Bacterial Biofilm Development Research Articles

Nanomaterials in the Management of Contact Lens-Associated Ocular Complication

Contact lens is the transparent hemisphere-shaped ophthalmic product worn by a large population all across the globe for improving vision. It is applied to the cornea of the eye not only for improving vision but also for the delivery of drugs. The contact lens has many benefits but it also causes several ophthalmic infections which include amoebic keratitis, fungal, bacterial, and viral keratitis. The development of bacterial biofilm in lens cases exacerbates the ocular infection after the application of contact lenses in the cornea. Contact lens often associated with discomfort and dry eye. Coating these contact lens with different nanomaterials and an antimicrobial agent is a very good approach to improve the quality and biological activity of lenses. Surface functionalization is the most commonly adopted, and versatile method to tailor the optical properties. This review highlights the new technology to achieve the sustained release of drugs from contact lens by controlling their chemical, mechanical, and optical properties..

Bioactive Coatings Based on Hydroxyapatite, Kanamycin, and Growth Factor for Biofilm Modulation.

The occurrence of opportunistic local infections and improper integration of metallic implants results in severe health conditions. Protective and tunable coatings represent an attractive and challenging selection for improving the metallic devices’ biofunctional performances to restore or replace bone tissue. Composite materials based on hydroxyapatite (HAp), Kanamycin (KAN), and fibroblast growth factor 2 (FGF2) are herein proposed as multifunctional coatings for hard tissue implants. The superior cytocompatibility of the obtained composite coatings was evidenced by performing proliferation and morphological assays on osteoblast cell cultures. The addition of FGF2 proved beneficial concerning the metabolic activity, adhesion, and spreading of cells. The KAN-embedded coatings exhibited significant inhibitory effects against bacterial biofilm development for at least two days, the results being superior in the case of Gram-positive pathogens. HAp-based coatings embedded with KAN and FGF2 protein are proposed as multifunctional materials with superior osseointegration potential and the ability to reduce device-associated infections.

Development, dynamics and control of antimicrobial-resistant bacterial biofilms: a review

Antimicrobial resistance is a major health issue inducing the inefficiency of antimicrobial drugs. Indeed, pathogenic microorganisms become resistant when frequently exposed to antimicrobial drugs. In particular, when bacteria are attached to a surface and expand as a biofilm, they become more resistant to antimicrobials because single or multiple bacterial species are embedded in a slimy extracellular polymeric substance that acts like a shield. Biofilms often contaminate medical devices and food industrial equipment, thus leading to infections and food spoilage. Here, we review the basics of biofilm development from a planktonic bacterium; signaling in biofilm and relationship with antimicrobial resistance; biofilm control and destruction strategies using quorum sensing inhibitors, ultrasound, acidic electrolyzed water, and enzymatic and combination killing; and emerging approaches, such as bacteriophage-mediated disruption and antimicrobial peptides to control bacterial biofilms.

Human Skin In Vitro Colonization Model for a Skin Wound Infected by Staphylococcus aureus Biofilm.

Biofilms provide an environment in which bacteria can survive adverse conditions such as nutrient or oxygen deficiencies, and antibiotic treatments. Bacterial survival of antibiotic treatments can often result in antimicrobial resistance, which can make treating infections substantially more difficult, increase the burden of healthcare costs, and hinder the healing of infected wounds. As Staphylococcus aureus is a bacterium that commonly causes skin infections, can be found in infected skin wounds, and is prone to developing antimicrobial resistance-especially within a biofilm microenvironment, the study and development of methodologies to treat infected wounds have become an important topic of research. To study the development of bacterial biofilm in a skin wound, this chapter discusses an in vitro model to access biofilm growth in an environment that mimics a human skin wound. This model serves as a tool to study the biofilm growth and efficacy of antibiotic use in an in vitro system that more closely resembles human skin tissue, rather than a polystyrene plate.

Selective delivery of silver nanoparticles for improved treatment of biofilm skin infection using bacteria-responsive microparticles loaded into dissolving microneedles

The treatment of infected chronic wounds has been hampered by development of bacterial biofilms and the low penetration of antibacterial compounds delivered by conventional dosage forms. Numerous bacterial biofilm formers have shown resistance to synthetic antibacterial agents. In this study, we explore the potential of silver nanoparticles (NPs) synthesized using green tea extract as antibiofilm agents against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) biofilms. Due to the toxicity of silver NPs, for the first time, silver NPs were incorporated into bacteria-responsive microparticles (MPs) prepared from poly (Ɛ-caprolactone) decorated with chitosan. The in vitro release of silver NPs from MPs increased up to 9-times in the presence of SA and PA, showing the selectivity of this approach. Incorporation of the MPs into dissolving microneedles (DMNs) could enhance the dermatokinetic profiles of silver NPs compared to DMNs containing silver NPs without MP formulations and conventional cream formulations. Furthermore, 100% of bacterial bioburdens were eradicated on ex vivo biofilm model in rat skin following 60 h of the administration of this system. The findings revealed here confirmed the feasibility of the loading of silver NPs into responsive MPs for improved antibiofilm activities when delivered using DMNs. Following on from these promising results, toxicity and in vivo pharmacodynamic studies should now be carried out in an appropriate model.

In Vitro Evaluation of Gentamicin or Vancomycin Containing Bone Graft Substitute in the Prevention of Orthopedic Implant-Related Infections.

Antibiotic-loaded bone graft substitutes are attractive clinical options and have been used for years either for prophylaxis or therapy for periprosthetic and fracture-related infections. Calcium sulfate and hydroxyapatite can be combined in an injectable and moldable bone graft substitute that provides dead space management with local release of high concentrations of antibiotics in a one-stage approach. With the aim to test preventive strategies against bone infections, a commercial hydroxyapatite/calcium sulfate bone graft substitute containing either gentamicin or vancomycin was tested against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, harboring different resistance determinants. The prevention of bacterial colonization and biofilm development by selected microorganisms was investigated along with the capability of the eluted antibiotics to select for antibiotic resistance. The addition of antibiotics drastically affected the ability of the selected strains to adhere to the tested compound. Furthermore, both the antibiotics eluted by the bone graft substitutes were able to negatively impair the biofilm maturation of all the staphylococcal strains. As expected, P. aeruginosa was significantly affected only by the gentamicin containing bone graft substitutes. Finally, the prolonged exposure to antibiotic-containing sulfate/hydroxyapatite discs did not lead to any stable or transient adaptations in either of the tested bacterial strains. No signs of the development of antibiotic resistance were found, which confirms the safety of this strategy for the prevention of infection in orthopedic surgery.

Quantitative analysis of the surficial and adhesion properties of the Gram-negative bacterial species Comamonas testosteroni modulated by c-di-GMP

Cyclic diguanylate monophosphate (c-di-GMP) is a ubiquitous intracellular secondary messenger which governs the transition from a bacterial cell’s planktonic state to biofilm formation by stimulating the production of a variety of exopolysaccharide material by the bacterial cell. A range of genes involved in c-di-GMP signaling in the Gram-negative species Comamonas testosteroni have been identified previously, yet the physical-chemical properties of the produced extracellular polymeric substances (EPS) and the bacterial adhesion characteristics regulated by c-di-GMP are not well understood. Here, we modulated the in vivo c-di-GMP levels of Comamonas testosteroni WDL7 through diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) gene editing. The strains and their adhesion properties were characterized by Fourier-transform infrared and two-dimensional correlation spectroscopy analysis (FTIR-2D CoS), contact angle and zeta potential measurements, atomic force microscopy (AFM) and extended-Derjaguin-Landau-Verwey-Overbeek (ExDLVO) analysis. Our results show that high c-di-GMP levels promoted the secretion of long-chain hydrophobic and electroneutral extracellular polysaccharides and proteins. The protein molecules on WDL7/pYedQ2 promoted the bacterial self-aggregation and adhesion onto negatively charged surfaces. In contrast, the reduction of intracellular c-di-GMP concentrations resulted in a nearly 80 % decrease in the adhesion of bacterial cells, although little change in the surface hydrophobicity or surface charge properties were observed for these cells relative to the wild type. These results indicate that the reduced adsorption of WDL7/YhjH that we observed may be caused by the flagellum-accelerated mobility at low c-di-GMP concentrations. Taken together, these results improve our mechanistic understanding of the effects of c-di-GMP in controlling bacterial physical-chemical properties and initial biofilm development.

Early and differential bacterial colonization on microplastics deployed into the effluents of wastewater treatment plants

Microbial colonization of microplastics (MPs) in aquatic ecosystems is a well-known phenomenon; however, there is insufficient knowledge of the early colonization phase. Wastewater treatment plant (WWTP) effluents have been proposed as important pathways for MPs entry and transport in aquatic environments and are hotspots of bacterial pathogens and antibiotic resistance genes (ARGs). This study aimed at characterizing bacterial communities in the early stage of biofilm formation on seven different types of MPs deployed in two different WWTPs effluents as well as measuring the relative abundance of two ARGs (sulI and tetM) on the tested MPs. Illumina Miseq sequencing of the 16S rRNA showed significant higher diversity of bacteria on MPs in comparison with free-living bacteria in the WWTP effluents. β-diversity analysis showed that the in situ environment (sampling site) and hydrophobicity, to a lesser extent, had a role in the early bacterial colonization phase. An early colonization phase MPs-core microbiome could be identified. Furthermore, specific core microbiomes for each type of polymer suggested that each type might select early attachment of bacteria. Although the tested WWTP effluent waters contained antibiotic resistant bacteria (ARBs) harboring the sulI and tetM ARGs, MPs concentrated ARBs harboring the sulI gene but not tetM. These results highlight the relevance of the early attachment phase in the development of bacterial biofilms on different types of MP polymers and the role that different types of polymers might have facilitating the attachment of specific bacteria, some of which might carry ARGs.

A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions

BackgroundStudying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions.ResultsUsing Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 minimal medium) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth rich medium ). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials.ConclusionThe platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

Synergistic Effect of the Cationic Peptide Hominin and a New Disinfectant Based on Isoquinoline on Formation of Biofilms in Multidrug-Resistant Staphylococci

The growing threat of proliferation of biofilm-forming hospital strains of coagulase-negative staphylococci resistant to antibiotics determines the need for an urgent search for new effective antibacterial compounds, as well as the development of methods for the combined use of traditional and alternative antibiotics. The article presents the results of a study of the combined effect of the drug «SA» — a new synthetic derivative of the alkaloid isoquinoline and a low-molecular-weight cationic peptide of the lantibiotic family hominin, which inhibits the development of bacterial biofilms of the clinical strain of Staphylococcus haemolyticus and its vancomycin-resistant isolates. It was found that combinations of these compounds have a synergistic effect that suppresses the formation of biofilms of both studied strains of staphylococci at reduced concentrations of these antibacterial compounds.

Illuminating the dynamics of biofilms.

This month’s Under the Lens discusses dual-view light-sheet microscopy and how its use has revealed the dynamics of bacterial biofilm development, a fundamental process found in bacteria.

Diabetic wound infection: A review on microbial population and infection control

A diabetic foot ulcer is one of the major complications of diabetes and it leads to lower extremity amputation in patients. This review explores the current research on microbial populations on diabetic wounds, and also treatment alternatives to combat the infection on chronic diabetic wounds. The microbial communities exist in diabetic wound infection are diverse. Microbes rarely survived in single species of planktonic cells. They usually exist in a complex polymicrobial biofilm population which consists of different types of microorganisms. Furthermore, the development of bacterial biofilm on the wound that usually consists of multidrug-resistant pathogens also delayed the wound healing. To overcome this problem, many types of modern wound dressing were developed including hydrocolloid, hydrogel, alginate and collagen wound dressing. Besides, modern biotechnological advancements such as cell therapy, bioengineered skin, dermal scaffolds, tissue-engineered artificial skin and growth factors were also employed to promote the recovery of the wound. In conclusion, diabetes mellitus is a major health care challenge worldwide. Diabetic patients are at risk for developing foot ulcer which ultimately leads to amputation; hence a safe and effective alternative treatment is required to improve diabetic patients’ quality life.

How to build a biofilm.

Two studies used advanced imaging techniques to study the formation and development of bacterial biofilms.

Activated Polyhydroxyalkanoate Meshes Prevent Bacterial Adhesion and Biofilm Development in Regenerative Medicine Applications.

Regenerative medicine has become an extremely valuable tool offering an alternative to conventional therapies for the repair and regeneration of tissues. The re-establishment of tissue and organ functions can be carried out by tissue engineering strategies or by using medical devices such as implants. However, with any material being implanted inside the human body, one of the conundrums that remains is the ease with which these materials can get contaminated by bacteria. Bacterial adhesion leads to the formation of mature, alive and complex three-dimensional biofilm structures, further infection of surrounding tissues and consequent development of complicated chronic infections. Hence, novel tissue engineering strategies delivering biofilm-targeted therapies, while at the same time allowing tissue formation are highly relevant. In this study our aim was to develop surface modified polyhydroxyalkanoate-based fiber meshes with enhanced bacterial anti-adhesive and juvenile biofilm disrupting properties for tissue regeneration purposes. Using reactive and amphiphilic star-shaped macromolecules as an additive to a polyhydroxyalkanoate spinning solution, a synthetic antimicrobial peptide, Amhelin, with strong bactericidal and anti-biofilm properties, and Dispersin B, an enzyme promoting the disruption of exopolysaccharides found in the biofilm matrix, were covalently conjugated to the fibers by addition to the solution before the spinning process. Staphylococcus epidermidis is one of the most problematic pathogens responsible for tissue-related infections. The initial antibacterial screening showed that Amhelin proved to be strongly bactericidal at 12 μg/ml and caused >50% reductions of biofilm formation at 6 μg/ml, while Dispersin B was found to disperse >70% of pre-formed biofilms at 3 μg/ml. Regarding the cytotoxicity of the agents toward L929 murine fibroblasts, a CC50 of 140 and 115 μg/ml was measured for Amhelin and Dispersin B, respectively. Optimization of the electrospinning process resulted in aligned fibers. Surface activated fibers with Amhelin and Dispersin B resulted in 83% reduction of adhered bacteria on the surface of the fibers. Additionally, the materials developed were found to be cytocompatible toward L929 murine fibroblasts. The strategy reported in this preliminary study suggests an alternative approach to prevent bacterial adhesion and, in turn biofilm formation, in materials used in regenerative medicine applications such as tissue engineering.

Analyzing the inhibitory effect of metabolic uncoupler on bacterial initial attachment and biofilm development and the underlying mechanism

Metabolic uncouplers inhibit biofilm and biofouling formation in membrane bioreactor (MBR) systems, which have been considered as a potential biofouling control alternative. To better understand the inhibitory mechanism of uncoupler on biofouling, this study investigated the impact of the uncoupler 3, 3′, 4′, 5-tetrachlorosalicylanilide (TCS) on biofilm formation of B. subtilis in different development stages. Significant reductions in both the initial bacterial attachment stage and the subsequent biofilm development stage were caused by TCS at 100 μg/L. The motility of B. subtilis in semisolid medium was inhibited by TCS, which explicitly explained the reduction in initial bacterial attachment. Meanwhile, a reduction of extracellular polymeric substance (EPS) secretion owing to TCS suggested why biofilm development was suppressed. In addition, the fluorescent materials in tight-bound EPS (TB-EPS) and loose-bound EPS (LB-EPS) of Bacillus subtilis cultured in different TCS concentrations were distinguished and quantified by three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC). The results of this study suggested that the biofilm inhibitory mechanism of the uncoupler was both a inhibition in bacterial motor ability and a reduction in EPS secretion.

Development and Diversity of Bacterial Biofilms in Response to Internal Tides, a Case Study off the Coast of Kuwait

Information pertaining to changes in abundance and composition of microbial communities on offshore platforms in relation to changes in environmental conditions due to internal tides are scarce. In this study, artificial substrata were deployed at two locations on a gently sloping seabed off the coast of Kuwait. The abiotic factors at the two sites were recorded spatially and temporally using time-series measurements and continuous turbulence profiling for 13 days. Results showed variations in water density between the upper and deeper waters with the pycnocline undulating between 6 and 15 m depth at both locations. In the water layer beneath the pycnocline, significant current shear due to the internal tides led to higher turbidity coupled with lower dissolved oxygen (DO) and chlorophyll a concentration. The microbiological data showed a significant decrease in the biofilm total biomass, bacterial counts and phototrophic biomass with increase in depth at both locations. The 16S rRNA amplicon sequence non-metric multidimensional (NMDS) scaling analysis revealed that biofouling bacterial communities affected by depth with Alphaproteobacteriaand Bacteroidetes members dominated upper and deeper water at both locations. The based-ordination analysis revealed that biofouling bacterial communities at 3 m were different than at 15 m, with a percentage of shared Operational taxonomic units (OTUs) ≤64% between locations and between depths. Despite the limitations of the study, the power of the employed system is demonstrated in the results that shed light on the significance of prevailing environmental conditions associated with internal tides in shaping the biofilm community in subtropical offshore water systems.

Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa.

Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors.

PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.

Design of fish processing equipment: exploring cleaning brush performance and material properties to minimize biofilm deposits

Abstract The development of bacterial biofilms in fish processing plants may facilitate the growth of human pathogenic bacteria such as Listeria monocytogenes and may therefore compromise food safety. In the Norwegian aquaculture industry, the cleaning of fish processing plants is the final process step during fish manufacturing processes. This article explores the efficiency of brushes of different designs in combination with using different cleaning methods in cleaning three types of material. A total of seven different brush designs (including those with soft, medium, and hard bristles; bristles also varied in length and density) and operational movements (linear versus rotational), were tested. It is believed that in the future, robots may clean fish processing plants, and equipping such robots with cleaning tools may significantly contribute to increasing food safety. This research investigates this future technology by investigating how tools that may be added to a robotic cleaning system will perform. The objective of verifying the performance of cleaning tools used in fish factories is to determine how effective they are in terms of the cleaning and removal of biofilm and how they perform with regard to cross-contamination. Test items of stainless steel, aluminum alloy and high-density polyethylene were experimentally inoculated with Pseudomonas fluorescence and Staphylococcus aureus, which were allowed to develop biofilms. Cleaning efficiency was analyzed spectrophotometrically by swabbing the cleaned test items and measuring the optical density at 600 nm in the swab eluates after overnight incubation. Cleaned test items and uncleaned controls were also stained with SYBR Green and photographed under ultraviolet light to evaluate biofilm removal efficiency by image analysis. Cleaning trials demonstrated that a rotating brush (240 rpm) performed better than brushes using a linear movement for the removal of biofilms on all material types, and that equipping robots with such tools may significantly improve cleaning performance. It is also found, however, that rotating brushes may introduce the risk of cross contamination due to splashing of bacteria into the air. The research findings and the methodology used for verification in this paper will serve as the foundation for future research on hygienic design and cleaning performance in the global fish processing industry.

A 3D soil-like nanostructured fabric for the development of bacterial biofilms for agricultural and environmental uses

Creation of beads-and-fibres 3D nanoscaffolds mimicking the typical architecture of soils at microscale and nanoscale for the development of bacterial biofilms for agricultural and environmental applications.