential for the elimination of biofilms.

Contaminación cruzada: Uso de tablas de cortar al manipular alimentos en el entorno doméstico

The objective of this work was to carry out a bibliographical review on the approach to cross contamination in the domestic environment, using cutting boards as a tool used in food handling in domestic households, serving as a vehicle for contamination, and as interest for study, in addition to important aspects related to the safety and handling of food at home. Foodborne diseases are defined as infectious or toxic diseases caused by the consumption of contaminated food or water, and the home is considered a place of origin for these diseases based on the food handled and ingested in the home itself. The main errors during food handling are related to the hygiene conditions in the places and surfaces where these foods are handled, with cross-contamination as the main factor responsible for the transfer of microorganisms from one material or contaminated food to another. Cutting surfaces are utensils widely used in the preparation and manipulation of food, whether raw or ready-to-eat and, as handlers are unaware of adequate measures for hygiene, storage, and conservation of these surfaces, the organic matter and microorganisms present in raw foods can remain adhered to them, serving as a source of constant contamination. The permanence and adhesion of microorganisms can occur on any type of surface with the production of biofilms. Biofilms can be defined as communities of sessile cells, consisting of one or more bacterial species, included in a matrix of extracellular polymers (exopolysaccharides - EPS) adhered to an inert or living surface. Biofilms can contribute to the permanence of microorganisms on cutting boards, which may compromise the microbiological quality of food handled on these surfaces, exposing people to biological risk through the biotransfer of pathogenic microorganisms. Studies show that these pathogenic microorganisms may have some degree of resistance to antibiotics and disinfectants used in disease control and the hygiene of this utensil, and exposure to pathogens associated with their degree of resistance raises concerns for the World Health Organization (WHO), which reveals a worrying scenario for public health.

The combinatorial applications of 1,4-naphthoquinone and tryptophan inhibit the biofilm formation of Staphylococcus aureus.

Microorganisms embedded within an extracellular polymeric matrix are known as biofilm. The extensive use of antibiotics to overcome the biofilm-linked challenges has led to the emergence of multidrug-resistant strains. Staphylococcus aureus is one such nosocomial pathogen that is known to cause biofilm-linked infections. Thus, novel strategies have been adopted in this study to inhibit the biofilm formation of S. aureus. Two natural compounds, namely, 1,4-naphthoquinone (a quinone derivative) and tryptophan (aromatic amino acid), have been chosen as they could independently show efficient antibiofilm activity. To enhance the antibiofilm potential, the two compounds were combined and tested against the same organism. Several experiments like crystal violet (CV) assay, protein estimation, extracellular polymeric substance (EPS) extraction, and estimation of metabolic activity confirmed that the combination of the two compounds could significantly inhibit the biofilm formation of S. aureus. To comprehend the underlying mechanism, efforts were further directed to understand whether the two compounds could inhibit biofilm formation by compromising the cell surface hydrophobicity of the bacteria. The results revealed that the cell surface hydrophobicity got reduced by ~ 49% when the compounds were applied together. Thus, the combinations could show enhanced antibiofilm activity by attenuating cell surface hydrophobicity. Further studies revealed that the selected concentrations of the compounds could disintegrate (~ 70%) the pre-existing biofilm of the test bacteria without showing any antimicrobial activity. Hence, the combined application of tryptophan and 1,4-naphthoquinone could be used to inhibit the biofilm threats of S. aureus.

Application of an Extracellular Matrix-Mimicking Fluorescent Polymer for the Detection of Proteolytic Venom Toxins

The cytotoxicity caused by snake venoms is a serious medical problem that greatly contributes to the morbidity observed in snakebite patients. The cytotoxic components found in snake venoms belong to a variety of toxin classes and may cause cytotoxic effects by targeting a range of molecular structures, including cellular membranes, the extracellular matrix (ECM) and the cytoskeleton. Here, we present a high-throughput assay (384-well plate) that monitors ECM degradation by snake venom toxins via the application of fluorescent versions of model ECM substrates, specifically gelatin and collagen type I. Both crude venoms and fractionated toxins of a selection of medically relevant viperid and elapid species, separated via size-exclusion chromatography, were studied using the self-quenching, fluorescently labelled ECM-polymer substrates. The viperid venoms showed significantly higher proteolytic degradation when compared to elapid venoms, although the venoms with higher snake venom metalloproteinase content did not necessarily exhibit stronger substrate degradation than those with a lower one. Gelatin was generally more readily cleaved than collagen type I. In the viperid venoms, which were subjected to fractionation by SEC, two (B. jararaca and C. rhodostoma, respectively) or three (E. ocellatus) active proteases were identified. Therefore, the assay allows the study of proteolytic activity towards the ECM in vitro for crude and fractionated venoms.

A199 BIOFILMS IN THE APPENDIX AND OTHER NON-INFLAMED SECTIONS OF THE COLON IN PEDIATRIC PATIENTS WITH INFLAMMATORY BOWEL DISEASES

Abstract Background Biofilms, aggregated bacteria colonizing the extracellular polymeric substances matrix, are associated with the mucosa of inflammatory bowel diseases (IBD) patients with some studies showing a mean density of the mucosal biofilms 2-fold higher in IBD patients than in controls. The appendix, which is a highly immune organ, seems to be involved in IBD pathogenesis. Purpose In this study we aimed to evaluate biofilms in the appendix and other non-inflamed regions of the colon in pediatric IBD patients. We hypothesized that biofilms composed of pathobionts in these sections could drive inflammation in IBD patients. Method Tissues from the appendix, peri-appendicular region, cecum, and ascending colon (ASC), collected from the resected colons of 9 pediatric IBD patients and one pediatric non-IBD patient, preserved in methanol Carnoy’s solution were processed and paraffin embedded. Combined fluorescence in situ hybridization (FISH) for biofilms (probe: EUB338) and immunofluorescence (IF) mucin staining (MUC2) identified biofilms and their location. Biofilm formation capacity of culturable bacteria (identified through 16S DNA Sanger sequencing) from these sections was measured. Interleukin (IL)-8 (pro-inflammatory chemokine) and IL-10 (anti-inflammatory cytokine) expressions were assessed by tissue qPCR. Result(s) FISH demonstrated biofilms in these sections, in close proximity to epithelial cells. We used a biofilm formation assay to assess the ability of the identified bacteria from these sections to form biofilms, illustrating their potential to colonize and evade host defense and potential antibiotics. We found that Enterococcus avium has a significantly higher ability to form biofilms than the negative control. IL-8 and IL-10 both had the highest expression level in the appendix and peri-appendicular region and the lowest in the ASC among all the patients, suggesting an active immune response. Mechanistic experiments are in progress to investigate the type of bacteria involved in the biofilms and their effects on the gut barrier integrity. Conclusion(s) Biofilms adjacent to the epithelium, especially in the appendix and peri-appendix, could interact with or invade epithelial cells. The elevated chemokine transcript level in the appendix could reflect the recruitment of immune cells to this section following bacterial invasion, resulting in the activation of the immune system. Identifying the bacteria involved in the biofilms and clarifying their characteristics will aid us in developing novel microbe-altering treatment strategies or personalized medicine. Disclosure of Interest None Declared