Veluchamy Prabhawathi

Antibiofilm properties of silver and gold incorporated PU, PCLm, PC and PMMA nanocomposites under two shear conditions.

Silver and gold nanoparticles (of average size ∼20–27 nm) were incorporated in PU (Polyurethane), PCLm (Polycaprolactam), PC (polycarbonate) and PMMA (Polymethylmethaacrylate) by swelling and casting methods under ambient conditions. In the latter method the nanoparticle would be present not only on the surface, but also inside the polymer. These nanoparticles were prepared initially by using a cosolvent, THF. PU and PCLm were dissolved and swollen with THF. PC and PMMA were dissolved in CHCl3 and here the cosolvent, THF, acted as an intermediate between water and CHCl3. FTIR indicated that the interaction between the polymer and the nanoparticle was through the functional group in the polymer. The formation of E.coli biofilm on these nanocomposites under low (in a Drip flow biofilm reactor) and high shear (in a Shaker) conditions indicated that the biofilm growth was higher (twice) in the former than in the latter (ratio of shear force = 15). A positive correlation between the contact angle (of the virgin surface) and the number of colonies, carbohydrate and protein attached on it were observed. Ag nanocomposites exhibited better antibiofilm properties than Au. Bacterial attachment was highest on PC and least on PU nanocomposite. Casting method appeared to be better than swelling method in reducing the attachment (by a factor of 2). Composites reduced growth of organisms by six orders of magnitude, and protein and carbohydrate by 2–5 times. This study indicates that these nanocomposites may be suitable for implant applications.

Antibacterial activity and QSAR of chalcones against biofilm-producing bacteria isolated from marine waters

Biofouling in the marine environment is a major problem. In this study, three marine organisms, namely Bacillus flexus (LD1), Pseudomonas fluorescens (MD3) and Vibrio natriegens (MD6), were isolated from biofilms formed on polymer and metal surfaces immersed in ocean water. Phylogenetic analysis of these three organisms indicated that they were good model systems for studying marine biofouling. The in vitro antifouling activity of 47 synthesized chalcone derivatives was investigated by estimating the minimum inhibitory concentration against these organisms using a twofold dilution technique. Compounds C-5, C-16, C-24, C-33, C-34 and C-37 were found to be the most active. In the majority of the cases it was found that these active compounds had hydroxyl substitutions. A quantitative structure-activity relationship (QSAR) was developed after dividing the total data into training and test sets. The statistical measures r 2, (>0.6) q 2 (>0.5) and the F-ratio were found to be satisfactory. Spatial, structural and electronic descriptors were found to be predominantly affecting the antibiofouling activity of these compounds. Among the spatial descriptors, Jurs descriptors showed their contribution in all the three antibacterial QSARs.

Design of a papain immobilized antimicrobial food package with curcumin as a crosslinker.

Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent.

Functionalized polycaprolactam as an active food package for antibiofilm activity and extended shelf life.

Papain is covalently crosslinked on polycaprolactam and tested as a wrapper for packaging cottage cheese, against E. coli biofilm. The bacterial count on neat polycaprolactam (NP) was 50×10(6)/ml on the 5th day which dramatically increased to 300×10(6) colony forming units (CFU)/ml by the end of 30th day. The corresponding CFU/ml on papain functionalized polycaprolactam (FP) was 10×10(2) on 5th day and 20×10(2) by the end of 30th day. Fourier transform infrared spectroscopic (FTIR) analysis of biofilm on NP showed the presence of polysaccharide, protein, lipid and metabolites which was three times reduced on FP. FT Raman spectroscopy showed the effect of papain on functional groups such as hydroxyl, amino, carbonyl, phosphoryl and aliphatic, leading to the inhibition of the biofilm. Motility, hydrophobicity and zeta potential of E. coli on NP and FP were 10.67 and 5.65 μm/s/V/cm; 88 and 20%; 8.93±2.09 and 2.65±0.52 mV respectively, thereby decreasing the biofilm forming ability of E. coli.

Antibiofilm properties of interfacially active lipase immobilized porous polycaprolactam prepared by LB technique.

Porous biomaterial is the preferred implant due to the interconnectivity of the pores. Chances of infection due to biofilm are also high in these biomaterials because of the presence of pores. Although biofilm in implants contributes to 80% of human infections [1], there are no commercially available natural therapeutics against it. In the current study, glutaraldehyde cross linked lipase was transferred onto a activated porous polycaprolactam surface using Langmuir-Blodgett deposition technique, and its thermostability, slimicidal, antibacterial, biocompatibility and surface properties were studied. There was a 20% increase in the activity of the covalently crosslinked lipase when compared to its free form. This immobilized surface was thermostable and retained activity and stability until 100°C. There was a 2 and 7 times reduction in carbohydrate and 9 and 5 times reduction in biofilm protein of Staphylococcus aureus and Escherichia coli respectively on lipase immobilized polycaprolactam (LIP) when compared to uncoated polycaprolactam (UP). The number of live bacterial colonies on LIP was four times less than on UP. Lipase acted on the cell wall of the bacteria leading to its death, which was confirmed from AFM, fluorescence microscopic images and amount of lactate dehydrogenase released. LIP allowed proliferation of more than 90% of 3T3 cells indicating that it was biocompatible. The fact that LIP exhibits antimicrobial property at the air-water interface to hydrophobic as well as hydrophilic bacteria along with lack of cytotoxicity makes it an ideal biomaterial for biofilm prevention in implants.

In vitro biocompatiblity of modified polycarbonate as a biomaterial

Nitrated and aminated polycarbonates were prepared chemically, characterized and tested in vitro as a possible biomaterial. Adhesion of Staphylococcus aureus NCIM 5021, Escherichia coli NCIM 2931 and Proteus vulgaris NCIM 2813 and the presence of carbohydrate, protein, CFU and ATP on these surfaces were examined. Cytotoxicity of these surfaces was investigated by growing L929 mouse fibroblast cells. NO2-PC was more hydrophilic than un-PC and reduced adhesion of bacterial protein and carbohydrate. NH2-PC was the most hydrophilic surface biofilm prevention and increased proliferation of the fibroblast cells. The motility of all the three organisms decreased on aminated surface when compared to that on the other two. This study indicated that reducing the surface hydrophobicity alone was not sufficient to develop a biocompatible material, but providing favorable surface functional groups was also a necessary criterion. A strong correlation was observed between the hydrophobicity of the polymer surface and the zeta potential of the organism with bacterial attachment (CFU/ml). A multi-linear regression model with these two parameters was able to fit the observed bacterial attachment data well.

A study on the long term effect of biofilm produced by biosurfactant producing microbe on medical implant

Low density polyethylene (LDPE) is used as a long term medical implant. Biofilm forming ability of two pathogenic microorganisms, namely, Bacillus subtilis (B. subtilis) and Pseudomonas aeruginosa (P. aeruginosa) on this polymer and the differences in the properties of these matrices are studied for a year. There are very few long term studies on biofilms formed on medical implants. After three months, colonies of B. subtilis were two times higher when compared to those of P. aeruginosa. And at the end of one year, they were two orders of magnitude higher than the later. The exopolysaccharide (EPS) and biosurfactant recovered from the polymer surface after three months were 21 and 10.4 μg/cm(2) for B. subtilis and 13 and 8.6 μg/cm(2) for P. aeruginosa. After one year, these were higher in B. subtilis (50 and 37.1 μg/cm(2), respectively) than in P. aeruginosa (34.1 and 31.8 μg/cm(2), respectively). B. subtilis consisted of protein controlling the community and sporulation development, while P. aeruginosa had either housekeeping or metabolic proteins. The EPS in the respective biofilm consisted of biosurfactants produced by B. subtilis (surfactins, m/z=1029 to 1134) and P. aeruginosa (rhamnolipids, m/z=568 to 705). Thermogravimetric analysis indicated that LDPE incubated with these organisms underwent a weight loss of 4 and 3% after three months and 11.1 and 9.2% after one year, respectively at 435 °C. Laccase and manganese peroxidase were detected in the biofilm which could be involved in the degradation. The biosurfactant of these microorganisms altered the hydrophobicity of the surface, favoring their attachment and proliferation.

Chalcone coating on cotton cloth – an approach to reduce attachment of live microbes

Drug resistant bacteria are a major threat to humans, especially those which mediate nosocomial infections. In this paper, three different 4-sulfonylmethyl chalcones are coated onto cotton cloths with acacia as the binder using a padding mangle to make them antibacterial. A 99% reduction in the adhesion of three slime producing organisms, namely Staphylococcus aureus NCIM5021, Escherichia coli NCIM2931 and Pseudomonas aeruginosa NCIM2901, on these surfaces was observed. The coated surfaces are more hydrophobic than the original one. The attachment of the bacteria (CFU ml-1) to the cloth is directly proportional (correlation coefficient, R = 0.58) to the hydrophobicity of the surface of the microorganism. The extent of bacterial attachment on the cloths (CFU ml-1) is not correlated with the minimum inhibitory concentration (MIC) of the chalcones (R = -0.1), but on the other hand it is negatively correlated with the hydrophilicity of the coated cloth (R = -0.52). This indicates that hydrophilic surfaces prevent bacterial attachment and hydrophobic organisms have a greater propensity to attach to hydrophobic surfaces than hydrophilic ones. A simple multi-linear regression model with the surface hydrophobicity of the organism and the hydrophilicity of the cloth is able to predict the extent of bacterial attachment. This study suggests that the coated cloths could find applications in hospital environments.

Design of antimicrobial polycaprolactam nanocomposite by immobilizing subtilisin conjugated Au/Ag core-shell nanoparticles for biomedical applications

Preparation of the gold core and silver shell NP (AuAgNP) is challenging because of the facile oxidation of silver. Here such a NP is carefully synthesized and conjugated with subtilisin to arrive at a stable spherical material of 120-130 nm in diameter (AuAgSNP). A biomaterial prepared by immobilizing AuAgSNP on polycaprolactam (PCL) exhibits antibiofilm properties against S. aureus and E. coli, but with lesser potency than the one prepared with bare AuAgNP. Subtilisin degrades the adhesive surface proteins of the bacteria thereby preventing the biofilm formation. Subtilisin conjugated AuAgSNP is not cytotoxic to 3T3 cells at its MIC, in contrast to AuAgNP. The presence of subtilisin promotes the fibroblast proliferation. This study indicates that AuAgSNP has antibacterial/antibiofilm activities as well as biocompatibility unlike NPs which are very cytotoxic to cells. Hence AuAgSNP can be used in medical implants and devices.