Devendra H. Dusane

Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters

Fifty Acinetobacter isolates were obtained from urinary tract infections and urinary catheter samples. Analytical profile index assays identified 47 isolates as Acinetobacter baumannii and three as Acinetobacter lwoffii. Six A. baumannii isolates (A1-A6) displayed hydrophobicity indices >70%. Twenty isolates exhibited lectin activity. Biofilm formation by these isolates was compared with those with low hydrophobicity index values (A45-A50). Biofilms on different surfaces were confirmed by light microscopy, epifluorescence microscopy and by obtaining scanning electron microscope images. Biofilm production was maximal at 30 °C, pH 7.0 in a medium with 5.0 g L(-1) NaCl, and its efficiency was reduced on urinary catheter surfaces at sub-minimum inhibitory concentration concentrations of colistin. Plasmid-mediated antibiotic resistance was observed in selected isolates of A. baumannii and experiments of conjugation and transformation showed the occurrence of gene transfer. Plasmid curing was used to examine the function of plasmids. Five plasmids of A. baumannii A3 were cured but no differences were observed between wild-type and plasmid-cured strains with respect to the biofilm formation capabilities. The prevalence of A. baumannii strains with biofilm mode of growth could explain their ability to persist in clinical environments and their role in device-related infections.

Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms

Removal of detrimental biofilms from surfaces exposed in the marine environment remains a challenge. A strain of Bacillus pumilus was isolated from the surface of titanium coupons immersed in seawater in the vicinity of Madras Atomic Power Station (MAPS) on the East coast of India. The bacterium formed extensive biofilms when compared to species such as Bacillus licheniformis, Pseudomonas aeruginosa PAO1 and Pseudomonas aureofaciens. A commercially available rhamnolipid was assessed for its ability to inhibit adhesion and disrupt pre-formed B. pumilus biofilms. The planktonic growth of B. pumilus cells was inhibited by concentrations >1.6mM. We studied the effect of various concentrations (0.05-100mM) of the rhamnolipid on adhesion of B. pumilus cells to polystyrene microtitre plates, wherein the effectiveness varied from 46 to 99%. Biofilms of B. pumilus were dislodged efficiently at sub-MIC concentrations, suggesting the role of surfactant activity in removing pre-formed biofilms. Scanning electron microscopy (SEM) confirmed the removal of biofilm-matrix components and disruption of biofilms by treatment with the rhamnolipid. The results suggest the possible use of rhamnolipids as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces.

Anti-biofilm potential of a glycolipid surfactant produced by a tropical marine strain of Serratia marcescens.

A tropical marine bacterium isolated from the hard coral, Symphyllia sp. was identified as Serratia marcescens on the basis of morphological, biochemical and 16S rDNA analysis. The bacterium showed antimicrobial activity towards the pathogens Candida albicans and Pseudomonas aeruginosa and the marine biofouling bacterium Bacillus pumilus. S. marcescens displayed biosurfactant activity as evidenced by drop collapse, blood hemolysis and surface tension reduction (52.0–27 mN m−1). The active compound was purified by solvent extraction and silicic acid chromatography. Characterization was by thin layer chromatography, gas chromatography mass spectroscopy (GC-MS), Fourier transform infrared (FTIR) spectroscopy and 1H as well as 13C nuclear magnetic resonance (NMR) analysis. The surfactant was found to be a glycolipid composed of glucose and palmitic acid. The glycolipid prevented adhesion of C. albicans BH, P. aeruginosa PAO1 and B. pumilus TiO1. The glycolipid also disrupted preformed biofilms of these cultures in microtitre plates. Confocal laser scanning microscopy and electron microscopy confirmed the effective removal of biofilms from glass surfaces. The glycolipid derived from S. marcescens could thus serve as a potential anti-biofilm agent.

Quorum sensing: implications on rhamnolipid biosurfactant production.

Abstract Quorum sensing (QS) has received significant attention in the past few decades. QS describes population density dependent cell to cell communication in bacteria using diffusible signal molecules. These signal molecules produced by bacterial cells, regulate various physiological processes important for social behavior and pathogenesis. One such process regulated by quorum sensing molecules is the production of a biosurfactant, rhamnolipid. Rhamnolipids are important microbially derived surface active agents produced by Pseudomonas spp. under the control of two interrelated quorum sensing systems; namely las and rhl. Rhamnolipids possess antibacterial, antifungal and antiviral properties. They are important in motility, cell to cell interactions, cellular differentiation and formation of water channels that Currently, biosurfactants are unable to compete economically with chemically synthesized compounds in the market due to high production costs. Once the genes required for biosurfactant production have been identified, they can be placed under the regulation of strong promoters in nonpathogenic, heterologous hosts to enhance production. The production of rhamnolipids could be increased by cloning both the rhlAB rhamnosyltransferase genes and the rhlRI quorum sensing system into a suitable bacterium such as E. coli or P. putida and facilitate rhamnolipid production. Biosurfactants can also be genetically engineered for different industrial applications assuming there is a strong understanding of both the genetics and the structure-function relationships of each component of the molecule. Genetic engineering of surfactin has already been reported, with recent papers describing the creation of novel peptide structures from the genetic recombination of several peptide synthetases. Recent application of dynamic metabolic engineering strategies for controlled gene expression could lower the cost of fermentation processes by increasing the product formation. Therefore, by integrating a genetic circuit into applications of metabolic engineering the biochemical production can be optimized. Furthermore, novel strategies could be designed on the basis of information obtained from the studies of quorum sensing and biosurfactants produced suggesting enormous practical applications.

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.

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.

Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis

Background Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. Methodology/Principal Findings B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. Conclusion/Significance We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant

BackgroundYarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison.ResultsThe methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB.ConclusionThe results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.

Alkaloids Modulate Motility, Biofilm Formation and Antibiotic Susceptibility of Uropathogenic Escherichia coli

Alkaloid-containing natural compounds have shown promise in the treatment of microbial infections. However, practical application of many of these compounds is pending a mechanistic understanding of their mode of action. We investigated the effect of two alkaloids, piperine (found in black pepper) and reserpine (found in Indian snakeroot), on the ability of the uropathogenic bacterium Escherichia coli CFT073 to colonize abiotic surfaces. Sub-inhibitory concentrations of both compounds (0.5 to 10 µg/mL) decreased bacterial swarming and swimming motilities and increased biofilm formation. qRT-PCR revealed a decrease in the expression of the flagellar gene (fliC) and motility genes (motA and motB) along with an increased expression of adhesin genes (fimA, papA, uvrY). Interestingly, piperine increased penetration of the antibiotics ciprofloxacin and azithromycin into E. coli CFT073 biofilms and consequently enhanced the ability of these antibiotics to disperse pre-established biofilms. The findings suggest that these alkaloids can potentially affect bacterial colonization by hampering bacterial motility and may aid in the treatment of infection by increasing antibiotic penetration in biofilms.

Biofilm formation by a biotechnologically important tropical marine yeast isolate, Yarrowia lipolytica NCIM 3589

Biofilm formation by Yarrowia lipolytica, a biotechnologically important fungus in microtitre plates, on glass slide surfaces and in flow cell was investigated. In microtitre plates, there was a short lag phase of adhesion followed by a period of rapid biofilm growth. The fungus formed extensive biofilms on glass slides, whereas in flow-cells a multicellular, three-dimensional microcolony structure was observed. The isolate formed biofilms in seawater and in fresh water media at neutral pH when grown in microtitre plates. The carbon sources differentially affected formation of biofilms in microtitre plates. Lactic acid, erythritol, glycerol, glucose and edible oils supported the formation of biofilms, while alkanes resulted in sub-optimal biofilm development. A variation in the morphology of the fungus was observed with different carbon sources. The results point to the possible existence of highly structured biofilms in varied ecological niches from where the yeast is isolated.