biofilms are associated with a variety of persistent infections as a result of their propensity to form and grow on foreign bodies .
compared with planktonic forms , organisms in biofilms exhibit increased resistance to the host immune system and antimicrobial therapy ; for this reason , the management of biofilm - associated infections is challenging .
today , many of these infections are definitively managed using medical device removal , an intervention that is both costly and inconvenient . given that biofilm - associated infections are difficult to manage , prevention strategies are ideal .
most preventive approaches utilize antimicrobials or antiseptics [ 48 ] ; however , considering that biofilms can survive in the presence of high concentrations of antimicrobial agents , new prophylactic strategies are needed .
chemical and mechanical strategies such as silver or gallium ions , cationic molecules , and other disinfectants have been studied as coatings of indwelling devices [ 912 ] .
substances with antibiofilm activity , such as lactoferrin or synthesized chalcones [ 1315 ] , as well as low acoustic energy [ 16 , 17 ] have shown some ability to prevent biofilm formation .
none of these strategies has , however , solved the clinical challenge of biofilm - associated infections .
the initial step of biofilm formation on medical devices involves adhesion of organisms to medical implant surfaces by electrostatic forces which are largely repulsive , as both are negatively charged .
direct current ( dc ) may augment repulsive electrostatic forces between organisms and medical implants [ 1922 ] .
in addition , dc may impact biofilm formation by changing physical conditions ( e.g. , temperature , ph ) at the implant surface and through the accumulation of products of oxidative stress [ 20 , 2327 ] .
previous studies have demonstrated that dc exhibits bactericidal activity against established biofilms [ 20 , 22 , 23 , 25 , 28 ] .
the bactericidal effect of dc against sessile cells suggests that this strategy may be useful to reduce biofilm formation . in a previous study
, we showed that 24 hours of 2,000  a dc reduced s. epidermidis biofilm formation . whether lower amperage of dc would also reduce biofilm formation and whether our findings with s. epidermidis generalize to other microorganisms are unknown .
the use of dc to reduce biofilm formation may provide a new strategy to prevent biofilm formation in clinical practice .
it has the potential benefit of eliminating the use of traditional antimicrobials and therefore decreasing the risk of selecting resistance to these agents .
herein , we examined the effect of different amperages and delivery durations of dc in reducing formation of biofilms of five bacterial and one fungal species .

 s. epidermidis xen 43 , staphylococcus aureus xen 30 , escherichia coli ( idrl-7029 , prosthetic hip infection clinical isolate ) , pseudomonas aeruginosa xen 5 , candida albicans ( gdh2346 , mouth infection clinical isolate ) , and propionibacterium acnes ( idrl-7676 , prosthetic shoulder infection clinical isolate ) were studied .
the xen strains were generous gifts of perkinelmer caliper life sciences ( formerly xenogen corp . ,
jyotsna chandra and mahmoud ghannoum ( university hospitals of cleveland and case western reserve university , cleveland , oh ) . experiments were performed using polycarbonate channeled chambers designed and fabricated by the mayo division of engineering ( figure 1 ) .
each chamber contained a groove into which a 12.5  1  mm teflon or titanium disc was inserted , positioned vertically .
cylindrical platinum electrodes , 1.5  55  mm , were placed in each chamber , 3  mm from the disc , with 1  cm of electrode extended above the chamber for the purpose of connecting the electrode to a current generator . a power source (
keithley 2400 sourcemeter ) or an 8-channel computer controlled current generator ( designed by mayo division of engineering ) was used to deliver direct current ( 200 or 500  a ) .
microorganisms were subcultured from frozen aliquots onto bbl trypticase soy agar with 5% sheep blood plates ( tsa ii , becton dickinson franklin lakes , nj ) and incubated overnight at 37c in 5% co2 .
one colony was added to 3  ml of trypticase soy broth ( tsb ) and grown for 1 - 2 hours at 37c on an orbital shaker .
the broth was adjusted to a 0.5 mcfarland standard and added to a previously described semisynthetic medium   supplemented with 64  ml of 1% glucose and tsb ( 10% ) to a final bacterial concentration of 10 colony forming units ( cfu)/ml . a continuous flow ( 3  ml / hour ) of the semisynthetic medium containing 10  cfu / ml test organism was delivered to the polycarbonate treatment chambers containing teflon or titanium discs . after 2 hours ( for gram - negative bacilli ) or 4 hours ( for gram - positive cocci ) , the semisynthetic medium containing the test organism was changed to a phosphate buffer ( 12.78  mg na2hpo4 , 6.15  mg kh2po4 , and 19.2  mg glucose in 1000  ml sterile water ) without bacteria , also flowing at 3  ml / hour .
dc ( 0 , 200 , or 500  a ) was delivered ( starting at the same time that semisynthetic medium with bacteria flow started ) for either 4 , 8 , 12 , 16 , 20 , or 24 hours of a 24-hour period when testing 500  a dc , or 12 , 24 , 36 , or 48 hours of a 48-hour period when testing 200  a dc , with 0  a controls tested at each time point .
testing was performed at 37c for gram - positive cocci and at room temperature for gram - negative bacilli .
after 24 hours when using 500  a or 48 hours when using 200  a , discs were aseptically removed from the test chambers , planktonic organisms rinsed off by gently dipping the discs into sterile saline , and the discs placed into sterile tubes containing 1  ml of sterile saline .
biofilm organisms were removed by vortexing and sonication in an ultrasound bath ( 40  khz , 320  mw / cm ) for 5 minutes .
the medium that remained in the chamber ( planktonic organisms ) after 24 or 48 hours was also quantitatively cultured .
biofilm results were expressed as log10  cfu / cm ; planktonic results were expressed as log10  cfu / ml .
c. albicans was subcultured from frozen aliquots and incubated for 48 hours at 30c in room air , only 0 and 500  a dc for 24 and 48 hours were tested , semisynthetic medium was changed to phosphate buffer after 4 hours , and experiments were conducted at room temperature . for p. acnes ,
the organism was subcultured from frozen aliquots and incubated for 72 hours at 37c under anaerobic conditions , only 0 and 500  a dc for 24 and 48 hours were tested , semisynthetic medium was changed to phosphate buffer after 4 hours , and experiments were conducted at 37c in an anaerobic chamber ( coy laboratory products , grass lake , mi ) .
in addition to performing experiments under aerobic conditions , s. epidermidis experiments were run under anaerobic conditions using 0 and 500  a dc for 24 hours .
we compared the difference between s. epidermidis and e. coli biofilm formation on titanium discs using 0 , 200 , and 500  a of dc for 12 and 24 hours and the treatment device and methods described above .
reductions in biofilm or planktonic cells were calculated comparing quantitative cultures of discs or surrounding fluid in chambers exposed and not exposed to electrical current .
was performed with each current delivery strategy and no current delivery using the wilcoxon rank sum test to determine if electricity reduced biofilm formation .
all tests were two - sided ; p values < 0.05 were considered statistically significant .
time- and dose - dependent reductions in biofilm formation on teflon discs were observed for s. epidermidis , s. aureus , e. coli , and p. aeruginosa , using 500 and 200  a ( figure 2 ) . for s. epidermidis , a 1  log10  cfu / cm reduction in biofilm formation
was observed starting at 8 hours of exposure to 500  a , with a 4  log10  cfu / cm reduction observed after 16 hours of exposure to 500  a or 24 hours of exposure to 200  a . for s. aureus
, there were 2  log10  cfu / cm reductions in biofilm formation with 12 or more hours of exposure to 200 and 500  a .
for e. coli , there were 1 and 4  log10  cfu / cm reductions in biofilm formation with 12 and 24 hours of exposure to 500  a , respectively ; a similar but smaller effect was observed with 200  a , with a 4  log10  cfu / cm reduction observed with 48 hours of exposure . for p. aeruginosa , a 1  log10  cfu / cm reduction in biofilm formation
was observed with 4 hours of exposure to 500  a or 36 hours of exposure to 200  a , with a 4  log10  cfu / cm reduction being observed after 24 hours of exposure to 500  a .
overall , significant reductions in biofilm formation were observed using 500  a for at least 12 hours ( p = 0.0495 ) and 200  a for at least 36 hours ( p < 0.05 ) for all four bacteria studied .
significant differences in amounts of planktonic cells were observed using 500  a for at least 12 hours ( p = 0.0495 ) and 200  a for at least 36 hours ( p = 0.0495 ) for all four bacteria studied . since dc reduced s. epidermidis , s. aureus , e. coli , and p. aeruginosa biofilm formation on teflon discs , we next tested whether this effect would be observed with yeast and an anaerobic bacterium on teflon discs , as well as with s. epidermidis and e. coli on titanium discs .
a 3  log10  cfu / cm reduction in c. albicans biofilm formation on teflon discs was detected after 24 hours of exposure to 500  a dc ( figure 3 ) .
there was no reduction in p. acnes biofilm formation with 48 hours of exposure to 500  a dc ( figure 4 ) , although there was a 1  log10  cfu / ml reduction in planktonic p. acnes with exposure to 500  a dc for 24 hours .
a 3  log10  cfu / cm reduction in s. epidermidis biofilm was observed with 24 hours of exposure to 500  a ( figure 4 ) . 1 and 2  log10  cfu / cm reductions in e. coli and s. epidermidis biofilm formation on titanium discs , respectively ,
were observed with 12 hours of exposure to 500  a ( p = 0.0495 )
. a 1  log10  cfu / cm reduction in biofilm formation was observed for both e. coli and s. epidermidis on titanium discs with 24 hours of exposure to 200  a ( p = 0.0495 ) .
the overall magnitude of the effect observed with titanium and teflon discs was similar for both bacteria , although e. coli ( means of 5 versus 6  log10  cfu / cm , p = 0.0009 ) but not s. epidermidis ( p = 0.0765 ) formed slightly less biofilm on untreated titanium than teflon discs .
results of these studies demonstrate that dc reduces staphylococcus species , e. coli , p. aeruginosa , and c. albicans biofilm formation . since these microorganisms are frequently involved in biofilm - associated infections , these findings are of potential clinical interest .
although our results are consistent with previous data showing a bactericidal effect of dc against sessile and planktonic cells [ 22 , 25 , 26 , 34 ] , previous studies have focused on treatment of established biofilms .
our results provide evidence that dc can reduce biofilm formation by staphylococci , gram - negative bacilli , and candida species .
overall , a reduction in biofilm formation was measureable within 12 hours of application of 500  a dc ; when applying 200  a of dc , an effect was observed after 36 hours of current application .
dc may reduce the formation of biofilms by preventing adherence of bacterial cells to surfaces , through augmentation of the noncovalent forces between organisms , and in our study teflon and titanium discs .
however , the decrease in the observed planktonic cell population suggests that there may be additional active mechanisms .
direct damage from dc to bacteria or yeast by electroporation and/or production of reactive oxygen species , as well as generation of other toxic substances , has been proposed .
chlorine has been identified as a toxic substance that plays a role in the bactericidal effect of electrical current against established biofilms .
the absence of an effect against p. acnes biofilm formation may be explained by the involvement of reactive oxygen species in the mechanism underlying the antibiofilm activity of electrical current .
the contribution of reactive oxygen species to this process is also supported by the decreased effect observed under anaerobic conditions with s. epidermidis .
differences in bactericidal effect have been described when using different electrode materials ; we observed less antibiofilm effect when using stainless steel compared with platinum electrodes ( data not shown ) .
although most of our experiments were performed using teflon discs , we demonstrated a similar effect using titanium discs , which is of clinical relevance since titanium is used in the construction of orthopedic implants .
further investigation is needed to determine the appropriate dose and time of administration of dc for reduction of biofilm formation .
future work could explore the capacity of cells to adhere to a surface that has been previously exposed to electrical current and intermittent dc administration .
overall , our results demonstrate that biofilm formation can be reduced using low dose dc .
potentially , this strategy could be used during surgery to prevent early infection and contamination of newly implanted foreign bodies .
 new strategies are needed for prevention of biofilm formation . 
 we have previously shown that 24  hr of 2,000  a of direct current ( dc ) reduces staphylococcus epidermidis biofilm formation in vitro . herein , we examined the effect of a lower amount of dc exposure on s. epidermidis , staphylococcus aureus , escherichia coli , pseudomonas aeruginosa , propionibacterium acnes , and candida albicans biofilm formation . 
 12  hr of 500  a dc decreased s. epidermidis , s. aureus , e. coli , and p. aeruginosa biofilm formation on teflon discs by 2 , 1 , 1 , and 2 log10 cfu / cm2 , respectively ( p < 0.05 ) . reductions in s. epidermidis , s. aureus , and e. coli biofilm formation were observed with as few as 12  hr of 200  a dc ( 2 , 2 and 0.4  log10  cfu / cm2 , resp . ) ; a 1  log10  cfu / cm2 reduction in p. aeruginosa biofilm formation was observed at 36  hr . 
 24  hr of 500  a dc decreased c. albicans biofilm formation on teflon discs by 2  log10  cfu / cm2 . 
 no reduction in p. acnes biofilm formation was observed . 
 1 and 2  log10  cfu / cm2 reductions in e. coli and s. epidermidis biofilm formation on titanium discs , respectively , were observed with 12  hr of exposure to 500  a . 
 electrical current is a potential strategy to reduce biofilm formation on medical biomaterials . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion