Purushottam V. Gawande

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0·1% tryptone, at 35 °C, and at inoculum concentration 2×107−2×108 spores 5 g−1 substrate; forA. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0·1% yeast extract, at 35 °C, and at an inoculum concentration of 2×107−2×108 spores 5 g−1 substrate. Under these conditions, A. terreus produced 68·9 IU ml−1 of xylanase, and A. niger, 74·5 IU ml−1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.

Antimicrobial and antibiofilm efficacy of triclosan and DispersinB combination.

OBJECTIVES The objectives of this study were to examine: (i) synergy of the combination of triclosan and DispersinB (DspB); (ii) in vitro efficacy and durability of triclosan + DspB-coated vascular catheters; and (iii) in vivo efficacy of triclosan + DspB-coated catheters compared with chlorhexidine-silver sulfadiazine (CH-SS)-coated and uncoated (control) vascular catheters in preventing colonization by Staphylococcus aureus. METHODS We investigated the potential synergistic antimicrobial and antibiofilm activity of triclosan and DspB by biofilm assays. The in vitro antimicrobial efficacy of triclosan + DspB-coated catheters was determined by microbial colonization assays. Antimicrobial durability of the coated catheters was tested by soaking segments in bovine serum for 7 days and determining antimicrobial activity, and by a serial plate transfer method. The in vivo efficacy of triclosan + DspB-coated catheters compared with CH-SS-coated and uncoated catheters was assessed by subcutaneous implantation of segments in a rabbit model of S. aureus infection. RESULTS The combination of triclosan and DspB showed synergistic antimicrobial and antibiofilm activity against S. aureus, Staphylococcus epidermidis and Escherichia coli, significantly reduced bacterial colonization (P < 0.05) and generally demonstrated a prolonged superior antimicrobial activity against clinical pathogens compared with CH-SS-coated catheters. Triclosan + DspB-coated and CH-SS-coated catheters exhibited equal in vivo efficacy (P <or= 0.05) in reducing colonization by S. aureus compared with uncoated catheters. CONCLUSIONS Catheters coated with the triclosan + DspB combination showed synergistic, broad-spectrum and durable antimicrobial activity. Furthermore, the in vivo efficacy of catheters coated with this unique antimicrobial/antibiofilm composition prompts clinical evaluation of such an innovative approach.

Antibiofilm Activity of GlmU Enzyme Inhibitors against Catheter-Associated Uropathogens

ABSTRACT The colonization of uropathogenic bacteria on urinary catheters resulting in biofilm formation frequently leads to the infection of surrounding tissue and often requires removal of the catheter. Infections associated with biofilms are difficult to treat since they may be more than 1,000 times more resistant to antibiotics than their planktonic counterparts. We have developed an antibiofilm composition comprising an N-acetyl-d-glucosamine-1-phosphate acetyltransferase (GlmU) inhibitor and protamine sulfate, a cationic polypeptide. The antibiofilm activity of GlmU inhibitors, such as iodoacetamide (IDA), N-ethyl maleimide (NEM), and NEM analogs, including N-phenyl maleimide, N,N′-(1,2-phenylene)dimaleimide (oPDM), and N-(1-pyrenyl)maleimide (PyrM), was tested against that of catheter-associated uropathogens. Both IDA and NEM inhibited biofilm formation in Escherichia coli. All NEM analogs showed antibiofilm activity against clinical isolates of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, and Enterococcus faecalis. The combination of oPDM with protamine sulfate (PS) enhanced its antibiofilm activity and reduced its effective concentration to as low as 12.5 μM. In addition, we found that the in vitro inhibitory activity of oPDM-plus-PS-coated silicone catheters against P. aeruginosa and S. epidermidis colonization was superior to that of catheters coated with silver hydrogel. Confocal scanning laser microscopy further confirmed that the oPDM-plus-PS-coated silicone catheters were almost free from bacterial colonization. Thus, a broad-spectrum antibiofilm composition comprising a GlmU inhibitor and protamine sulfate shows promise for use in anti-infective coatings for medical devices, including urinary catheters.

Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S-100

Aspergillus sp. 5 (strain 5) and Aspergillus sp. 44 (strain 44) produced xylanase (34.3 and 32.7 IU ml-1, respectively) with very low levels of cellulases when grown on 1% wheat bran medium. Xylanase was non-covalently immobilized on Eudragit S-100 for saccharification. The system retained 70 and 80% of strain 5 and strain 44 xylanase activity, respectively. On immobilization, optimum temperature of activity broadened between 50 and 60 degrees C as compared to 50 degrees C in the case of the free enzymes. No significant shift in the pH optima was observed on immobilization. However, immobilization increased enzyme stability mainly by decreasing the temperature sensitivity to the inactivation reaction. The K(m) values increased from 5.6 to 8.3 mg ml-1 for strain 5 xylanase and 7.0 to 9.0 mg ml-1 for strain 44 xylanase. Enzymatic saccharification of xylan and wheat bran was improved on xylanase immobilization. Immobilized xylanase from both the strains produced three times more sugar as compared to free xylanase. In repeated batch saccharification studies immobilized xylanase was recycled three times without loss of enzyme activity.

Efficacy of combination of chlorhexidine and protamine sulphate against device-associated pathogens

OBJECTIVES The objectives of this study were to examine: (i) the potential in vitro synergy of combining protamine sulphate (PS) with chlorhexidine (CHX); (ii) the in vitro spectrum and durability of antimicrobial activity of CHX + PS-coated catheters; and (iii) the in vivo efficacy of CHX + PS-coated catheters in comparison with silver-hydrogel-coated and uncoated catheters. METHODS The potential synergistic antimicrobial and antibiofilm activities of CHX and PS were investigated in vitro by the MIC and biofilm assays. The spectrum and durability of antimicrobial activity of CHX + PS-coated catheters were studied in vitro by using a serial plate transfer method. The in vivo efficacy of CHX + PS-coated catheters was assessed in a rabbit model against Escherichia coli. RESULTS In vitro studies showed that the combination of CHX + PS has a synergistic inhibitory effect on E. coli and provides a significant synergistic antibiofilm and antimicrobial activity against E. coli, Pseudomonas aeruginosa and Staphylococcus epidermidis. Furthermore, catheters coated with CHX + PS provided a broad-spectrum and enduring in vitro antimicrobial activity over a 10 day period. The in vivo efficacy study demonstrated that subcutaneously implanted CHX + PS-coated catheters in rabbits are significantly less likely to become colonized (2/28 = 7%) than either silver-hydrogel-coated (25/28 = 89%; P < 0.001) or uncoated catheters (18/28 = 64%; P < 0.001) by E. coli. CONCLUSIONS The synergistic, broad-spectrum and durable in vitro activity of the CHX + PS combination and the robust in vivo efficacy of catheters coated with this unique composition encourage clinical evaluation of this innovative approach.

Inoculation onto Solid Surfaces Protects Salmonella spp. during Acid Challenge: a Model Study Using Polyethersulfone Membranes

ABSTRACT Salmonellae are the most frequently reported cause of outbreaks of food-borne gastroenteritis in the United States. In clinical trials, the oral infective dose (ID) for healthy volunteers was estimated to be approximately 1 million cells. However, in reports from various outbreaks, the ID of Salmonella species associated with solid foods was estimated to be as few as 100 cells. We found that fresh-cut produce surfaces not only provided suitable solid support for pathogen attachment but also played a critical role in increasing the acid tolerance of the pathogen. However the acidic nature of certain produce played no role in making salmonellae resistant to stomach acidity. Inoculation onto fresh-cut produce surfaces, as well as onto inert surfaces, such as polyethersulfone membranes and tissue paper, increased the survival of salmonellae during acid challenge (50 mM Na-citrate, pH 3.0; 37°C; 2 h) by 4 to 5 log units. Acid challenge experiments using cells inoculated onto polyethersulfone membranes provided a model system suitable for studying the underlying fundamentals of the protection that occurs when Salmonella strains are associated with solid foods. The surface-associated acid protection, which was observed in several Salmonella strains, required de novo protein synthesis and was independent of stationary-phase sigma transcription factor.

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

Aims:  To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter‐associated bacteria.

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms.

ABSTRACT We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast.

Aim:  To determine the effect of sodium bicarbonate (SB), sodium metaperiodate (SMP) and sodium dodecyl sulfate (SDS) combination on biofilm formation and dispersal in dental unit waterline (DUWL)‐associated bacteria and yeast.

Purification of Aspergillus sp xylanase by precipitation with an anionic polymer Eudragit S100

The separation of xylanases from the crude culture filtrates of Aspergillus sp 5 and Aspergillus sp 44 was carried out using affinity precipitation with a commercially available enteric polymer Eudragit S100. With affinity precipitation the yield of enzyme was 85.3, 82.7% with 10.8, 4.08-folds (specific activity of ammonium sulphate precipitate was taken as 100%) increases in the specific activity of Aspergillus sp 5 and Aspergillus sp 44, respectively. A comparison of SDS-PAGE electrophoretic patterns of the ammonium sulphate and purified enzyme by affinity precipitation showed significant purification of the enzyme. Zymogram analysis revealed recovery of three and two forms of xylanases from Aspergillus sp 5 and Aspergillus sp 44, respectively.