Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds

Enzymatic reduction of azo and indigoid compounds

A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40°C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N∼1∼,N∼1∼-dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H2O2) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy

Effect of Mono and Di-rhamnolipids on Biofilms Pre-formed by Bacillus subtilis BBK006.

Different microbial inhibition strategies based on the planktonic bacterial physiology have been known to have limited efficacy on the growth of biofilms communities. This problem can be exacerbated by the emergence of increasingly resistant clinical strains. Biosurfactants have merited renewed interest in both clinical and hygienic sectors due to their potential to disperse microbial biofilms. In this work, we explore the aspects of Bacillus subtilis BBK006 biofilms and examine the contribution of biologically derived surface-active agents (rhamnolipids) to the disruption or inhibition of microbial biofilms produced by Bacillus subtilis BBK006. The ability of mono-rhamnolipids (Rha-C10-C10) produced by Pseudomonas aeruginosa ATCC 9027 and the di-rhamnolipids (Rha-Rha-C14-C14) produced by Burkholderia thailandensis E264, and phosphate-buffered saline to disrupt biofilm of Bacillus subtilis BBK006 was evaluated. The biofilm produced by Bacillus subtilis BBK006 was more sensitive to the di-rhamnolipids (0.4 g/L) produced by Burkholderia thailandensis than the mono-rhamnolipids (0.4 g/L) produced by Pseudomonas aeruginosa ATCC 9027. Rhamnolipids are biologically produced compounds safe for human use. This makes them ideal candidates for use in new generations of bacterial dispersal agents and useful for use as adjuvants for existing microbial suppression or eradication strategies

Inhibitory effects of lipopeptides and glycolipids on C. albicans - Staphylococcus spp. dual-species biofilms

Microbial biofilms strongly resist host immune responses and antimicrobial treatments and are frequently responsible for chronic infections in peri-implant tissues. Biosurfactants (BSs) have recently gained prominence as a new generation of anti-adhesive and antimicrobial agents with great biocompatibility and were recently suggested for coating implantable materials in order to improve their anti-biofilm properties. In this study, the anti-biofilm activity of lipopeptide AC7BS, rhamnolipid R89BS and sophorolipid SL18 was evaluated against clinically relevant fungal/bacterial dual-species biofilms (Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis) through quantitative and qualitative in vitro tests. C. albicans - S. aureus and C. albicans - S. epidermidis cultures were able to produce a dense biofilm on the surface of the polystyrene plates and on medical-grade silicone discs. All tested BSs demonstrated an effective inhibitory activity against dual-species biofilms formation in terms of total biomass, cell metabolic activity, microstructural architecture and cell viability, up to 72h on both these surfaces. In co-incubation conditions, in which BSs were tested in free soluble form, rhamnolipid R89BS (0.05 mg/ml) was the most effective among the tested biosurfactants against the formation of both dual-species biofilms, reducing on average 94% and 95% of biofilm biomass and metabolic activity at 72h of incubation, respectively. Similarly, rhamnolipid R89BS silicone surface coating proved to be the most effective in inhibiting the formation of both dual-species biofilms, with average reductions of 93% and 90%, respectively. Scanning Electron Microscopy observations showed areas of treated surfaces that were free of microbial cells or in which thinner and less structured biofilms were present, compared to controls. The obtained results endorse the idea that coating of implant surfaces with BSs may be a promising strategy for the prevention of C. albicans -Staphylococcus spp. colonization on medical devices, and can potentially contribute to the reduction of the high economic efforts undertaken by healthcare systems for the treatment of these complex fungal-bacterial infections

Biomedical and therapeutic applications of biosurfactants

During the last years, several applications of biosurfactants with medical purposes have been reported. Biosurfactants are considered relevant molecules for applications in combating many diseases and as therapeutic agents due to their antibacterial, antifungal and antiviral activities. Furthermore, their role as anti-adhesive agents against several pathogens illustrate their utility as suitable anti-adhesive coating agents for medical insertional materials leading to a reduction of a large number of hospital infections without the use of synthetic drugs and chemicals. Biomedical and therapeutic perspectives of biosurfactants applications are presented and discussed in this chapter

Potential therapeutic applications of microbial surface-activecompounds

Numerous investigations of microbial surface-active compounds or biosurfactants over the past two decades have led to the discovery of many interesting physicochemical and biological properties including antimicrobial, anti-biofilm and therapeutic among many other pharmaceutical and medical applications. Microbial control and inhibition strategies involving the use of antibiotics are becoming continually challenged due to the emergence of resistant strains mostly embedded within biofilm formations that are difficult to eradicate. Different aspects of antimicrobial and anti-biofilm control are becoming issues of increasing importance in clinical, hygiene, therapeutic and other applications. Biosurfactants research has resulted in increasing interest into their ability to inhibit microbial activity and disperse microbial biofilms in addition to being mostly nontoxic and stable at extremes conditions. Some biosurfactants are now in use in clinical, food and environmental fields, whilst others remain under investigation and development. The dispersal properties of biosurfactants have been shown to rival that of conventional inhibitory agents against bacterial, fungal and yeast biofilms as well as viral membrane structures. This presents them as potential candidates for future uses in new generations of antimicrobial agents or as adjuvants to other antibiotics and use as preservatives for microbial suppression and eradication strategies

Reduced TCA cycle rates at high hydrostatic pressure hinder hydrocarbon degradation and obligate oil degraders in natural, deep-sea microbial communities

Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO2 production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea