Prolonged inhibitory effects against planktonic growth,adherence, and biofilm formation of pathogens causing ventilator-associated pneumonia using a novel polyamide/silver nanoparticle composite-coated endotracheal tube

Abstract

Microbial cells can rapidly form biofilm on endotracheal tubes (ETT) causing ventilator-associated pneumonia, a serious complication in patients receiving mechanical ventilation. A novel polyamide with a good balance of hydrophilic/hydrophobic moieties was used for the embedment of green-reduction silver nanoparticles (AgNPs) for the composite-coated ETT. The films were conformal with a thickness of ～ 17?±?3?µm accommodating high loading of 60?±?35?nm spherical-shaped AgNPs. The coated ETT resulted in a significant difference in reducing both planktonic growth and microbial adhesion of single and mixed-species cultures, compared with uncoated ETT (p?<?0.05). A time-kill assay demonstrated rapid bactericidal effects of the coating on bacterial growth and cell adhesion to ETT surface. Biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus, commonly encountered pathogens, was inhibited by > 96% after incubation for 72?h. Polyamide/AgNP composite-coated ETT provided a broad-spectrum activity against both Gram-positive and Gram-negative bacteria as well as Candida albicans and prolonged antimicrobial activity.     

Effect of biofouling on anodized and sol-gel treated titanium surfaces: a comparative study

Anodization and sol-gel treatments of titanium (Ti) were evaluated as biofilm control measures on surfaces exposed to seawater exposed to ultraviolet light. Anodized and sol-gel treated specimens were characterized using Raman spectroscopy to confirm the presence of TiO₂. The single anatase phase was observed at the anodized surfaces whereas the anatase/rutile mixed phase was detected on the sol-gel coated surfaces. After exposure of the specimens to seawater, biofilms were characterized by total viable counts, and epifluorescence and Raman microscopy. These techniques confirmed the reduction in biofilm formation on both the anodized and sol-gel coated Ti specimens compared to the untreated specimens. Biofilm control by anodization was found to be more effective than by sol-gel treatment of the specimens. The higher particle size and the inhomogeneity at the sol-gel coated surfaces produced less effective biofilm control.     

Fast corroding,thin magnesium coating displays antibacterial effects and low cytotoxicity

Bacterial colonisation and biofilm formation are characteristics of implant-associated infections. In search of candidates for improved prosthetic materials, fast corroding Mg-based coatings on titanium surfaces were examined for their cytotoxic and antimicrobial properties. Human osteoblasts and Staphylococcus epidermidis were each cultured on cylindrical Ti samples coated with a thin layer of Mg/Mg₄₅Zn₅Ca, applied via magnetron sputtering. Uncoated titanium samples served as controls. S. epidermidis was quantified by counting colony forming units. The biofilm-bound fraction was isolated via ultrasonic treatment, and the planktonic fraction via centrifugation. Biofilm-bound S. epidermidis was significantly decreased by approximately four to five orders of magnitude in both Mg- and Mg₄₅Zn₅Ca-coated samples after seven days compared to the control. The osteoblast viability was within the tolerance threshold of 70% stated in DIN EN ISO 10993-5:2009-10 for Mg (~80%) but not for Mg₄₅Zn₅Ca (~25%). Accordingly, Mg-coated titanium was identified as a promising candidate for an implant material with antibacterial properties and low cytotoxicity levels. The approach of exploiting fast corrosion contrasts with existing methods, which have generally focused on reducing corrosion.     

Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols

doi: 10.1111/j.1741‐2358.2011.00485.x
Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols Objective: The aim of this study was to investigate Candida albicans biofilm formation on denture liners and to analyse the efficacy of cleaning protocols. Material and methods: Specimens were prepared from four silicone‐based soft denture liners. After artificial ageing and surface free energy determination, specimens were incubated with saliva (2 h) and Candida albicans ATCC 10231 for either short‐ (2.5 h) or long‐term (24 h) biofilm formation. Adherent cells were determined either after incubation of specimens with Candida albicans or after treatment with different denture cleaning protocols. Statistical analysis was performed using one‐way anova and the Games–Howell test (α = 0.05). Results: For both short‐ and long‐term biofilm formation, similar amounts of Candida albicans cells were found on the surface of the different liners (p = 0.295 and 0.178, respectively). For both short‐ and long‐term biofilm formation, the highest cleaning efficacy was observed for sodium hypochlorite (NaOCl; p < 0.01). The efficacy of the chemical denture cleaner in removing long‐term Candida albicans biofilms was significantly lower than the efficacy of removal by brushing (p < 0.001). Conclusion: Different silicone‐based soft denture liners yield similar Candida albicans biofilm formation on their surface. The highest efficacy for the removal of Candida albicans biofilms was identified for NaOCl. Chemical denture cleaners appear to have rather low efficacy to remove mature Candida albicans biofilms.     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Effect of biofouling on anodized and sol-gel treated titanium surfaces: a comparative study

Anodization and sol-gel treatments of titanium (Ti) were evaluated as biofilm control measures on surfaces exposed to seawater exposed to ultraviolet light. Anodized and sol-gel treated specimens were characterized using Raman spectroscopy to confirm the presence of TiO(2). The single anatase phase was observed at the anodized surfaces whereas the anatase/rutile mixed phase was detected on the sol-gel coated surfaces. After exposure of the specimens to seawater, biofilms were characterized by total viable counts, and epifluorescence and Raman microscopy. These techniques confirmed the reduction in biofilm formation on both the anodized and sol-gel coated Ti specimens compared to the untreated specimens. Biofilm control by anodization was found to be more effective than by sol-gel treatment of the specimens. The higher particle size and the inhomogeneity at the sol-gel coated surfaces produced less effective biofilm control.     

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.     

Resveratrol,pterostilbene, and baicalein: plant-derived anti-biofilm agents

Microbial adhesion to surfaces and the subsequent biofilm formation may result in contamination in food industry and in healthcare-associated infections and may significantly affect postoperative care. Some plants produce substances with antioxidant and antimicrobial properties that are able to inhibit the growth of food-borne pathogens. The aim of our study was to evaluate antimicrobial and anti-biofilm effect of baicalein, resveratrol, and pterostilbene on Candida albicans, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. We determined the minimum inhibitory concentrations (MIC), the minimum adhesion inhibitory concentration (MAIC), and the minimum biofilm eradication concentration (MBEC) by crystal violet and XTT determination. Resveratrol and pterostilbene have been shown to inhibit the formation of biofilms as well as to disrupt preformed biofilms. Our results suggest that resveratrol and pterostilbene appear potentially very useful to control and inhibit biofilm contaminations by Candida albicans, Staphylococcus epidermidis, and Escherichia coli in the food industry.     

Improvisation and Evaluation of Laterosporulin Coated Titanium Surfaces for dental Applications: An In Vitro Investigation

Despite recent improvement in implant survival rates, there remains a significant demand for enhancing the long-term clinical efficacy of titanium (Ti) implants, particularly for the prevention of peri-implantitis. Bioactive substances such as antimicrobial peptides are emerging as effective alternatives for contemporary antimicrobial agents used in dental health care. Current research work was focused to use laterosporulins that are non-haemolytic cationic antimicrobial peptides from Brevibacillus spp. for coating commercially available Ti discs. The coated Ti surfaces were evaluated in vitro for biofilm formation by two dental plaque isolates Streptococcus gordonii strain DIGK25 and S. mutans strain DIGK119 as representatives of commensal and pathogenic streptococci respectively. The biofilm inhibition was ascertained with replicated experiments on hydroxyapatite discs and confirmed by florescence microscopy. The laterosporulin coated Ti discs showed significantly reduced biofilm formation by oral streptococci and displayed promising potential to enhance the antibacterial surface properties. Such improvised Ti surfaces may curb the menace of oral streptococcal biofilm formation on dental implants and the associated implant failures.     

Disruption of fungal and bacterial biofilms by lauroyl glucose

Aim: The ability of enzymatically synthesized lauroyl glucose to disrupt fungal (Candida albicans, Candida lipolytica) and bacterial (Pseudomonas aeruginosa PAO1, Pseudomonas aureofaciens) biofilms was investigated. Methods and Results: Preformed biofilms of C. albicans and C. lipolytica in polystyrene microtitre plates were disrupted upto 45% and 65%, respectively, while P. aeruginosa and P. aureofaciens biofilms were disrupted by 51% and 57%. Precoating of the microtitre wells with lauroyl glucose affected cell attachment and biofilm growth of all the cultures to a lesser extent. With C. albicans and C. lipolytica, there was 11% and 32% decrease in the development of biofilms, respectively. With P. aeruginosa and P. aureofaciens, the reduction was 21% and 12% after 48 h. Lauroyl glucose effectively inhibited the formation of biofilms on glass slide surfaces when added along with the inoculum. Analysis by confocal laser scanning microscopy showed that the growth of the biofilms was lesser as compared with the control experiments. Lauroyl glucose displayed minimum inhibitory concentration values >500 μg ml^?1 for the test cultures and was comparable to that obtained with acetyl salicylate. Conclusion: Lauroyl glucose reduces biofilm growth of all the four test cultures on polystyrene and glass surfaces. Significance and Impact of the Study: This report is a novel application of the enzymatically synthesized, environmental‐friendly nonionic surfactant.     

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.     

Insights into biological activity of ureidoamides with primaquine and amino acid moieties

Primaquine (PQ) ureidoamides 5a–f were screened for antimicrobial, biofilm eradication and antioxidative activities. Susceptibility of the tested microbial species towards tested compounds showed species- and compound-dependent activity. N-(diphenylmethyl)-2-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-4-methylpentanamide (5a) and 2-(4-chlorophenyl)-N-(diphenylmethyl)-2-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]acetamide (5d) showed antibacterial activity against S. aureus strains (MIC?=?6.5?µg/ml). Further, compounds 5c and 5d had weak antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. None of the tested compounds showed a wide spectrum of antifungal activity. In contrast, most of the compounds exerted strong activity in a biofilm eradication assay against E. coli, P. aeruginosa and Candida albicans, comparable to or even higher than gentamycin, amphotericin B or parent PQ. The most active compounds were 5a and 5b. Tested compounds were inactive against biofilm formation by C. parapsylosis, Enterococcus faecalis, C. tropicalis and C. krusei. Compounds 5b–f significantly inhibited lipid peroxidation (80–99%), whereas compound 5c presented interesting LOX inhibition.     

Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm

Abstract

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48?h biofilm was reduced by 2.10 log cfu cm^?2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ～2.5 log cfu cm^?2 of viable cells remaining after 105?min cleaning by water at 65?°C, compared to 4.5 log cfu cm^?2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste     

Do oral biofilms influence the wear and corrosion behavior of titanium?

The main aim of this work was to study the simultaneous wear-corrosion of titanium (Ti) in the presence of biofilms composed of Streptococcus mutans and Candida albicans. Both organisms were separately grown in specific growth media, and then mixed in a medium supplemented with a high sucrose concentration. Corrosion and tribocorrosion tests were performed after 48 h and 216 h of biofilm growth. Electrochemical corrosion tests indicated a decrease in the corrosion resistance of Ti in the presence of the biofilms although the TiO₂ film presented the characteristics of a compact oxide film. While the open circuit potential of Ti indicated a tendency to corrosion in the presence of the biofilms, tribocorrosion tests revealed a low friction on biofilm covered Ti. The properties of the biofilms were similar to those of the lubricant agents used to decrease the wear rate of materials. However, the pH-lowering promoted by microbial species, can lead to corrosion of Ti-based oral rehabilitation systems.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Evaluation of the ability of C. albicans to form biofilm in the presence of phage-resistant phenotypes of P. aeruginosa

Pseudomonas aeruginosa and Candida albicans are disparate microbial species, but both are known to be opportunistic pathogens frequently associated with nosocomial infections. The aim of this study was to provide a better understanding of the interactions between these microorganisms in dual-species biofilms. Several bacteriophage-resistant P. aeruginosa phenotypes have been isolated and were used in dual-species mixed-biofilm studies. Twenty-four and 48 h mixed-biofilms were formed using the isolated phenotypes of phage-resistant P. aeruginosa and these were compared with similar experiments using other P. aeruginosa strains with a defined lipopolysaccharide (LPS) deficiency based on chromosomal knockout of specific LPS biosynthetic genes. Overall, the results showed that the variants of phage-resistant P. aeruginosa and LPS mutants were both less effective in inhibiting the growth of C. albicans in mixed-biofilms compared to the wild-type strains of P. aeruginosa. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. In conclusion, the ability of strains of P. aeruginosa to inhibit the formation of a biofilm of C. albicans appears to be correlated with the LPS chain lengths of phenotypes of P. aeruginosa, suggesting that LPS has a suppressive effect on the growth of C. albicans.     

Comparative Effect of Chlorhexidine and Some Mouthrinses on Bacterial Biofilm Formation on Titanium Surface

The aim of the present study was to evaluate the effectiveness of chlorhexidine digluconate (CHX) and commonly used mouthrinses to single- and poly-species biofilms by S. mutans, S. aureus and P. aeruginosa, on titanium discs of grade IV. The formation of single- and poly-species biofilms at 16.5, 40.5 and 64.5-h incubation on titanium surface was evaluated by plate count (CFU ml⁻¹) before and after exposure to CHX and four mouthrinses (Curasept, Listerine, Meridol and Buccagel) and expressed as percentage of Inhibitory Activity (IA%). The application of the different anti-plaque formulations on biofilm can reduce the adhesion of bacteria to titanium surface with different degrees. The higher efficacy was observed for Listerine that shows IA% = 100 on the biofilm formed by S. mutans at 16.5 h. Log count of CFU was dependent to culture time and four mouthrinses for S. mutans and S. aureus, whilst was not dependent to culture time but to mouthrinses for P. aeruginosa. In general, the efficacy was particularly lesser to poly-species biofilms; no statistical differences were evidenced between all the mouthrinses and CHX as control group. The tested mouthrinses, compared to reference CHX 0.2%, have demonstrated a significant lower antibacterial activity than Listerine towards the experimental biofilms. This “in vitro” biofilm model should prove extremely useful for pre-clinical testing of anti-plaque agents, which inhibit biofilm formation, can prevent subsequent implant failure.     

Titanium Surface Priming with Phase-Transited Lysozyme to Establish a Silver Nanoparticle-Loaded Chitosan/Hyaluronic Acid Antibacterial Multilayer via Layer-by-Layer Self-Assembly

Objectives

The formation of biofilm around implants, which is induced by immediate bacterial colonization after installation, is the primary cause of post-operation infection. Initial surface modification is usually required to incorporate antibacterial agents on titanium (Ti) surfaces to inhibit biofilm formation. However, simple and effective priming methods are still lacking for the development of an initial functional layer as a base for subsequent coatings on titanium surfaces. The purpose of our work was to establish a novel initial layer on Ti surfaces using phase-transited lysozyme (PTL), on which multilayer coatings can incorporate silver nanoparticles (AgNP) using chitosan (CS) and hyaluronic acid (HA) via a layer-by-layer (LbL) self-assembly technique.

Methods

In this study, the surfaces of Ti substrates were primed by dipping into a mixture of lysozyme and tris(2-carboxyethyl)phosphine (TCEP) to obtain PTL-functionalized Ti substrates. The subsequent alternating coatings of HA and chitosan loaded with AgNP onto the precursor layer of PTL were carried out via LbL self-assembly to construct multilayer coatings on Ti substrates.

Results

The results of SEM and XPS indicated that the necklace-like PTL and self-assembled multilayer were successfully immobilized on the Ti substrates. The multilayer coatings loaded with AgNP can kill planktonic and adherent bacteria to 100% during the first 4 days. The antibacterial efficacy of the samples against planktonic and adherent bacteria achieved 65%-90% after 14 days. The sustained release of Ag over 14 days can prevent bacterial invasion until mucosa healing. Although the AgNP-containing structure showed some cytotoxicity, the toxicity can be reduced by controlling the Ag release rate and concentration.

Conclusions

The PTL priming method provides a promising strategy for fabricating long-term antibacterial multilayer coatings on titanium surfaces via the LbL self-assembly technique, which is effective in preventing implant-associated infections in the early stage.