Biosynthesis of silver nanoparticle using Bacillus licheniformis culture-supernatant for combating pathogenic biofilms.

Bacterial biofilms pose a significant threat to healthcare due to their recalcitrance to antibiotics and disinfectants. This study explores the anti-biofilm potential of Bacillus licheniformis cell-free culture supernatant (CFS) and its derived silver nanoparticles (bSNPs) against Staphylococcus aureus and Pseudomonas aeruginosa. The CFS exhibited potent anti-biofilm activity against both bacterial species, even at low concentrations, while devoid of significant bactericidal effects, mitigating resistance risks. Characterization studies revealed the non-proteinaceous nature and thermal stability of the CFS's anti-biofilm agent, suggesting a robust and heat-resistant structure. Green synthesis of bSNPs from CFS resulted in nanoparticles with significant anti-biofilm properties, particularly against P. aeruginosa, indicating differences in susceptibility between the bacterial species. Epifluorescence microscopy confirmed bSNPs' ability to inhibit and partially disrupt biofilm formation without inducing cellular lysis. The study highlights the potential of B. licheniformis CFS and bSNPs as promising biofilm control agents, offering insights into their mechanisms of action and broad-spectrum efficacy. Further research elucidating the underlying molecular mechanisms and identifying specific bioactive compounds is warranted for the translation of these findings into clinically relevant applications for combating biofilm-associated infections.

Regulating bacterial biofilms in food and biomedicine: unraveling mechanisms and Innovating strategies.

Bacterial biofilm has brought a lot of intractable problems in food and biomedicine areas. Conventional biofilm control mainly focuses on inactivation and removal of biofilm. However, with robust construction and enhanced resistance, the established biofilm is extremely difficult to eradicate. According to the mechanism of biofilm development, biofilm formation can be modulated by intervening in the key factors and regulatory systems. Therefore, regulation of biofilm formation has been proposed as an alternative way for effective biofilm control. This review aims to provide insights into the regulation of biofilm formation in food and biomedicine. The underlying mechanisms for early-stage biofilm establishment are summarized based on the key factors and correlated regulatory networks. Recent developments and applications of novel regulatory strategies such as anti/pro-biofilm agents, nanomaterials, functionalized surface materials and physical strategies are also discussed. The current review indicates that these innovative methods have contributed to effective biofilm control in a smart, safe and eco-friendly way. However, standard methodology for regulating biofilm formation in practical use is still missing. As biofilm formation in real-world systems could be far more complicated, further studies and interdisciplinary collaboration are still needed for simulation and experiments in the industry and other open systems.

Effects of antimicrobials over sessile and planktonic microbiota associated with an industrial cooling water system.

The bacterial community from a cooling water system was investigated through culture-dependent and independent strategies, and the responses of planktonic and sessile bacteria (grown in glass slides and stainless-steel coupons) to antimicrobials of industrial and clinical use were assessed. The morphotypes with higher biofilm-forming potential were Pseudoxanthomonas sp., Rheinheimera sp., Aeromonas sp. and Staphylococcus sp., and the first also exhibited lower susceptibility to all antibiotics and biocides tested. 16S rRNA high throughput sequencing indicated that Pseudomonadota (77.1% on average, sd 11.1%), Bacteroidota (8.4, sd 5.7%), and Planctomycetota (3.0, sd 1.3%) were the most abundant phyla. KEGG orthologs associated with antibiotics and biocide resistance were abundant in all samples. Although the minimum inhibitory and bactericidal concentrations were generally higher for biofilms, morphotypes in planktonic form also showed high levels of resistance, which could be associated with biofilm cells passing into the planktonic phase. Overall, monochloramine was the most effective biocide.

Biofilm control strategies in the light of biofilm-forming microorganisms.

Biofilm is a complex consortium of microorganisms attached to biotic or abiotic surfaces and live in self-produced or acquired extracellular polymeric substances (EPSs). EPSs are mainly formed by lipids, polysaccharides, proteins, and extracellular DNAs. The adherence to the surface of microbial communities is seen in food, medical, dental, industrial, and environmental fields. Biofilm development in food processing areas challenges food hygiene, and human health. In addition, bacterial attachment and biofilm formation on medical implants inside human tissue can cause multiple critical chronic infections. More than 30 years of international research on the mechanisms of biofilm formation have been underway to address concerns about bacterial biofilm infections. Antibiofilm strategies contain cold atmospheric plasma, nanotechnological, phage-based, antimicrobial peptides, and quorum sensing inhibition. In the last years, the studies on environmentally-friendly techniques such as essential oils and bacteriophages have been intensified to reduce microbial growth. However, the mechanisms of the biofilm matrix formation are still unclear. This review aims to discuss the latest antibiofilm therapeutic strategies against biofilm-forming bacteria.

Effectiveness and mechanisms of essential oils for biofilm control on food-contact surfaces: An updated review.

Microbial biofilms represent a constant source of contamination in the food industry, being also a real threat for human health. In fact, most of biofilm-producing bacteria are becoming resistant to sanitizers, thus arousing the interest in natural alternatives to prevent biofilm formation on foods and food-contact surfaces. In particular, studies on biofilm control by essential oils (EOs) application are increasing, being EOs characterized by unique mixtures of compounds able to impair the mechanisms of biofilm development. This review reports the anti-biofilm properties of EOs in bacterial biofilm control (inhibition, removal and prevention of biofilm dispersion) on food-contact surfaces. The relationship between EOs effect and composition, concentration, involved bacteria, and surfaces is discussed, and the possible sites of action are also elucidated. The findings prove the high biofilm controlling capability of EOs through the regulation of genes and proteins implicated in motility, Quorum Sensing and exopolysaccharides (EPS) matrix. Moreover, incorporation in nanosized delivery systems, formulation of blends and combination of EOs with other strategies can increase their anti-biofilm activity. This review provides an overview of the current knowledge of the EOs effectiveness in controlling bacterial biofilm on food-contact surfaces, providing valuable information for improving EOs use as sanitizers in food industries.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Bacterial Inactivation and Biofilm Disruption through Indigenous Prophage Activation Using Low-Intensity Cold Atmospheric Plasma.

Biofilms can be pervasive and problematic in water treatment and distribution systems but are difficult to eradicate due to hindered penetration of antimicrobial chemicals. Here, we demonstrate that indigenous prophages activated by low-intensity plasma have the potential for efficient bacterial inactivation and biofilm disruption. Specifically, low-intensity plasma treatment (i.e., 35.20 W) elevated the intracellular oxidative reactive species (ROS) levels by 184%, resulting in the activation of prophage lambda (λ) within antibiotic-resistant Escherichia coli K-12 (lambda+) [E. coli (λ+)]. The phage activation efficiency was 6.50-fold higher than the conventional mitomycin C induction. Following a cascading effect, the activated phages were released upon the lysis of E. coli (λ+), which propagated further and lysed phage-susceptible E. coli K-12 (lambda-) [E. coli (λ-)] within the biofilm. Bacterial intracellular ROS analysis and ROS scavenger tests revealed the importance of plasma-generated ROS (e.g., •OH, 1O2, and •O2-) and associated intracellular oxidative stress on prophage activation. In a mixed-species biofilm on a permeable membrane surface, our "inside-out" strategy could inactivate total bacteria by 49% and increase the membrane flux by 4.33-fold. Furthermore, the metagenomic analysis revealed that the decrease in bacterial abundance was closely associated with the increase in phage levels. As a proof-of-concept, this is the first demonstration of indigenous prophage activations by low-intensity plasma for antibiotic-resistant bacterial inactivation and biofilm eradication, which opens up a new avenue for managing associated microbial problems.

Bacteriophages: A weapon against mixed-species biofilms in the food processing environment.

Mixed-species biofilms represent the most frequent actual lifestyles of microorganisms in food processing environments, and they are usually more resistant to control methods than single-species biofilms. The persistence of biofilms formed by foodborne pathogens is believed to cause serious human diseases. These challenges have encouraged researchers to search for novel, natural methods that are more effective towards mixed-species biofilms. Recently, the use of bacteriophages to control mixed-species biofilms have grown significantly in the food industry as an alternative to conventional methods. This review highlights a comprehensive introduction of mixed-species biofilms formed by foodborne pathogens and their enhanced resistance to anti-biofilm removal strategies. Additionally, several methods for controlling mixed-species biofilms briefly focused on applying bacteriophages in the food industry have also been discussed. This article concludes by suggesting that using bacteriophage, combined with other 'green' methods, could effectively control mixed-species biofilms in the food industry.

Effectiveness of selected essential oils and one hydrolate to prevent and remove Listeria monocytogenes biofilms on polystyrene and stainless steel food-contact surfaces.

AIMS: This study aimed to evaluate the effectiveness of selected essential oils (EOs) and hydrolates (Hs) against Listeria monocytogenes biofilms on polystyrene (PS) and stainless steel (SS) surfaces. METHODS AND RESULTS: Among others, Origanum hirtum EO, Corydothymus capitatus EO and Citrus aurantium H were selected to treat L. monocytogenes biofilms during and after biofilm formation. Sub-minimum inhibitory concentrations (MICs) of C. capitatus EO (0.31 µl/ml) showed the highest inhibiting effect against biofilm formation on PS, while on SS no significant differences between the EOs were observed (43.7%-88.7% inhibition). Overall, the tested biosanitizers showed limited activity as biofilm removal agents. Although generally less effective, C. aurantium H exhibited good biofilm inhibition performance at 62.5 µl/ml, particularly on PS. Confocal laser scanning microscopy proved that sub-MICs of the biosanitizers drastically changed L. monocytogenes biofilm architecture, with bacterial cells elongation in the presence of C. capitatus EO. CONCLUSIONS: Our findings suggest that the tested EOs and H are able to control Listeria biofilms, particularly preventing biofilm formation on both materials. Considering its mild aroma and hydrophilicity, the H exhibited promising perspectives of application. SIGNIFICANCE AND IMPACT OF STUDY: This study raises the possibility of applying EOs and Hs to control biofilms on different surfaces in the food industry.

Photoacoustic removal of Enterococcus faecalis biofilms from titanium surface with an Er:YAG laser using super short pulses.

Biofilms that grow on implant surfaces pose a great risk and challenge for the dental implant survival. In this work, we have applied Er:YAG photoacoustic irrigation using super short pulses (Er:YAG-SSP) to remove biofilms from the titanium surfaces in the non-contact mode. Mature Enterococcus faecalis biofilms were treated with saline solution, chlorhexidine, and hydrogen peroxide, or photoacoustically with Er:YAG-SSP for 10 or 60 s. The number of total and viable bacteria as well as biofilm surface coverage was determined prior and after different treatments. Er:YAG-SSP photoacoustic treatment significantly increases the biofilm removal rate compared to saline or chemically treated biofilms. Up to 92% of biofilm-covered surface can be cleaned in non-contact mode during 10 s without the use of abrasives or chemicals. In addition, Er:YAG-SSP photoacoustic irrigation significantly decreases the number of viable bacteria that remained on the titanium surface. Within the limitations of the present in vitro model, the ER:YAG-SSP seems to constitute an efficient therapeutic option for quick debridement and decontamination of titanium implants without using abrasives or chemicals.

Effects of stabilized hypochlorous acid on oral biofilm bacteria.

BACKGROUND: Caries and periodontitis are amongst the most prevalent diseases worldwide, leading to pain and loss of oral function for those affected. Prevention relies heavily on mechanical removal of dental plaque biofilms but for populations where this is not achievable, alternative plaque control methods are required. With concerns over undesirable side-effects and potential bacterial resistance due to the use of chlorhexidine gluconate (CHX), new antimicrobial substances for oral use are greatly needed. Here we have investigated the antimicrobial effect of hypochlorous acid (HOCl), stabilized with acetic acid (HAc), on oral biofilms and compared it to that of CHX. Possible adverse effects of stabilized HOCl on hydroxyapatite surfaces were also examined. METHODS: Single- and mixed-species biofilms of six common oral bacteria (Streptococcus mutans, Streptococcus gordonii, Actinomyces odontolyticus, Veillonella parvula, Parvimonas micra and Porphyromonas gingivalis) within a flow-cell model were exposed to HOCl stabilized with 0.14% or 2% HAc, pH 4.6, as well as HOCl or HAc alone. Biofilm viability was assessed in situ using confocal laser scanning microscopy following LIVE/DEAD® BacLight™ staining. In-situ quartz crystal microbalance with dissipation (QCM-D) was used to study erosion of hydroxyapatite (HA) surfaces by stabilized HOCl. RESULTS: Low concentrations of HOCl (5 ppm), stabilized with 0.14% or 2% HAc, significantly reduced viability in multi-species biofilms representing supra- and sub-gingival oral communities, after 5 min, without causing erosion of HA surfaces. No equivalent antimicrobial effect was seen for CHX. Gram-positive and Gram-negative bacteria showed no significant differential suceptibility to stabilized HOCl. CONCLUSIONS: At low concentrations and with exposure times which could be achieved through oral rinsing, HOCl stabilized with HAc had a robust antimicrobial activity on oral biofilms, without causing erosion of HA surfaces or affecting viability of oral keratinocytes. This substance thus appears to offer potential for prevention and/or treatment of oral biofilm-mediated diseases.

Affordable oral health care: dental biofilm disruption using chloroplast made enzymes with chewing gum delivery.

Current approaches for oral health care rely on procedures that are unaffordable to impoverished populations, whereas aerosolized droplets in the dental clinic and poor oral hygiene may contribute to spread of several infectious diseases including COVID-19, requiring new solutions for dental biofilm/plaque treatment at home. Plant cells have been used to produce monoclonal antibodies or antimicrobial peptides for topical applications to decrease colonization of pathogenic microbes on dental surface. Therefore, we investigated an affordable method for dental biofilm disruption by expressing lipase, dextranase or mutanase in plant cells via the chloroplast genome. Antibiotic resistance gene used to engineer foreign genes into the chloroplast genome were subsequently removed using direct repeats flanking the aadA gene and enzymes were successfully expressed in marker-free lettuce transplastomic lines. Equivalent enzyme units of plant-derived lipase performed better than purified commercial enzymes against biofilms, specifically targeting fungal hyphae formation. Combination of lipase with dextranase and mutanase suppressed biofilm development by degrading the biofilm matrix, with concomitant reduction of bacterial and fungal accumulation. In chewing gum tablets formulated with freeze-dried plant cells, expressed protein was stable up to 3 years at ambient temperature and was efficiently released in a time-dependent manner using a mechanical chewing simulator device. Development of edible plant cells expressing enzymes eliminates the need for purification and cold-chain transportation, providing a potential translatable therapeutic approach. Biofilm disruption through plant enzymes and chewing gum-based delivery offers an effective and affordable dental biofilm control at home particularly for populations with minimal oral care access.

Ways to control harmful biofilms: prevention, inhibition, and eradication.

Biofilms are complex microbial architectures that encase microbial cells in a matrix comprising self-produced extracellular polymeric substances. Microorganisms living in biofilms are much more resistant to hostile environments than their planktonic counterparts and exhibit enhanced resistance against the microbicides. From the human perspective, biofilms can be classified into beneficial, neutral, and harmful. Harmful biofilms impact food safety, cause plant and animal diseases, and threaten medical fields, making it urgent to develop effective and robust strategies to control harmful biofilms. In this review, we discuss various strategies to control biofilm formation on infected tissues, implants, and medical devices. We classify the current strategies into three main categories: (i) changing the properties of susceptible surfaces to prevent biofilm formation; (ii) regulating signalling pathways to inhibit biofilm formation; (iii) applying external forces to eradicate the biofilm. We hope this review would motivate the development of innovative and effective strategies for controlling harmful biofilms.

Current and future perspectives for controlling Vibrio biofilms in the seafood industry: a comprehensive review.

The contamination of seafood with Vibrio species can have severe repercussions in the seafood industry. Vibrio species can form mature biofilms and persist on the surface of several seafoods such as crabs, oysters, mussels, and shrimp, for extended duration. Several conventional approaches have been employed to inhibit the growth of planktonic cells and prevent the formation of Vibrio biofilms. Since Vibrio biofilms are mostly resistant to these control measures, novel alternative methods need to be urgently developed. In this review, we propose environmentally friendly approaches to suppress Vibrio biofilm formation using a hypothesized mechanism of action.

PHENOLIC COMPOUND-DERIVED NATURAL ANTIMICROBIALS ARE LESS EFFECTIVE IN DENTAL BIOFILM CONTROL COMPARED TO CHLORHEXIDINE.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Efficacy of natural antimicrobials derived from phenolic compounds in the control of biofilm in children and adolescents compared to synthetic antimicrobials: A systematic review and meta-analysis. Martins ML, Ribeiro-Lages MB, Masterson D, Magno MB, Cavalcanti YW, Maia LC, Fonseca-Gonçalves A. Arch Oral Biol 2020;118:104844. SOURCE OF FUNDING: Government. This study was financially supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brazil (CAPES) through the grant number 001. TYPE OF STUDY/DESIGN: Systematic review with meta-analysis of data.

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4).

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O2 -based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm2 membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 µm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m-2 ·d-1 maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.

Mixed-species biofilms in the food industry: Current knowledge and novel control strategies.

Attachment of microorganisms to food contact surfaces and the subsequent formation of biofilms may cause equipment damage, food spoilage and even diseases. Mixed-species biofilms are ubiquitous in the food industry and they generally exhibit higher resistance to disinfectants and antimicrobials compared to single-species biofilms. The physiology and metabolic activity of microorganisms in mixed-species biofilms are however rather complicated to study, and despite targeted research efforts, the potential role of mixed-species biofilms in food industry is still rather unexplored. In this review, we summarize recent studies in the context of bacterial social interactions in mixed-species biofilms, resistance to disinfectants, detection methods, and potential novel strategies to control the formation of mixed-species biofilms for enhanced food safety and food quality.

Antibacterial rGO-CuO-Ag film with contact- and release-based inactivation properties.

To reduce the high operational costs of water treatment because of membrane biofouling, next-generation materials are being developed to counteract microbial growth. These modern anti-biofouling strategies are based on new membrane materials or membrane surface modifications. In this study, antimicrobial films comprising rGO, rGO-CuO, rGO-Ag, and rGO-CuO-Ag were synthesized, evaluated, and tested for potential biofouling control using Pseudomonas aeruginosa PAO1 as the model bacterium. The combined rGO-CuO-Ag film displayed enhanced reduction (10-log reduction) in biofouling in comparison to the rGO film (control), followed by the rGO-Ag film (8-log reduction) and rGO-CuO film (0-log reduction). This demonstrated that the use of mixed antimicrobial agents is more effective in reducing biofouling than that of a single agent. The rGO-CuO-Ag film exhibited consistent, controlled, and moderate release of silver (Ag) ions. The release of Ag ions produced a long-lasting antimicrobial effect. These results underscore the potential applications of combined antimicrobial surface-based agents in practice and further research.

Influence of surface copper content on Stenotrophomonas maltophilia biofilm control using chlorine and mechanical stress.

This work aimed to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. Stenotrophomonas maltophilia isolated from drinking water was used as a model microorganism and biofilms were developed in a rotating cylinder reactor using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10 mg l-1 of free chlorine for 10 min, an increased shear stress (a fluid velocity of 1.5 m s-1 for 30s), and a combination of both treatments. These shock treatments were not effective in biofilm control. The benefits from the use of copper surfaces was found essentially in reducing the numbers of non-damaged cells. Copper materials demonstrated better performance in biofilm prevention than chlorine. In general, copper alloys may have a positive public health impact by reducing the number of non-damaged cells in the water delivered after chlorine exposure.

Efficacy of adjunctive anti-plaque chemical agents in managing gingivitis: A systematic review and network meta-analyses.

AIM: The aim of this network meta-analysis (NMA) was to compare the efficacy of different oral hygiene products for chemical biofilm control, in 6-month home-use, randomized clinical trials (RCTs), in terms of changes in gingival index (GI). MATERIAL AND METHODS: Six-month RCTs assessing GI were identified and screened for inclusion. Relevant information was extracted, and quality and potential risk of bias were estimated. Mean differences between baseline and end were calculated to obtain standardized mean differences (SMDs). NMA protocols were applied to assess direct and indirect comparisons among products using Löe & Silness GI, modified GI and gingival severity index. RESULTS: Fifty-three papers were included, 19 studies for mouth rinses, 32 for dentifrices, comprising data from 5,775 and 2,682 subjects, respectively. When ranking treatments, similar results were observed for all tested dentifrices, with the lowest effect observed for sanguinarine and baking soda. For mouth rinses, essential oils, triclosan-copolymer, chlorhexidine (at concentrations ≥ 0.10%) and cetylpyridinium chloride (>0.05%) demonstrated the greatest effect. CONCLUSION: Although NMA revealed significant differences when comparing placebo versus some active agents, when comparing among active agents, no differences were found for dentifrices, while mouth rinses containing essential oils showed the greatest effect on GI scores.