BACKGROUND
biofilms, which are formed by the majority of microorganisms in natural environments, are structures with low sensitivity to drugs  <cit> .
many laboratories are synthesizing or isolating new compounds preventing the formation of biofilms or causing their elimination  <cit> .
adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds.
biosurfactants are promising compounds often showing antimicrobial and antiadhesive properties and sometimes penetrating and removing mature biofilms  <cit> .
microbial surfactants-amphiphilic, surface-active, secondary metabolites of bacteria or fungi ranging from low-molecular-mass glycolipids, sophorolipids, rhamnolipids and lipopeptides, to high-molecular-mass proteins, lipopolysaccharides and lipoproteins  <cit> -can interact with interfaces and inhibit the adhesion of microorganisms to different surfaces.
they are an alternative to synthetic surface-active agents because of their low toxicity and biodegradability  <cit> .
another mechanism of biosurfactant action is the permeabilization of bacterial cells.
the rhamnolipid secreted by pseudomonas sp.
s- <dig> permeabilized gram-negative and gram-positive cells, but a strong inhibition of growth was observed only in the case of gram-positive bacteria  <cit> .
biofilm disruption was observed after the addition of rhamnolipids from pseudomonas aeruginosa  <cit>  and lipopeptide from bacillus spp.
<cit> .
a particular group of biosurfactants, lipopeptides, can act as antibiotics and also as antiviral  <cit>  and antitumor agents  <cit> .
surfactin from bacillus subtilis can interact with the plasma membranes of bacterial and fungal cells leading to their disruption  <cit> .
the effects of biosurfactants on decreased microbial adhesion and detachment from different surfaces can be conveniently utilized in many fields, from medicine to various branches of industry, e.g., antimicrobial or antitumor activities  <cit>  and their surface activity and antiadhesive properties can be suitable for preventing microbial colonization of implants or urethral catheters.
microbial surfactants from lactobacillus fermentum and lactobacillus acidophilus adsorbed on glass, reduced the number of adhering uropathogenic cells of enterococcus faecalis by 77%  <cit> .
a surfactant released by streptococcus thermophilus has been used for fouling control of heat-exchanger plates in pasteurizers as it retards the colonization of other thermophilic strains of streptococcus responsible for fouling  <cit> .
within the collection of  <dig> morphologically distinct bacteria isolated from water and ground provided from the wild environment around the university of wroclaw scientific base in the arctic archipelago of svalbard  <cit> , pseudomonas fluorescens bd <dig> was identified as strain which strongly reduces surface tension of its culture supernatant  <cit> .
we purified and identified the chemical structure of two new p. fluorescens bd <dig> biosurfactants, pseudofactin i and ii  <cit> .
both compounds are cyclic lipopeptides with a palmitic acid connected to the terminal amino group of an octapeptide.
the c-terminal carboxylic group of the last amino acid  forms a lactone with the hydroxyl of thr <dig>  the biosurfactant was found to be stable within the range from -20°c to 100°c, had the minimum surface tension  and the critical micelle concentration   <cit> .
emulsification activity and stability of pseudofactin ii was greater than that of the synthetic surfactants such as tween  <dig> and triton x- <dig>
the aim of this paper was to assess how the pseudofactin ii influences the adhesion and biofilm formation of microorganisms such as escherichia coli, e. faecalis, enterococcus hirae, staphylococcus epidermidis, proteus mirabilis, vibrio ordalii, vibrio harveyi and candida albicans found in gastrointestinal and urinary tract.
since the effects of a surfactant may differ depending on both the type of the microorganism and the type of surface it adheres to, we tested its action on the adherence of the above pathogenic microorganisms to three types of surfaces, polystyrene, glass  and silicone .
methods
microorganisms and culture conditions
p. fluorescens bd <dig> strain was obtained from freshwater from the arctic archipelago of svalbard  <cit>  and maintained on the mineral salts medium msm 2so <dig>   <dig>  g/l sodium citrate 2h2o, and  <dig>  g/l mgso <dig> h2o) with 2% d-glucose.
the antimicrobial and antiadhesive properties of pseudofactin ii were tested on several pathogenic strains that colonize animals gastrointestinal tract or medical devices.
e. coli atcc  <dig>  e. coli atcc  <dig>  e. coli 17- <dig> , e. faecalis atcc  <dig>  e. faecalis ja/ <dig> , e. hirae atcc  <dig>  s. epidermidis kctc  <dig>  <cit> , p. mirabilis atcc  <dig> were grown at 37°c and v. harveyi atcc  <dig>  v. ordalii kccm  <dig> were grown at 28°c in lb medium .
two fungal strains, c. albicans atcc  <dig> and c. albicans sc <dig>  <cit> , were grown in a  <dig>  g/l yeast nitrogen base , broth  containing 2% d-glucose for adhesion tests.
to prevent filamentation of c. albicans, pre-culture was incubated at 28°c, while experiments with biofilms were performed at 37°c.
rpmi- <dig> medium  was used for candida biofilms formation.
isolation and purification of pseudofactin ii
pseudofactin ii produced by p. fluorescens bd <dig> was obtained by extraction of cell free supernatant by ethyl acetate and evaporation of the extract under vacuum.
the crude biosurfactant was separated by rp-hplc in the same manner as reported earlier  <cit> .
purified pseudofactin ii fraction was dried and stored at -20°c for further studies.
analytical rp-hplc  of purified pseudofactin ii showed that its purity was > 99%.
antimicrobial assays
the antimicrobial activity of isolated pseudofactin ii was determined by the microdilution method in 96-well flat-bottomed plastic microplates .
briefly,  <dig> μl volumes of sterile double strength lb  or ynb  medium were dispensed into the wells of a 96-well microplate.
subsequently,  <dig> μl volumes of pseudofactin ii  solution in phosphate-buffered saline  were added to the microplate wells and mixed with the medium.
negative and growth control wells did not contain biosurfactant.
all wells  were inoculated with  <dig> μl of overnight bacterial or yeast cultures  in lb or ynb medium respectively, and the microplates were incubated for  <dig> h at 37°c or 28°c for bacterial or yeast cultures, respectively.
after  <dig> h of incubation, the optical density at  <dig> nm of each well was measured using an asys uvm  <dig>  microplate reader.
the growth inhibition percentages at different pseudofactin ii concentrations for each microorganism were calculated as:
%growthinhibition=1-odtodc× <dig>
where odt represents the optical density of the well with a given pseudofactin ii concentration and odc is the optical density of the control well .
assays were carried out three times in three replicates.
preadhesion treatment with pseudofactin ii
inhibition of microbial adhesion by pseudofactin ii was tested in 96-well plates .
briefly, the wells of a sterile 96-well flat-bottom plate were filled with  <dig> μl of  <dig> - <dig>  mg/ml pseudofactin ii dissolved in pbs.
the plates were incubated for  <dig> h at 37°c on a rotary shaker  at  <dig> rpm and subsequently washed twice with pbs.
negative control  wells contained pseudofactin ii at the highest concentration tested  while positive control wells contained pbs buffer only.
the overnight cultures of microbial strains were centrifuged, washed twice with pbs  and re-suspended in pbs to an optical density od <dig> =  <dig>  for bacterial and od <dig> =  <dig>  for candida strains.
the highest adhesion without pseudofactin ii were observed at these optical densities .
a  <dig> μl aliquot of a washed microbial suspension was added and incubated in the wells.
after a  <dig> h incubation at 37°c in a rotary shaker  at  <dig> rpm nonadherent cells were removed by three washes with pbs.
then the plates were stained with  <dig> % crystal-violet for  <dig> min and again washed three times with pbs.
the adherent microorganisms were permeabilized and the dye was resolubilized with  <dig> μl of isopropanol- <dig>  n hcl and  <dig> μl of  <dig> % sds per well.
crystal violet optical density readings of each well were taken at  <dig> nm on the asys uvm  <dig>  microplate.
pseudofactin ii did not affect the absorption of negative control .
the microbial adhesion inhibition was calculated as growth inhibition.
assays were carried out three times in three replicates.
postadhesion treatment with pseudofactin ii
the 96-well flat-bottomed plates were incubated for  <dig> h on a rotary shaker  at  <dig> rpm with  <dig> μl of bacterial suspension  and candida suspension  in pbs at 37°c.
unattached microbial cells were removed by washing the wells three times with pbs.
next,  <dig> μl of  <dig> - <dig>  mg/ml pseudofactin ii was added to each well and incubated at 37°c for  <dig> h on a rotary shaker  at  <dig> rpm.
control wells contained only pbs.
the plates were washed three times, adherent cells were fixed with  <dig> μl of  <dig> % crystal violet for  <dig> min and again washed three times with pbs.
the adherent microorganisms were permeabilized and the dye was resolubilized with  <dig> μl of isopropanol- <dig>  n hcl and  <dig> μl of  <dig> % sds per well.
the crystal violet optical density of each well was measured at  <dig> nm using the microplate reader.
assays were carried out three times in three replicates.
the microbial adhesion dislodging percentages at different pseudofactin ii concentrations for each microorganism were calculated as:
%growthinhibition=1-odtodc× <dig>
where odt represents the optical density of the well with a given pseudofactin ii concentration and odc the optical density of the control well .
assays were carried out three times in three replicates.
confocal laser scanning microscopy
confocal laser scanning microscopy  was used for visualizing the formation of bacterial and candida biofilms in the absence or presence of pseudofactin ii  in the culture medium.
bacterial and yeast biofilms were formed on thermanox plastic coverslips , glass microscopic coverslips  and segments of silicone urethral catheters  placed in wells of 24-well plates  containing lb medium for bacteria and rpmi- <dig> medium for yeast.
inocula were prepared as follows:  <dig> h old overnight cultures were harvested and re-suspended at normalized dilutions .
five hundred microliters inocula were injected into the wells with the coverslips and incubated for  <dig> h at 37°c.
after this time, the coverslips were washed with pbs for  <dig> min.
then, the bacterial biofilms were stained for  <dig> min at 37°c with  <dig> ml of  <dig> % live/dead baclight viability stain  dissolved in pbs, and pbs-containing concanavalin a-alexa fluor  <dig>  conjugate  for candida biofilms.
the stained biofilms were visualized by clsm with an olympus fluoview  <dig>  microscope.
the clsm used an argon ion laser at 480- <dig> nm for excitation and a 500- <dig> nm band pass filter for emission.
clsm images were processed by olympus fluoview  <dig> software.
assays were carried out two times.
representative images are presented on figure  <dig>
biofilm formation in urethral catheters
the uropathogenic strains e. coli, e. faecalis, e. hirae and c. albicans were used in these tests.
ten microliter volumes of overnight cultures of e. coli atcc  <dig>  e. faecalis atcc  <dig>  e. hirae atcc  <dig> were added into  <dig> μl of fresh lb medium, and the same volume of c. albicans sc <dig> was added into  <dig> μl of fresh rpmi- <dig> medium.
to the medium was added  <dig> μl pseudofactin ii  solution in lb medium  and rpmi- <dig> medium for c. albicans and  <dig> cm long segments of sterile silicone urethral catheters .
the catheters were incubated at 37°c overnight.
the cultures were removed and the catheters were washed with distilled water.
after washing,  <dig> μl of crystal violet  was added to the catheters for  <dig> min.
the stained biofilms were rinsed three times with distilled water and allowed to dry at room temperature for  <dig> min before examination.
in a parallel experiment the catheters were pretreated with pseudofactin ii by being placed in a tube with  <dig> μl of  <dig>  mg/ml pseudofactin ii dissolved in pbs, incubated for  <dig> h at 37°c and subsequently washed twice with pbs.
then the experiment was carried out as in the case of adding pseudofactin ii into the growth medium.
assays were carried out two times.
representative images are presented on figure  <dig>  this experiment was carried out under dynamic conditions using a peristaltic pump, where the flow of culture with or without pseudofactin ii trough urethral catheters was  <dig> ml/h.
RESULTS
antimicrobial activity of pseudofactin ii
lipopeptides have typical amphiphilic structure of a surfactant, where the hydrophobic moiety is a hydroxyl or α-alkyl-β-hydroxy fatty acid  and the hydrophilic moiety is a short chain or a cyclic peptide  <cit> .
instead, the hydrophobic moiety of pseudofactin ii contains palmitic acid, which is a saturated fatty acid having no hydroxyl group.
rhodofactin, another lipopeptide with palmitic acid, has been described by peng et al.
<cit> ; however, contrary to pseudofactin ii it has a short peptide chain which does not form lactone ring.
its antimicrobial activity has not yet been described.
the antimicrobial activity of pseudofactin ii isolated from p. fluorescens bd <dig> was evaluated at concentrations from  <dig>  to  <dig>  mg/ml .
at  <dig>  mg/ml the agent caused a total growth inhibition of s. epidermidis kctc  <dig> and considerable growth inhibition of p. mirabilis atcc  <dig> , e. coli atcc  <dig> and e. coli 17- <dig> , e. hirae atcc  <dig> .
in contrast to surfactin or iturin, produced by b. subtilis  <cit> , lichenysin from bacillus licheniformis  <cit>  or polymyxin b and e from bacillus polymyxa  <cit> , pseudofactin ii showed much weaker dose dependent antimicrobial activity against most strains tested in this work .
only for two vibrio strains pseudofactin ii completely inhibited the growth in the lowest tested concentration, thus they were not used in further experiments .
this may be due to its unique chemical structure different from any currently known lipopeptides, which features a hydrophobic alkyl chain without a hydroxyl moiety attached to cyclic peptide.
antiadhesive and cell-dislodging activity of pseudofactin ii on polystyrene surfaces
adhesion of pathogenic microorganisms to solid surfaces or to infection sites has been found to be inhibited by biosurfactants capable of modifying the physico-chemical properties of the surface to reduce adhesion and biofilm formation on a given biomaterial.
pseudofactin ii was found to possess antiadhesive activity against all tested microorganisms.
the pretreatment of polystyrene surfaces with pseudofactin ii significantly decreased the adhesion of all bacteria and yeast, and this antiadhesive effect was concentration-dependent .
the highest reduction of adhesion  was observed for c. albicans sc  <dig>  c. albicans atcc  <dig>  p. mirabilis atcc  <dig> and e. coli atcc  <dig>  the dislodging effect of pseudofactin ii on preformed biofilms on untreated surfaces was lower than the preventive effect of pretreatment and was in the range of 26-70% for  <dig>  mg/ml pseudofactin ii .
pbs was used as control and set at 0% as no microbial inhibition occurs.
values ± confidence interval, n = 9
negative controls  were set at 0%.
values ± confidence interval, n = 9
the adhesion of pathogenic bacteria to polystyrene surfaces was inhibited by two lipopeptide biosurfactants produced by b. subtilis and b. licheniformis  <cit> , and adhesion of listeria monocytogenes to polystyrene microplates was reduced by 84% on pretreating the surface with surfactin , and by 82% when it was treated with purified rhamnolipid   <cit> .
gudina et al.
<cit>  characterized the anti-adhesive activity of biosurfactants against several microorganisms including gram-positive and gram-negative bacteria.
this biosurfactant at concentration  <dig> mg/ml showed high anti-adhesive activity against staphylococcus aureus , s. epidermidis , streptococcus agalactiae  and low anti-adhesive activity against p. aeruginosa  and e. coli .
coating with pseudofactin ii was effective above critical micelle concentration   <cit> .
our results suggest that when the surface is covered by pseudofactin ii micelles attached to polystyrene by van der waals forces, the adhesion is inhibited more strongly than it is with monomers.
pseudofactin ii reduces biofilm formation on polystyrene, glass and silicone
biofilms are defined as microorganisms attached to a diverse range of biotic and abiotic surfaces and proliferating on them.
the human body and medical devices or implants including: urinary catheters, voice prostheses, orthopedic implants, ocular prostheses and contact lenses are exposed to adhesion and biofilm formation by many opportunistic microorganisms.
thus we have tested the influence of pseudofactin ii on biofilm formation on different materials.
the activity of pseudofactin ii against biofilm formation was visualized by confocal laser scanning microscopy .
the biofilm growth of e. coli, e. faecalis, e. hirae and c. albicans on polystyrene, glass and silicone from urethral catheters is shown in figures 1a-d, 1i-l and 1r-u, respectively.
the biosurfactant inhibited biofilm formation at the concentration  <dig>  mg/ml on polystyrene, glass and silicone surfaces .
e. faecalis atcc  <dig> adhesion to all tested surfaces is less intensive than others strains .
in fact, the adhesion of this strain to  <dig> wells plate was between  <dig> to 4-fold weaker than others tested bacterial strains .
this effect may be due to small amount of adhesion proteins on e. faecalis atcc  <dig> strain.
pseudofactin in the tested concentration inhibited mixed biofilm formation , similarly as for the single strain .
in our experiments all the tested gram-negative and gram-positive bacteria showed decrease of adhesion.
the results of the present study indicate that pseudofactin ii have potential to be used for efficient removal and inhibition of biofilms for pathogenic microorganisms.
rivardo et al.
<cit>  demonstrated that biosurfactants obtained from bacillus spp.
were able to inhibit biofilm formation for two pathogenic strains e. coli at 97% and s. aureus at 90%, respectively.
irie et al.
<cit>  demonstrated that rhamnolipids produced by p. aeruginosa were able to disperse biofilm for bordetella bronchiseptica.
pseudofactin ii prevents biofilm formation in urethral catheters
to test biofilm formation on medical device, silicone urethral catheters,  <dig> cm segments of the catheters were incubated with e. coli atcc  <dig>  e. faecalis atcc  <dig>  e. hirae atcc  <dig> and c. albicans sc  <dig>  e. coli, e. faecalis and e. hirae formed biofilms mainly at the air-liquid interface, while the biofilm formed by c. albicans was dispersed along the whole growth surface .
even though the pseudofactin ii present in the growth medium , was at the concentration of  <dig>  mg/ml which did not significantly affect the growth of the tested microbial cultures, biofilm formation was nearly completely prevented.
the pretreatment of silicone urethral catheters with pseudofactin ii prior to inoculation with medium was just as effective as including the biosurfactant in the growth medium .
we observed the similar effect in dynamic conditions for urethral catheters using a flow of  <dig> ml/h .
earlier reports noted an inhibition of biofilms formed by several microorganisms, e.g.
salmonella typhimurium, s. enterica, e. coli and p. mirabilis on vinyl urethral catheters by a surfactin produced by b. subtilis  <cit> .
our results show that pseudofactin ii is promising compound for inhibition and disruption of biofilms and has potential applications in medicine.
CONCLUSIONS
the biosurfactant pseudofactin ii, produced by p. fluorescens bd <dig> strain and purified by hplc, showed antiadhesive activity against several pathogenic microorganisms, such as e. coli, e. faecalis, e. hirae, s. epidermidis, p. mirabilis and c. albicans, which are potential biofilm formers on catheters, implants and internal prostheses.
up to 99% prevention of c. albicans sc  <dig> adhesion could be achieved by  <dig>  mg/ml pseudofactin ii.
confocal laser scanning microscopy confirmed the action of pseudofactin ii as an inhibitor of biofilm formation.
in addition, pseudofactin ii dispersed preformed biofilms.
due to its surface tension properties and lack of hemolytic activity , pseudofactin ii can be used as a surface coating agent against microbial colonization of different surfaces, e.g.
implants or urethral catheters.
authors' contributions
tj carried out experiments, ml participated in the design of the study, data analysis, coordination and helped to draft the manuscript, ak conceived the experiments and draft the manuscript.
all authors read and approved the final manuscript.

 biofilms, which are formed by the majority of microorganisms in natural environments, are structures with low sensitivity to drugs. many laboratories are synthesizing or isolating new compounds preventing the formation of biofilms or causing their elimination. adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. biosurfactants are promising compounds often showing antimicrobial and antiadhesive properties and sometimes penetrating and removing mature biofilms. microbial surfactants-amphiphilic, surface-active, secondary metabolites of bacteria or fungi ranging from low-molecular-mass glycolipids, sophorolipids, rhamnolipids and lipopeptides, to high-molecular-mass proteins, lipopolysaccharides and lipoproteins -can interact with interfaces and inhibit the adhesion of microorganisms to different surfaces. they are an alternative to synthetic surface-active agents because of their low toxicity and biodegradability. another mechanism of biosurfactant action is the permeabilization of bacterial cells. the rhamnolipid secreted by pseudomonas sp. s- permeabilized gram-negative and gram-positive cells, but a strong inhibition of growth was observed only in the case of gram-positive bacteria. biofilm disruption was observed after the addition of rhamnolipids from pseudomonas aeruginosa and lipopeptide from bacillus spp.. a particular group of biosurfactants, lipopeptides, can act as antibiotics and also as antiviral and antitumor agents. surfactin from bacillus subtilis can interact with the plasma membranes of bacterial and fungal cells leading to their disruption. the effects of biosurfactants on decreased microbial adhesion and detachment from different surfaces can be conveniently utilized in many fields, from medicine to various branches of industry, e.g., antimicrobial or antitumor activities and their surface activity and antiadhesive properties can be suitable for preventing microbial colonization of implants or urethral catheters. microbial surfactants from lactobacillus fermentum and lactobacillus acidophilus adsorbed on glass, reduced the number of adhering uropathogenic cells of enterococcus faecalis by 77%. a surfactant released by streptococcus thermophilus has been used for fouling control of heat-exchanger plates in pasteurizers as it retards the colonization of other thermophilic strains of streptococcus responsible for fouling. within the collection of morphologically distinct bacteria isolated from water and ground provided from the wild environment around the university of wroclaw scientific base in the arctic archipelago of svalbard, pseudomonas fluorescens bd was identified as strain which strongly reduces surface tension of its culture supernatant. we purified and identified the chemical structure of two new p. fluorescens bd biosurfactants, pseudofactin i and ii. both compounds are cyclic lipopeptides with a palmitic acid connected to the terminal amino group of an octapeptide. the c-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of thr the biosurfactant was found to be stable within the range from -20°c to 100°c, had the minimum surface tension and the critical micelle concentration. emulsification activity and stability of pseudofactin ii was greater than that of the synthetic surfactants such as tween and triton x- the aim of this paper was to assess how the pseudofactin ii influences the adhesion and biofilm formation of microorganisms such as escherichia coli, e. faecalis, enterococcus hirae, staphylococcus epidermidis, proteus mirabilis, vibrio ordalii, vibrio harveyi and candida albicans found in gastrointestinal and urinary tract. since the effects of a surfactant may differ depending on both the type of the microorganism and the type of surface it adheres to, we tested its action on the adherence of the above pathogenic microorganisms to three types of surfaces, polystyrene, glass and silicone. methods microorganisms and culture conditions p. fluorescens bd strain was obtained from freshwater from the arctic archipelago of svalbard and maintained on the mineral salts medium msm 2so g/l sodium citrate 2h2o, and g/l mgso h2o) with 2% d-glucose. the antimicrobial and antiadhesive properties of pseudofactin ii were tested on several pathogenic strains that colonize animals gastrointestinal tract or medical devices. e. coli atcc e. coli atcc e. coli 17-, e. faecalis atcc e. faecalis ja/, e. hirae atcc s. epidermidis kctc, p. mirabilis atcc were grown at 37°c and v. harveyi atcc v. ordalii kccm were grown at 28°c in lb medium. two fungal strains, c. albicans atcc and c. albicans sc, were grown in a g/l yeast nitrogen base, broth containing 2% d-glucose for adhesion tests. to prevent filamentation of c. albicans, pre-culture was incubated at 28°c, while experiments with biofilms were performed at 37°c. rpmi- medium was used for candida biofilms formation. isolation and purification of pseudofactin ii pseudofactin ii produced by p. fluorescens bd was obtained by extraction of cell free supernatant by ethyl acetate and evaporation of the extract under vacuum. the crude biosurfactant was separated by rp-hplc in the same manner as reported earlier. purified pseudofactin ii fraction was dried and stored at -20°c for further studies. analytical rp-hplc of purified pseudofactin ii showed that its purity was > 99%. antimicrobial assays the antimicrobial activity of isolated pseudofactin ii was determined by the microdilution method in 96-well flat-bottomed plastic microplates. briefly, μl volumes of sterile double strength lb or ynb medium were dispensed into the wells of a 96-well microplate. subsequently, μl volumes of pseudofactin ii solution in phosphate-buffered saline were added to the microplate wells and mixed with the medium. negative and growth control wells did not contain biosurfactant. all wells were inoculated with μl of overnight bacterial or yeast cultures in lb or ynb medium respectively, and the microplates were incubated for h at 37°c or 28°c for bacterial or yeast cultures, respectively. after h of incubation, the optical density at nm of each well was measured using an asys uvm microplate reader. the growth inhibition percentages at different pseudofactin ii concentrations for each microorganism were calculated as: %growthinhibition=1-odtodc× where odt represents the optical density of the well with a given pseudofactin ii concentration and odc is the optical density of the control well. assays were carried out three times in three replicates. preadhesion treatment with pseudofactin ii inhibition of microbial adhesion by pseudofactin ii was tested in 96-well plates. briefly, the wells of a sterile 96-well flat-bottom plate were filled with μl of - mg/ml pseudofactin ii dissolved in pbs. the plates were incubated for h at 37°c on a rotary shaker at rpm and subsequently washed twice with pbs. negative control wells contained pseudofactin ii at the highest concentration tested while positive control wells contained pbs buffer only. the overnight cultures of microbial strains were centrifuged, washed twice with pbs and re-suspended in pbs to an optical density od = for bacterial and od = for candida strains. the highest adhesion without pseudofactin ii were observed at these optical densities. a μl aliquot of a washed microbial suspension was added and incubated in the wells. after a h incubation at 37°c in a rotary shaker at rpm nonadherent cells were removed by three washes with pbs. then the plates were stained with % crystal-violet for min and again washed three times with pbs. the adherent microorganisms were permeabilized and the dye was resolubilized with μl of isopropanol- n hcl and μl of % sds per well. crystal violet optical density readings of each well were taken at nm on the asys uvm microplate. pseudofactin ii did not affect the absorption of negative control. the microbial adhesion inhibition was calculated as growth inhibition. assays were carried out three times in three replicates. postadhesion treatment with pseudofactin ii the 96-well flat-bottomed plates were incubated for h on a rotary shaker at rpm with μl of bacterial suspension and candida suspension in pbs at 37°c. unattached microbial cells were removed by washing the wells three times with pbs. next, μl of - mg/ml pseudofactin ii was added to each well and incubated at 37°c for h on a rotary shaker at rpm. control wells contained only pbs. the plates were washed three times, adherent cells were fixed with μl of % crystal violet for min and again washed three times with pbs. the adherent microorganisms were permeabilized and the dye was resolubilized with μl of isopropanol- n hcl and μl of % sds per well. the crystal violet optical density of each well was measured at nm using the microplate reader. assays were carried out three times in three replicates. the microbial adhesion dislodging percentages at different pseudofactin ii concentrations for each microorganism were calculated as: %growthinhibition=1-odtodc× where odt represents the optical density of the well with a given pseudofactin ii concentration and odc the optical density of the control well. assays were carried out three times in three replicates. confocal laser scanning microscopy confocal laser scanning microscopy was used for visualizing the formation of bacterial and candida biofilms in the absence or presence of pseudofactin ii in the culture medium. bacterial and yeast biofilms were formed on thermanox plastic coverslips, glass microscopic coverslips and segments of silicone urethral catheters placed in wells of 24-well plates containing lb medium for bacteria and rpmi- medium for yeast. inocula were prepared as follows: h old overnight cultures were harvested and re-suspended at normalized dilutions. five hundred microliters inocula were injected into the wells with the coverslips and incubated for h at 37°c. after this time, the coverslips were washed with pbs for min. then, the bacterial biofilms were stained for min at 37°c with ml of % live/dead baclight viability stain dissolved in pbs, and pbs-containing concanavalin a-alexa fluor conjugate for candida biofilms. the stained biofilms were visualized by clsm with an olympus fluoview microscope. the clsm used an argon ion laser at 480- nm for excitation and a 500- nm band pass filter for emission. clsm images were processed by olympus fluoview software. assays were carried out two times. representative images are presented on figure biofilm formation in urethral catheters the uropathogenic strains e. coli, e. faecalis, e. hirae and c. albicans were used in these tests. ten microliter volumes of overnight cultures of e. coli atcc e. faecalis atcc e. hirae atcc were added into μl of fresh lb medium, and the same volume of c. albicans sc was added into μl of fresh rpmi- medium. to the medium was added μl pseudofactin ii solution in lb medium and rpmi- medium for c. albicans and cm long segments of sterile silicone urethral catheters. the catheters were incubated at 37°c overnight. the cultures were removed and the catheters were washed with distilled water. after washing, μl of crystal violet was added to the catheters for min. the stained biofilms were rinsed three times with distilled water and allowed to dry at room temperature for min before examination. in a parallel experiment the catheters were pretreated with pseudofactin ii by being placed in a tube with μl of mg/ml pseudofactin ii dissolved in pbs, incubated for h at 37°c and subsequently washed twice with pbs. then the experiment was carried out as in the case of adding pseudofactin ii into the growth medium. assays were carried out two times. representative images are presented on figure this experiment was carried out under dynamic conditions using a peristaltic pump, where the flow of culture with or without pseudofactin ii trough urethral catheters was ml/h.  antimicrobial activity of pseudofactin ii lipopeptides have typical amphiphilic structure of a surfactant, where the hydrophobic moiety is a hydroxyl or α-alkyl-β-hydroxy fatty acid and the hydrophilic moiety is a short chain or a cyclic peptide. instead, the hydrophobic moiety of pseudofactin ii contains palmitic acid, which is a saturated fatty acid having no hydroxyl group. rhodofactin, another lipopeptide with palmitic acid, has been described by peng et al.; however, contrary to pseudofactin ii it has a short peptide chain which does not form lactone ring. its antimicrobial activity has not yet been described. the antimicrobial activity of pseudofactin ii isolated from p. fluorescens bd was evaluated at concentrations from to mg/ml. at mg/ml the agent caused a total growth inhibition of s. epidermidis kctc and considerable growth inhibition of p. mirabilis atcc, e. coli atcc and e. coli 17-, e. hirae atcc. in contrast to surfactin or iturin, produced by b. subtilis, lichenysin from bacillus licheniformis or polymyxin b and e from bacillus polymyxa, pseudofactin ii showed much weaker dose dependent antimicrobial activity against most strains tested in this work. only for two vibrio strains pseudofactin ii completely inhibited the growth in the lowest tested concentration, thus they were not used in further experiments. this may be due to its unique chemical structure different from any currently known lipopeptides, which features a hydrophobic alkyl chain without a hydroxyl moiety attached to cyclic peptide. antiadhesive and cell-dislodging activity of pseudofactin ii on polystyrene surfaces adhesion of pathogenic microorganisms to solid surfaces or to infection sites has been found to be inhibited by biosurfactants capable of modifying the physico-chemical properties of the surface to reduce adhesion and biofilm formation on a given biomaterial. pseudofactin ii was found to possess antiadhesive activity against all tested microorganisms. the pretreatment of polystyrene surfaces with pseudofactin ii significantly decreased the adhesion of all bacteria and yeast, and this antiadhesive effect was concentration-dependent. the highest reduction of adhesion was observed for c. albicans sc c. albicans atcc p. mirabilis atcc and e. coli atcc the dislodging effect of pseudofactin ii on preformed biofilms on untreated surfaces was lower than the preventive effect of pretreatment and was in the range of 26-70% for mg/ml pseudofactin ii. pbs was used as control and set at 0% as no microbial inhibition occurs. values ± confidence interval, n = 9 negative controls were set at 0%. values ± confidence interval, n = 9 the adhesion of pathogenic bacteria to polystyrene surfaces was inhibited by two lipopeptide biosurfactants produced by b. subtilis and b. licheniformis, and adhesion of listeria monocytogenes to polystyrene microplates was reduced by 84% on pretreating the surface with surfactin, and by 82% when it was treated with purified rhamnolipid. gudina et al. characterized the anti-adhesive activity of biosurfactants against several microorganisms including gram-positive and gram-negative bacteria. this biosurfactant at concentration mg/ml showed high anti-adhesive activity against staphylococcus aureus, s. epidermidis, streptococcus agalactiae and low anti-adhesive activity against p. aeruginosa and e. coli. coating with pseudofactin ii was effective above critical micelle concentration. our results suggest that when the surface is covered by pseudofactin ii micelles attached to polystyrene by van der waals forces, the adhesion is inhibited more strongly than it is with monomers. pseudofactin ii reduces biofilm formation on polystyrene, glass and silicone biofilms are defined as microorganisms attached to a diverse range of biotic and abiotic surfaces and proliferating on them. the human body and medical devices or implants including: urinary catheters, voice prostheses, orthopedic implants, ocular prostheses and contact lenses are exposed to adhesion and biofilm formation by many opportunistic microorganisms. thus we have tested the influence of pseudofactin ii on biofilm formation on different materials. the activity of pseudofactin ii against biofilm formation was visualized by confocal laser scanning microscopy. the biofilm growth of e. coli, e. faecalis, e. hirae and c. albicans on polystyrene, glass and silicone from urethral catheters is shown in figures 1a-d, 1i-l and 1r-u, respectively. the biosurfactant inhibited biofilm formation at the concentration mg/ml on polystyrene, glass and silicone surfaces. e. faecalis atcc adhesion to all tested surfaces is less intensive than others strains. in fact, the adhesion of this strain to wells plate was between to 4-fold weaker than others tested bacterial strains. this effect may be due to small amount of adhesion proteins on e. faecalis atcc strain. pseudofactin in the tested concentration inhibited mixed biofilm formation, similarly as for the single strain. in our experiments all the tested gram-negative and gram-positive bacteria showed decrease of adhesion. the results of the present study indicate that pseudofactin ii have potential to be used for efficient removal and inhibition of biofilms for pathogenic microorganisms. rivardo et al. demonstrated that biosurfactants obtained from bacillus spp. were able to inhibit biofilm formation for two pathogenic strains e. coli at 97% and s. aureus at 90%, respectively. irie et al. demonstrated that rhamnolipids produced by p. aeruginosa were able to disperse biofilm for bordetella bronchiseptica. pseudofactin ii prevents biofilm formation in urethral catheters to test biofilm formation on medical device, silicone urethral catheters, cm segments of the catheters were incubated with e. coli atcc e. faecalis atcc e. hirae atcc and c. albicans sc e. coli, e. faecalis and e. hirae formed biofilms mainly at the air-liquid interface, while the biofilm formed by c. albicans was dispersed along the whole growth surface. even though the pseudofactin ii present in the growth medium, was at the concentration of mg/ml which did not significantly affect the growth of the tested microbial cultures, biofilm formation was nearly completely prevented. the pretreatment of silicone urethral catheters with pseudofactin ii prior to inoculation with medium was just as effective as including the biosurfactant in the growth medium. we observed the similar effect in dynamic conditions for urethral catheters using a flow of ml/h. earlier reports noted an inhibition of biofilms formed by several microorganisms, e.g. salmonella typhimurium, s. enterica, e. coli and p. mirabilis on vinyl urethral catheters by a surfactin produced by b. subtilis. our results show that pseudofactin ii is promising compound for inhibition and disruption of biofilms and has potential applications in medicine.  the biosurfactant pseudofactin ii, produced by p. fluorescens bd strain and purified by hplc, showed antiadhesive activity against several pathogenic microorganisms, such as e. coli, e. faecalis, e. hirae, s. epidermidis, p. mirabilis and c. albicans, which are potential biofilm formers on catheters, implants and internal prostheses. up to 99% prevention of c. albicans sc adhesion could be achieved by mg/ml pseudofactin ii. confocal laser scanning microscopy confirmed the action of pseudofactin ii as an inhibitor of biofilm formation. in addition, pseudofactin ii dispersed preformed biofilms. due to its surface tension properties and lack of hemolytic activity, pseudofactin ii can be used as a surface coating agent against microbial colonization of different surfaces, e.g. implants or urethral catheters. authors' contributions tj carried out experiments, ml participated in the design of the study, data analysis, coordination and helped to draft the manuscript, ak conceived the experiments and draft the manuscript. all authors read and approved the final manuscript. 
pseudofactin ii lowered the adhesion to three types of surfaces of bacterial strains of five species: escherichia coli, enterococcus faecalis, enterococcus hirae, staphylococcus epidermidis, proteus mirabilis and two candida albicans strains. adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. pseudofactin ii is a novel compound identified as cyclic lipopeptide with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. pseudofactin ii also caused a marked inhibition of the initial adhesion of e. faecalis, e. coli, e. hirae and c. albicans strains to silicone urethral catheters. the c-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of thr pseudofactin ii showed antiadhesive activity against several pathogenic microorganisms which are potential biofilm formers on catheters, implants and internal prostheses. pretreatment of a polystyrene surface with mg/ml pseudofactin ii inhibited bacterial adhesion by 36-90% and that of c. albicans by 92-99%. the highest concentration tested caused a total growth inhibition of s. epidermidis, partial inhibition of other bacteria and 8-9% inhibition of c. albicans growth. pseudofactin ii can be used as a disinfectant or surface coating agent against microbial colonization of different surfaces, e.g. up to 99% prevention could be achieved by mg/ml pseudofactin ii. pseudofactin ii is a recently identified biosurfactant secreted by pseudomonas fluorescens bd the strain obtained from freshwater from the arctic archipelago of svalbard. in this study the role and applications of pseudofactin ii as a antiadhesive compound has been investigated from medicinal and therapeutic perspectives. the same concentration of pseudofactin ii dislodged 26-70% of preexisting biofilms grown on previously untreated surfaces. in addition, pseudofactin ii dispersed preformed biofilms. glass, polystyrene, silicone modifies its hydrophobicity, interfering with the microbial adhesion and desorption processes.
 pseudofactin ii is a recently identified biosurfactant secreted by pseudomonas fluorescens bd the strain obtained from freshwater from the arctic archipelago of svalbard. pseudofactin ii is a novel compound identified as cyclic lipopeptide with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. the c-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of thr adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. adsorption of biosurfactants to a surface e.g. glass, polystyrene, silicone modifies its hydrophobicity, interfering with the microbial adhesion and desorption processes. in this study the role and applications of pseudofactin ii as a antiadhesive compound has been investigated from medicinal and therapeutic perspectives.  pseudofactin ii lowered the adhesion to three types of surfaces of bacterial strains of five species: escherichia coli, enterococcus faecalis, enterococcus hirae, staphylococcus epidermidis, proteus mirabilis and two candida albicans strains. pretreatment of a polystyrene surface with mg/ml pseudofactin ii inhibited bacterial adhesion by 36-90% and that of c. albicans by 92-99%. the same concentration of pseudofactin ii dislodged 26-70% of preexisting biofilms grown on previously untreated surfaces. pseudofactin ii also caused a marked inhibition of the initial adhesion of e. faecalis, e. coli, e. hirae and c. albicans strains to silicone urethral catheters. the highest concentration tested caused a total growth inhibition of s. epidermidis, partial inhibition of other bacteria and 8-9% inhibition of c. albicans growth.  pseudofactin ii showed antiadhesive activity against several pathogenic microorganisms which are potential biofilm formers on catheters, implants and internal prostheses. up to 99% prevention could be achieved by mg/ml pseudofactin ii. in addition, pseudofactin ii dispersed preformed biofilms. pseudofactin ii can be used as a disinfectant or surface coating agent against microbial colonization of different surfaces, e.g. implants or urethral catheters. biosurfactantlipopeptideadhesionbiofilmuropathogenic microorganisms 