Polymeric Biofilm Research Articles

Aggregatibacter actinomycetemcomitans Dispersin B: The Quintessential Antibiofilm Enzyme.

The extracellular matrix of most bacterial biofilms contains polysaccharides, proteins, and nucleic acids. These biopolymers have been shown to mediate fundamental biofilm-related phenotypes including surface attachment, intercellular adhesion, and biocide resistance. Enzymes that degrade polymeric biofilm matrix components, including glycoside hydrolases, proteases, and nucleases, are useful tools for studying the structure and function of biofilm matrix components and are also being investigated as potential antibiofilm agents for clinical use. Dispersin B is a well-studied, broad-spectrum antibiofilm glycoside hydrolase produced by Aggregatibacter actinomycetemcomitans. Dispersin B degrades poly-N-acetylglucosamine, a biofilm matrix polysaccharide that mediates biofilm formation, stress tolerance, and biocide resistance in numerous Gram-negative and Gram-positive pathogens. Dispersin B has been shown to inhibit biofilm and pellicle formation; detach preformed biofilms; disaggregate bacterial flocs; sensitize preformed biofilms to detachment by enzymes, detergents, and metal chelators; and sensitize preformed biofilms to killing by antiseptics, antibiotics, bacteriophages, macrophages, and predatory bacteria. This review summarizes the results of nearly 100 in vitro and in vivo studies that have been carried out on dispersin B since its discovery 20 years ago. These include investigations into the biological function of the enzyme, its structure and mechanism of action, and its in vitro and in vivo antibiofilm activities against numerous bacterial species. Also discussed are potential clinical applications of dispersin B.

Accumulation of 5-methyltetrahydrofolate and other bioactive compounds, in the course of fermentation of green tea (Camellia sinensis) kombucha

Kombucha is a potential probiotic tea-based drink with increasing worldwide consumption. Studies on this probiotic beverage are growing rapidly, especially about micronutrients and microbial population. As such, the present study performed the molecular identification of the microorganism and evaluated 5-methyltetrahydrofolate content by HPLC-DAD, phenolic compounds, flavonoids, carotenoids, antioxidant activity by spectrophotometric methods, and physicochemical composition of green tea kombucha on fermentation days 1, 3, 7, 14, and 21. DNA sequencing identified the Microbacterium genus as predominant. However, was unable to safely determine the species level because of the rRNA 16S gene sequence similarity between four species M. ureisolvens, M. yannicii, M. chocolatum e M. atlanticum. The concentration of 5-methyltetrahydrofolate found on the third day was 39.12 ± 1.32 μg/mL (liquid) and 45.78 ± 8.42 μg/mL (polymeric biofilm); On the twenty-first day it was 50.87 ± 3.56 μg/mL (liquid) and 54.88 ± 3.89 μg/mL (polymeric biofilm). Total phenolic compounds increased with fermentation; however, flavonoids and carotenoids were degraded by the process. The information on 5-methyltetrahydrofolate is unprecedented and highly relevant for food guidelines, since related deficiencies can lead to fetal malformation in the first three months of pregnancy. Lastly, the best fermentation time to obtain 5-methyltetrahydrofolate and others bioactive compounds is between days 7–14. Further analyses are also encouraged to understand the bioavailability of the vitamin.

New artificial hematophagy system with attractive polymeric biofilm for maintenance of Culex quinquefasciatus (Diptera: Culicidae) in the laboratory

BackgroundMaintaining mosquito colonies in the laboratory requires a blood supply so that females' oocytes can mature and oviposition can take place. In this study, a new artificial hematophagy system for colonization and maintenance of Culex quinquefasciatus in the laboratory was developed and tested.MethodsWe developed an attractive polymeric biofilm including 25% l-lactic acid for use as a membrane in an artificial hematophagy system and compared the feeding rate of females with Parafilm-M®. We also evaluated the oviposition rate, larval survival and adult emergence of females fed through the attractive biofilm.ResultsThe average percentage of female Cx. quinquefasciatus fed through the attractive biofilm was 87%, while only 20% became engorged with Parafilm-M® (p < 0.0001). Feeding through the attractive biofilm developed in this study produced high levels of evaluated biological parameters; the percentage of egg laying by females that underwent artificial hematophagy through the biofilm was 90%, with an average of 158 eggs per raft. From these eggs, 97% of the larvae hatched, of which 95% reached the pupal stage. The adult emergence rate corresponded to 93% of pupae.ConclusionsInsects fed with attractant through the biofilm system had a higher engorgement rate compared to those fed through Parafilm-M®. Our study is preliminary and suggests that polymeric biofilm has great potential for artificially feeding mosquitoes in the laboratory. Based on this research, new studies will be carried out with biofilm and different systems.Graphical

Design, fabrication, evaluation, and in vitro study of green biomaterial and antibacterial polymeric biofilms of polyvinyl alcohol/tannic acid/CuO/ SiO2

In this study, a three-component biofilm for rapid wound dressing consisting of polyvinyl alcohol (PVA)/tannic acid (TA)/with CuO/SiO2 with different percentages (0, 5, 10, and 15 wt% NPs) is evaluated. In addition to controlling bleeding and absorption of blood and wound secretions, it protects the damaged tissue from the attack of microbes. It protects against viruses and thus reduces the treatment time. Analysis of biofilms morphology is performed by Field emission scanning electron microscopy (FE-SEM), phases in biofilms were analyzed by X-ray diffraction (XRD) analysis, chemical bonds, and functional groups are analyzed by Fourier transform infrared (FTIR) spectroscopy, and mechanical tests are performed to evaluate the strength of the samples. The thermogravimetric analysis (TGA) is applied to estimate the thermal stability of the biopolymer films with various percentages of CuO/SiO2 nanoparticles. Also, antibacterial test, bioactivity of the biofilms, the percentage of swelling ratio, and porosity of the samples were examined by immersing the samples in simulated body fluid (SBF) and Phosphate-buffered saline (PBS) for 14 days in vitro. The composite makeup of the TA/PVA sample, comprising 15 wt % CuO/SiO2 and containing 15 wt% of nanoparticles, exhibited superior heat resistance compared to other samples by an increase of 50 °C. This improvement can be attributed to the nanoparticles reaching their saturation point. The swelling ratio was assessed in both SBF and PBS, and in both instances, the sample increased by up to 10 wt% before decreasing, indicating the saturation of the nanoparticles.

Theranostic gold-in-gold cage nanoparticles enable photothermal ablation and photoacoustic imaging in biofilm-associated infection models.

Biofilms are structured communities of microbial cells embedded in a self-produced matrix of extracellular polymeric substances. Biofilms are associated with many health issues in humans, including chronic wound infections and tooth decay. Current antimicrobials are often incapable of disrupting the polymeric biofilm matrix and reaching the bacteria within. Alternative approaches are needed. Here, we described a complex structure of a dextran-coated gold-in-gold cage nanoparticle that enabled photoacoustic and photothermal properties for biofilm detection and treatment. Activation of these nanoparticles with a near infrared laser could selectively detect and kill biofilm bacteria with precise spatial control and in a short timeframe. We observed a strong biocidal effect against both Streptococcus mutans and Staphylococcus aureus biofilms in mouse models of oral plaque and wound infections, respectively. These effects were over 100 times greater than those seen with chlorhexidine, a conventional antimicrobial agent. Moreover, this approach did not adversely affect surrounding tissues. We concluded that photothermal ablation using theranostic nanoparticles is a rapid, precise, and nontoxic method to detect and treat biofilm-associated infections.

Preparation of gallic acid functionalized polyvinyl alcohol/β‐cyclodextrin‐silver nanoparticles cast film as an antimicrobial and antioxidant agent

AbstractIn recent studies, researchers have been dedicated to the synthesis of silver nanoparticles (AgNPs) that can be adjusted in size, owing to their unique physical and chemical properties. In this study, AgNPs with well‐defined and stable characteristics are successfully functionalized with Gallic acid (GA) within a polymer matrix consisting of polyvinyl alcohol/β‐cyclodextrin polymeric biofilms (BFs) using a casting approach. To evaluate the particle size, functional groups, and crystallinity of the AgNPs, various techniques including UV, FT‐IR, TEM, and XRD are employed. The prepared BF films (BFs) are comprehensively analyzed for mechanical strength, color ratio, swelling ratio, surface morphology, elemental ratio, thermal behavior, and water wettability using stress–strain analysis, spectrophotometer color lab, SEM, EDAX, TGA, and contact angle techniques. Furthermore, the antimicrobial properties of the BFs are also investigated, along with their antioxidant activity assessed through the DPPH radical assay. The multifunctional BFs exhibited promising potential as antimicrobial agents for food preservation.

BIOSORPTION CHARACTERISTICS OF HG(II) INTO BIOFILM

Biosorption is an alternative technology developed to overcome pollution, including heavy metal contamination in aquatic ecosystems. The accuracy of choosing a biosorbent strongly influences the success of biosorption. The organisms that are widely developed as biosorbents are microbes. Microbes live in ecosystems by forming biofilms. Therefore, knowledge related to the mechanism of heavy metal adsorption, such as Hg(II) by biofilms, will contribute to the development ofbiofilm-based biosorption. This study aims to analyze the biosorption characteristics of Hg(II) bybiofilms taken from aquatic ecosystems. The methods used are kinetic adsorption analysis, isotherm adsorption analysis, and FTIR spectra analysis of biofilms. This study indicates that the adsorption process of Hg(II) into the biofilm is a physicochemical process. The adsorption sites that bind Hg(II) are negatively charged sites resulting from the ionization of functional groups such as carboxyl groups present in polymeric biofilms. According to this study, biofilm is an excellent biosorbent to adsorb pollutants such as Hg(II) from aquatic ecosystems.

The Polymeric Matrix Composition of Vibrio cholerae Biofilms Modulate Resistance to Silver Nanoparticles Prepared by Hydrothermal Synthesis.

Biofilms represent the dominant microbial lifestyle in nature. These complex microbial communities in which bacteria are embedded in a self-produced protective polymeric extracellular matrix, display an enhanced resistance to antimicrobials and thus represent a major health challenge. Although nanoparticles have proven to be effective against bacteria, the interactions between nanoparticles and the polymeric biofilm matrix are still unclear. In this work, silver nanoparticles (AgNPs) were used on mature biofilms formed by the pathogen Vibrio cholerae, and their effects on the biofilm microstructure were evaluated. Bacteria cells within mature biofilms showed an increased tolerance to AgNPs, with their elimination requiring a concentration nine times higher than planktonic cells. Mutant strains not able to form a pellicle biofilm were four times more susceptible to AgNPs than the wild-type strain forming a strong biofilm. Moreover, electron microscopy analysis revealed that AgNPs interacted with the extracellular matrix components and disrupted its microstructure. Finally, two major proteins, Bap1 and RbmA, appeared to mediate the biofilm bacterial resistance to AgNPs. This work highlights the role of the polymeric biofilm matrix composition in resistance to AgNPs. It underlines how crucial it is to understand and characterize the interactions between nanoparticles and the biofilm matrix, in order to design appropriate metallic nanoparticles efficient against bacterial biofilms.

Staphylococcus aureus Biofilm: Morphology, Genetics, Pathogenesis and Treatment Strategies.

Staphylococcus aureus is a nosocomial bacterium causing different infectious diseases, ranging from skin and soft tissue infections to more serious and life-threatening infections such as septicaemia. S. aureus forms a complex structure of extracellular polymeric biofilm that provides a fully secured and functional environment for the formation of microcolonies, their sustenance and recolonization of sessile cells after its dispersal. Staphylococcus aureus biofilm protects the cells against hostile conditions, i.e., changes in temperature, limitations or deprivation of nutrients and dehydration, and, more importantly, protects the cells against antibacterial drugs. Drugs are increasingly becoming partially or fully inactive against S. aureus as they are either less penetrable or totally impenetrable due to the presence of biofilms surrounding the bacterial cells. Other factors, such as evasion of innate host immune system, genome plasticity and adaptability through gene evolution and exchange of genetic material, also contribute to the ineffectiveness of antibacterial drugs. This increasing tolerance to antibiotics has contributed to the emergence and rise of antimicrobial resistance (AMR), a serious problem that has resulted in increased morbidity and mortality of human and animal populations globally, in addition to causing huge financial losses to the global economy. The purpose of this review is to highlight different aspects of S. aureus biofilm formation and its overall architecture, individual biofilm constituents, clinical implications and role in pathogenesis and drug resistance. The review also discusses different techniques used in the qualitative and quantitative investigation of S. aureus biofilm and various strategies that can be employed to inhibit and eradicate S. aureus biofilm.

An Innovative Method for the Recycling of Waste Carbohydrate-Based Flours.

This work represents an innovative study that, for the first time, explores the possibility to use waste flours to produce thermoplastic polymeric bio-films. To the best of our knowledge, this is the first time that waste flours, derived from bakeries, pizzerias or pasta factories, have been proposed for the production of bio-polymers, as a replacement of neat starch. To this aim, durum waste flour derived from a pasta factory, soft waste flour derived from pizzerias and neat maize starch used as control material were firstly analyzed from dimensional, morphological and chemical points of view. Afterwards, waste flour films were produced by the addition of a nature-based plasticizer, glycerol. Mechanical characterization of the plasticized thermoplastic films, produced by compression molding, evidenced low performances, even in the case of the neat maize starch. In order to improve the mechanical properties, the possibility to include polylactic acid and cardanol-based plasticizer was also investigated. Mass transport properties of all the produced bio-films were investigated by measuring their water vapor permeability and hygroscopic absorption. The durability properties of the bio-films were assessed by accelerated ageing tests, while the bio-degradability of the waste-based films was evaluated by measuring the solubility and the degradation in water. The physicochemical analyses of the novel bio-films evidenced good mechanical properties; specifically, the waste-based films showed a lower hygroscopic absorption and water solubility than those of the blends containing neat starch.

Fish Scale Derived Hydroxyapatite reinforced Poly (Lactic acid) Polymeric Bio-films: Possibilities for Sealing/locking the Internal Fixation Devices

Polylactic acid (PLA) and Hydroxyapatite (HAp) each have various applications in bone repair materials. In this paper,hydroxyapatite reinforced polylactic acid biofilmswere processed through twinscrew blended cum solvent casting method.PLA/HAp films were uniformly distributed and smooth surface were obtained.Thehydroxyapatites were fabricated from fish scales biowastes in a cost effective manner through chemical cum heat treatment method.The observation through energy dispersive X-ray spectroscopy confirms the presence of major element responsible for Hydroxyapatite.The surface morphology through field emission scanning electron microscope reveals the information of HAp distribution in the PLA uniformly.The particle size of the HAp was analyzed through Delsa Nano. The presence of corresponding functional group was identified by analyzing fourier transform infrared spectroscopy. The thermal stability and crystallization temperatures were analyzed by differential scanning calorimeter cum thermo gravimetric analysis. The surface wettability in order to confirm to hydrophilic nature were analyzed by using contact angle measurement. The results were concluded based upon the experiments and found that addition of HAp leads to convert the biofilms in hydrophilic in nature to make more biocompatibility. The thermal stability of the biofilms was also improved due to addition of Hydroxyapatite particles.

Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology.

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other abiotic surfaces (polystyrene and stainless steel). Therefore, this RTCA technology opens new opportunities in the biofilm research arena and its application could be further explored for other bacterial genera as well as for different bio-active molecules.

Detection of biofilm in bronchoalveolar lavage from children with non‐cystic fibrosis bronchiectasis

The presence of Pseudomonas aeruginosa biofilms in lower airway specimens from cystic fibrosis (CF) patients is well established. To date, biofilm has not been demonstrated in bronchoalveolar lavage (BAL) from people with non-CF bronchiectasis. The aim of this study was to determine (i) if biofilm was present in BAL from children with and without bronchiectasis, and (ii) if biofilm detection differed between sequentially collected BAL. Testing for biofilm in two sequentially collected BAL from children with and without bronchiectasis was done using BacLight™ live-dead staining and lectin staining for extracellular polymeric biofilm matrices. Bacterial culture and cytological measures were performed on the first and second lavages, respectively. Clinically important BAL infection was defined as >104 cfu of respiratory pathogens/ml BAL. Biofilm was detected in BAL from seven of eight (87.5%) children with bronchiectasis (aged 0.8-6.9 years), but was not detected in any of three controls (aged 1.3-8.6 years). The biofilms contained both live and dead bacteria irrespective of antibiotic use prior to bronchoscopy. Biofilm was detected more frequently in the second lavage than the first. Three of the seven biofilm-positive BAL were culture-positive for respiratory pathogens at clinically important levels. Biofilm is present in BAL from children with non-CF bronchiectasis even when BAL-defined clinically important infection was absent. Studies to characterize lower airway biofilms and determine how biofilm contributes to bronchiectasis disease progression and treatment outcomes are necessary. Pediatr Pulmonol. 2015; 50:284-292. © 2014 Wiley Periodicals, Inc.

Abstract 1678: Systemic gallium nitrate therapy results in sustained sputum concentrations that may be therapeutic for biofilm-associated pulmonary bacterial infections

Abstract Background: The formation of polymeric biofilms represents an important defense mechanism evolved by planktonic bacteria. Bacteria living in biofilms may resist killing by standard antiinfective agents despite nominal in vitro sensitivity owing in part to reduced drug penetration. Elemental iron is an essential nutrient for most infectious organisms. Agents that sequester iron have been tested experimentally with initially favorable results in serious infections, including Pseudomonas aeruginosa. Kaneko et al (J Clin Invest 117;877, 2007) proposed the use of ionic gallium as a potential antiinfective that interferes with iron metabolism and showed that gallium solutions (0.5-10 μM; 1 μg/ml=14.5 μM) disrupted biofilms produced by P. aeruginosa and resulted in collapse of resistant bacterial colonies. They speculated that an aerosolized gallium solution might be useful for P. aeruginosa pulmonary infections. Due to variability of drug dispersion within diseased pulmonary tissue, we speculated that systemic therapy with a gallium-containing solution could achieve potentially therapeutic levels in sputum of infected patients. Methods: A 31-year old man with end-stage cystic fibrosis lung disease (FEV1 20%) and highly resistant P. aeruginosa was treated on an investigational basis with several cycles of gallium nitrate (Ganite®), an agent for the treatment of hypercalcemia of malignancy. The drug was given by continuous IV infusion for 5 days at doses ranging from approximately 100 to 250 mg/m2/d. In 2 cycles, serial samples of expectorated sputum and whole blood or plasma were obtained and assayed for gallium concentration by ICP/MS. Informed consent and ethical approval were obtained. Results: After the first drug infusion (150 mg/m2/d for 5 days), sputum gallium concentration from a single sample was unexpectedly high (2.2 μg/ml) relative to previously reported blood concentrations. Approximately 1 month later, a sputum concentration was still measurable (0.32 μg/ml), and the patient was retreated at a dose of 200 mg/m2/d for 5 days. Paired sputum samples on day 5 showed gallium levels of 2.13 and 2.65 μg/ml, respectively, whereas simultaneous measurements of gallium in plasma and whole blood were 1.35 and 1.64 μg/ml, respectively. Despite a previously reported plasma T1/2 ∼ 12 h, serial measurements after the drug infusion showed a progressive increase in sputum gallium concentration on days 6, 7, 9, and 11 (1.83, 2.04, 4.35, and 4.78 μg/ml, respectively). Conclusions: Gallium appears to be preferentially concentrated in infected sputum. This effect increases and remains sustained post therapy, suggesting that transport across a gradient can be maintained for a prolonged period. Systemic gallium therapy achieves sputum drug levels that may be therapeutic for highly resistant bacteria associated with biofilm formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1678.