Integrative proteomics and metabolomics approach to elucidate the antimicrobial effect of simvastatin on Escherichia coli.

Globally, simvastatin is one of the most commonly used statin drugs. Its antimicrobial properties have been investigated against various pathogens. However, its effect on biological processes in bacteria has been unclear. This study focused on altered biological and metabolic processes at protein and metabolite levels induced by simvastatin. MS-based proteomics and metabolomics were used to investigate the altered proteins and metabolites between experimental groups. Proteomics results showed that simvastatin induced various antimicrobial targets such as chaperon protein DnaK and cell division protein FtsZ. Metabolomics results revealed phenotypic changes in cells under simvastatin stress. Integrated proteomics and metabolomics result indicated that various metabolic processes were altered to adapt to stress conditions. Energy metabolism (glycolysis, tricarboxylic acid cycle, etc.), amino acid synthesis and ribosomal proteins, and purine and pyrimidine synthesis were induced by the effect of simvastatin. This study will contribute to the understanding of antimicrobial properties of statin drugs.

Sub-Inhibitory Concentrations of Ciprofloxacin Alone and Combinations with Plant-Derived Compounds against P. aeruginosa Biofilms and Their Effects on the Metabolomic Profile of P. aeruginosa Biofilms.

INTRODUCTION: Alternative anti-biofilm agents are needed to combat Pseudomonas aeruginosa infections. The mechanisms behind these new agents also need to be revealed at a molecular level. MATERIALS AND METHODS: The anti-biofilm effects of 10 plant-derived compounds on P. aeruginosa biofilms were investigated using minimum biofilm eradication concentration (MBEC) and virulence assays. The effects of ciprofloxacin and compound combinations on P. aeruginosa in mono and triple biofilms were compared. A metabolomic approach and qRT-PCR were applied to the biofilms treated with ciprofloxacin in combination with baicalein, esculin hydrate, curcumin, and cinnamaldehyde at sub-minimal biofilm inhibitory concentration (MBIC) concentrations to highlight the specific metabolic shifts between the biofilms and to determine the quorum sensing gene expressions, respectively. RESULTS: The combinations of ciprofloxacin with curcumin, baicalein, esculetin, and cinnamaldehyde showed more reduced MBICs than ciprofloxacin alone. The quorum sensing genes were downregulated in the presence of curcumin and cinnamaldehyde, while upregulated in the presence of baicalein and esculin hydrate rather than for ciprofloxacin alone. The combinations exhibited different killing effects on P. aeruginosa in mono and triple biofilms without affecting its virulence. The findings of the decreased metabolite levels related to pyrimidine and lipopolysaccharide synthesis and to down-regulated alginate and lasI expressions strongly indicate the role of multifactorial mechanisms for curcumin-mediated P. aeruginosa growth inhibition. CONCLUSIONS: The use of curcumin, baicalein, esculetin, and cinnamaldehyde with ciprofloxacin will help fight against P. aeruginosa biofilms. To the best of our knowledge, this is the first study of its kind to define the effect of plant-based compounds as possible anti-biofilm agents with low MBICs for the treatment of P. aeruginosa biofilms through metabolomic pathways.

Mucoadhesive bilayered buccal platform for antifungal drug delivery into the oral cavity.

A drug delivery technology comprising a mucoadhesive bilayered buccally anchored tablet containing natamycin was developed. The concept was to anchor the tablet to the buccal tissue and allow controlled release of the drug through the matrix into the mouth. Carbomer (Carbopol ® 974 P NF) was used to formulate the mucoadhesive layer. Hydroxypropyl methylcellulose (HPMC) (Methocel® K4M) at 10, 15, 20, and 40% w/w was used for the drug-containing layer. Natamycin, an amphoteric macrolide antifungal agent, was incorporated into the formulations. In addition, tablets containing erythrosine as a marker were prepared in order to examine the distribution and retention of the dye in the oral cavity. As expected, the in vitro analysis showed that the concentration of natamycin released decreased with the increasing proportion of HPMC in the formulation. A small volunteer study was conducted using the tablets containing 10% and 20% HPMC to quantitate the patterns of distribution of the drug released into the oral cavity (upper right buccal vestibule, lower right and left buccal vestibules, and sublingual region). The mucoadhesive bilayered buccal tablet formulation provided a unidirectional release of the drug from the tablet into the oral cavity in a prolonged release fashion, maintaining drug concentration above the MIC value (2 µg/mL) for Candida albicans. The amount of the drug in the sublingual region was found to be lowest when compared with other regions, which is due to the higher flow of saliva in this region. Graphical abstract.

Evaluation of Antimicrobial Durability and Anti-Biofilm Effects in Urinary Catheters Against Enterococcus faecalis Clinical Isolates and Reference Strains.

BACKGROUND: Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans biofilms are major causes of catheter-associated urinary tract infections. Antimicrobial-coated or impregnated urinary catheters are seen as a possible way to prevent these infections. AIMS: To determine the biofilm-forming ability of 89 E. faecalis isolates from urinary tract infections and to compare several urinary catheters for antimicrobial durability and the inhibitory effects on biofilm formation of different laboratory strains and clinical isolates of E. faecalis. STUDY DESIGN: In vitro experimental study. METHODS: The biofilm forming ability of E. faecalis isolates was determined by the crystal violet staining and plate counting methods. For comparison of urinary catheters, biofilms of 45 E. faecalis isolates from the catheter samples of hospitalized patients and five laboratory strains of E. coli ATCC25922, S. epidermidis ATCC35984, P. aeruginosa ATCC27853, E. faecalis ATCC29212 and C. albicans ATCC90028 were formed on the catheters in 24-well tissue culture plates. Scanning electron microscopy analysis was performed to observe biofilms. RESULTS: All 89 E. faecalis isolates were found to be biofilm positive. Nitrofurazone-impregnated catheters significantly reduced the cell counts of E. faecalis isolates and completely inhibited the formation of P. aeruginosa and S. epidermidis biofilms compared with the others. Regarding reduction of biofilm cell counts, a hydrophilic-coated catheter was more effective against P. aeruginosa, whereas a silver-coated catheter was found to be more effective against S. epidermidis. The nitrofurazone-impregnated catheter had the best antimicrobial durability. CONCLUSION: Urine isolates of E. faecalis had considerable ability with respect to biofilm formation. The nitrofurazone-impregnated catheter was the most effective against all tested bacteria; however, the effect of a hydrophilic or silver-coated catheter depends on the species present in it.

Design and evaluation of gamma-sterilized vancomycin hydrochloride-loaded poly(ɛ-caprolactone) microspheres for the treatment of biofilm-based medical device-related osteomyelitis.

CONTEXT: There is a great necessity to find and use accomplished terminal sterilization technique for industrial manufacturing, research and development studies. Gamma (γ)-sterilization has been commonly employed for wide range of products as indicated by the pharmacopoeias. However, carefully examination should be performed prior to administration since γ-radiation can cause changes in drug and polymer excipients. No information is available in literature about γ-sterilization effects on vancomycin HCl-loaded poly (ɛ-caprolactone) (PCL) microspheres. OBJECTIVE: Formulations were developed using a different preparation approach for the treatment of medical device-related osteomyelitis, and γ-sterilization effects on the physicochemical characterization of the formulations were examined. METHODS: Water-in-oil-in-water (w/o/w) emulsion technique using polyvinyl alcohol (PVA) in inner and outer phase was applied to prepare formulations. Physicochemical properties of the formulations were investigated before and after γ-sterilization and the antibacterial activity against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were measured. RESULTS: The particle size of the nonsterilized formulations were between 58 and 134 µm. 60% or 20% of vancomycin HCl were released from 42.500 Mn or 70.000-90.000 Mn PCL microspheres, respectively, in 24 h. No difference was observed in the particle size, drug-loading efficiency, morphology, in vitro release and antimicrobial activity of the formulations after γ-sterilization (p > 0.05).

Polyethyleneimine brushes effectively inhibit encrustation on polyurethane ureteral stents both in dynamic bioreactor and in vivo.

Polyurethane (PU) ureteral stents have been widely used as biomedical devices to aid the flow of the urine. Due to the biofilm formation and encrustation complications it has been hindered their long term clinical usage. To overcome these complications, in this study, cationic polyethyleneimine (PEI) brushes grafted on PU stents and their performances were tested both in a dynamic biofilm reactor system (in vitro) and in a rat model (in vivo). Thus, we hypothesized that PEI brushes inhibit bacterial adhesion owing to the dynamic motion of brushes in liquid environment. In addition, cationic structure of PEI disrupts the membrane and so kills the bacteria on time of contact. Cationic PEI brushes decreased the biofilm formation up to 2 orders of magnitude and approximately 50% of encrustation amount in respect to unmodified PU, in vitro. In addition, according to Atomic Absorption Spectroscopy (AAS) results, approximately 90% of encrustation was inhibited on in vivo animal models. Decrease in encrustation was clearly observed on the stents obtained from rat model, by Scanning Electron Microscopy (SEM). Also, histological evaluations showed that; PEI brush grafting decreased host tissue inflammation in close relation to decrease in biofilm formation and encrustation. As a results; dual effect of anti-adhesive and contact-killing antibacterial strategy showed high efficiency on PEI brushes grafted PU stents both in vitro and in vivo.

An investigation of the bactericidal activity of chlorhexidine digluconateagainst multidrug-resistant hospital isolates.

BACKGROUND/AIM: Hospital infections are among the most prominent medical problems around the world. Using proper biocides in an appropriate way is critically important in overcoming this problem. Several reports have suggested that microorganisms may develop resistance or reduce their susceptibility to biocides, similar to the case with antibiotics. In this study we aimed to determine the antimicrobial activity of chlorhexidine digluconate against clinical isolates. MATERIALS AND METHODS: The susceptibility of 120 hospital isolated strains of 7 bacterial genera against chlorhexidine digluconate was determined by agar dilution test, using minimum inhibitory concentration (MIC) values and the EN 1040 Basic Bactericidal Activity Test to determine the bactericidal activity. According to MIC values, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were found to be less susceptible to chlorhexidine digluconate. RESULTS: Quantitative suspension test results showed that 4% chlorhexidine digluconate was effective against antibiotic resistant and susceptible bacteria after 5 min of contact time and can be safely used in our hospital. However, concentrations below 4% chlorhexidine digluconate caused a decrease in bactericidal activity, especially for Staphylococcus aureus and P. aeruginosa. CONCLUSION: It is crucial to use biocides at appropriate concentrations and to perform surveillance studies to trace resistance or low susceptibility patterns of S. aureus, P. aeruginosa, and other hospital isolates.

Designing of dynamic polyethyleneimine (PEI) brushes on polyurethane (PU) ureteral stents to prevent infections.

Permanent antibacterial coatings have been developed by brush-like polyethyleneimine (PEI) on polyurethane (PU) ureteral stents since bacterial adhesion and biofilm formation with the following encrustation on stent surface limit their long term usage. In order to control or prevent bacterial infections; PEI chains with two different molecular weights (Mn: 1800 or 60,000 Da) were covalently attached on the polyurethane (PU) surface by "grafting to" approach to obtain a brush-like structure. Then, PEI brushes were alkylated with bromohexane to enhance the disruption of bacterial membranes with increasing polycationic character. X-ray Photoelectron and Infrared Spectroscopy investigations confirmed that PEI grafting and alkylation steps were performed successfully. Surface roughness in dry state increased dramatically from 65.8 nm to 277.7 nm and 145.2 nm for short chain PEI and long chain PEI grafted samples, respectively. Both low and high molecular weight PEI grafts exhibited a brush-like structure and potent antibacterial activity by lowering the adherence of Klebsiella pneumonia, Escherichia coli and Proteus mirabilis species up to two orders of magnitude without any cytotoxic effect on L929 and G/G cells. Thus, permanent bactericidal activity was achieved by the contact-active strategy of dynamic PEI brush-like structures on polyurethane ureteral stent.