infections of medical prosthetic devices in trauma and orthopaedic surgery are difficult to treat with conventional therapies like antibiotics and surgery , and can lead to severe consequences , such as removal of the implant with functional loss of the affected limb [ 18 , 26 ] . the most frequently isolated bacteria causing these implant- and patient - related infections are s. aureus and the coagulase - negative s. epidermidis [ 1 , 11 ] .
less common are pseudomonas aeruginosa , klebsiella oxytoca , enterococcus faecalis , and enterobacter cloacae . in prosthesis - associated infections ,
the causative bacteria form biofilms composed mainly of a polysaccharide matrix . the most important difference between bacteria in biofilms and planktonic microorganisms that can explain the alteration in antibiotic resistance is the metabolic state of biofilm bacteria .
the genotype and phenotype of bacteria in biofilms and the protein expression are altered and the oxygen concentration in biofilms is limited .
these changes result in metabolic quiescence , which could be overcome by providing alternative electron acceptors of fermentable substrates [ 3 , 24 ] .
current antimicrobial therapies frequently can not adequately treat biofilm - associated infections [ 8 , 9 , 24 , 26 ] and therefore it is important to develop new treatment approaches to reduce biofilm formation ( bf ) on biomaterials .
the most important development in the treatment of infectious diseases during the last century was the discovery of penicillin by fleming in 1928 . at approximately the same time , maggot debridement therapy ( mdt ) was introduced by william s. baer , an orthopaedic surgeon who worked at johns hopkins hospital in baltimore , maryland .
although baer showed the beneficial effects of maggots of the lucilia sericata , mdt essentially disappeared after the discovery of penicillin , which could control numerous types of severe infections . because of increasing antibiotic resistance , larval therapy was reintroduced in the 1980s as a treatment for severely infected wounds
maggots now are used successfully in various patient clinics [ 13 , 25 ] . in 2004 ,
mdt was approved by the us food and drug administration ( 510[k ] # 33391 ) . in our clinical practice ,
we use maggots captured in small nylon bags , consisting of a 2-mm thin foam layer of polyvinyl alcohol ( biobag ; biomonde gmbh , barsbttel , germany ) .
we have observed healing of wound infections suspect for bf using maggots in such bags . in an earlier pilot study
however , the current research investigated , for the first time , the influence of a range of concentrations of es on mature 7-day - old biofilms formed by new , commonly isolated bacterial species on different biomaterials .
if maggot es reduce bf of bacteria that frequently are isolated from biofilm - associated infections , es could provide us with a new treatment possibility .
therefore , we asked ( 1 ) whether five bacterial species ( s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae ) , were able to form biofilms on polyethylene ( pe ) , titanium ( ti ) , and/or surgical stainless steel ( sss ) , and if so , whether ( 2 ) the quantity of bf differed among the bacterial species , ( 3 ) the quantity of bf depended on the biomaterial surface , and ( 4 ) the quantity of biofilms of the biofilm - forming bacteria differed among 3 , 5 , 7 , or 9  days of incubation time
. finally , we hypothesized ( 5 ) sterile maggot es could decrease the growth of the bacterial biofilms on the commonly used biomaterials sss , ti , and pe .
first , we investigated bf of five different , clinical isolated species on comb - like models of pe , ti , and sss suspending in a 96-well microtiter plate with nutrient medium and bacteria ( fig .  1 ) .
two of the five tested bacterial species formed visible biofilms that could be quantified and these bacteria were used to investigate the second to fifth research questions .
the dependent variable in the second to fifth research questions was the quantity of formed biofilm ; bf was compared among the two biofilm - forming species , among the different biomaterials sss , ti , and pe and between various incubation times on days 5 , 7 , and 9 .
bf  =  biofilm formation ; pe  =  polyethylene ; ti  =  titanium ; sss  =  surgical stainless steel ; es  =  excretions and secretions .
bf  =  biofilm formation ; pe  =  polyethylene ; ti  =  titanium ; sss  =  surgical stainless steel ; es  =  excretions and secretions .
the custom - made , sterile comb - like models of regularly available orthopaedic implant materials , consisting of eight prongs were produced according to our design .
these models of sss , ti , and pe ( litos gmbh , hamburg , germany ) were made to fit in a 96-well microtiter plate .
s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae all were isolated from infected prosthetic devices of patients in our clinic .
briefly , s. aureus and s. epidermidis were grown overnight in tryptic soy broth ( tsb ) medium ( becton , dickinson & co , franklin lakes , nj ) and k. oxytoca , e. faecalis , and e. cloacae in brain heart infusion ( bhi ) medium ( becton , dickinson & co ) at 37c .
a stationary phase culture was made in either tsb or bhi to a density of mcfarland 0.5 corresponding with 1.5    10 colony - forming units ( cfus ) per l .
the inoculum was controlled by inoculating nutrient agar plates ( biotrading benelux bv , mijdrecht , the netherlands ) with 10-l volumes of serial 1:10 dilutions of the bacterial suspension and counting the formed cfus after incubation overnight at 37c .
the bacterial suspension was diluted to a final inoculum of 2.5    10 bacteria per ml and a volume of 100 l of this suspension was pipetted into 24 wells of a sterile 96-well flat - bottomed microtiter plate ( greiner bio - one bv , monroe , nc ) .
one comb of each biomaterial was placed in eight of the wells and four of these microtiter plates with combs were incubated for 3 , 5 , 7 , or 9  days at 37c to form biofilms on the devices .
control combs were suspended in nutrient medium . after the incubation time , each comb was washed under slow - running distilled water at room temperature for 30 seconds to remove planktonic cells .
all comb models were stained for 15 minutes with 1% crystal violet , based on the methods of pitts et al .   and stepanovic et al . .
each comb was suspended in eight wells of a new microtiter plate filled with 270 l ethanol absolute to absorb the crystal violet .
the plates were left for 48 hours at room temperature to allow the stained biofilm bacteria to elute with ethanol .
all stained biofilm bacteria are soluble in ethanol , however the biofilm on pe could not be eluted entirely in ethanol ( maximum time tested was 96 hours ) , but we standardized the time of elution to 48 hours to guarantee reliable interpretation of the results .
after gentle shaking of the microtiter plate , a volume of 150 l of the ethanol and crystal violet solution was pipetted into a new plate and the optical density ( od ) of this solution was measured at a wavelength of 595  nm and is representative for the quantity of biofilm formation .
biofilms of each bacterial species were formed at least in octuplicate , so that one comb was tested with each of the eight prongs suspended in a separate well with nutrient medium and bacterial suspension .
sterile maggot es of instar-3 larvae ( biomonde gmbh ) were collected as described previously .
briefly , the maggots were incubated in sterile tubes for 1 hour at 35c in darkness .
then , es were removed by pipette , divided in aliquots , and stored at 80c .
one es pool was collected from at least 1000 maggots , which resulted in approximately 1200 l es per hour .
the protein concentration was determined using the pierce bicinchoninic acid protein assay kit ( pierce biotechnology , rockford , il ) .
absolute od values of the control bf after 7  days without es are reported in results . to compute absolute and relative od , we first subtracted the material - specific background absorbance .
the measured od values of the biofilm experiments were converted to percentages using the following formula:1\documentclass[12pt]{minimal } 
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				 \begin{document}$$ \left [ { { \text{od}}\,\left ( { \text{biofilm } } \right ) \times 100\% } \right]/{\text{od}}\,\left ( { \text{control } } \right ) $ $ \end{document}where od ( biofilm ) refers to either bf measured on day 3 , 5 or 9 ( without es ) or bf measured after the addition of increasing concentrations of es and where od ( control ) is the bf after 7  days on the negative control prongs . in all experiments ,
biofilms were allowed to form on 13 combs of each of the materials for 7  days , two other combs were suspended in nutrient medium without bacteria as negative controls and represented the material - specific background absorbance .
two other combs were suspended in a new microtiter plate with medium and incubated another 24 hours to allow additional growth of the biofilm ( the 8-day - old biofilms ) ; nine combs were suspended in a logarithmic range of concentrations of es , diluted in nutrient medium : 0.31 , 0.93 , 2.78 , 8.33 , 25 , and 75  g es per well .
all wells were incubated another 24 hours at 37c and then we quantified the biofilm as described previously .
biofilm inhibition ( or prevention ) by es was defined as bf that was lower than 8-day - old biofilms which grew further after day 7 .
bf that was lower than 7-day - old bf after incubation in es was defined as biofilm breakdown .
the first research question was not analyzed statistically because the results were obtained by observation only .
for the second and the last research questions , student s t test for independent groups was used .
the means of the 7-day - old biofilms of the biofilm - forming bacterial species were compared to analyze whether bf depended on the bacterial species .
furthermore , the mean bf with es ( per concentration ) and the mean of the 8-day - old biofilm without es were compared . for the third research question ,
one - way anova was used and followed by the least significance difference post hoc test .
all analyses  met the assumption of homogeneity of variances ( levene s statistic : p  >  0.05 ) . to analyze whether the bf depended on the material surface ,
the mean quantity of a 7-day - old biofilm was compared among the materials for each bacterium .
the mean quantities of biofilms formed on days 5 , 7 , and 9 for each bacterium were analyzed using repeated - measures anova followed by the bonferroni correction to investigate whether the bf changed among these 3  days .
all analyses met the assumption of sphericity ( mauchly s test : p  >  0.05 ) .
all statistical analyses were performed using spss for windows , version 14.0 ( spss inc , chicago , il ) .
all tested bacterial species were able to form visible biofilms on pe , sss , and ti , however biofilms of k. oxytoca , e. faecalis , and e. cloacae could barely be observed , in contrast to the clearly visible biofilms of s. aureus and s. epidermidis . quantities of biofilm formed by k. oxytoca , e. faecalis , and e. cloacae were insufficient to be measured .
therefore , we focused on bf by s. aureus and s. epidermidis and continued the experiments with these two bacterial species .
no differences in quantity were seen on pe ( p  =  0.582 ) , sss ( p  =  0.051 ) , or ti ( p  =  0.178 ) ( fig .  2).fig . 
2a  bthe ods reflect the quantities of 7-day - old biofilms produced by ( a ) s. aureus and ( b ) s. epidermidis on pe , sss , and ti and answer the second and the third research questions , respectively , whether the quantity of bf differed among s. aureus and s. epidermidis and whether bf depended on the biomaterial surface .
both bacteria formed equal quantities of bf and showed pe had the highest bf followed by sss and ti with the lowest quantity of biofilm . * for these comparisons
the ods reflect the quantities of 7-day - old biofilms produced by ( a ) s. aureus and ( b ) s. epidermidis on pe , sss , and ti and answer the second and the third research questions , respectively , whether the quantity of bf differed among s. aureus and s. epidermidis and whether bf depended on the biomaterial surface .
both bacteria formed equal quantities of bf and showed pe had the highest bf followed by sss and ti with the lowest quantity of biofilm .
bf by s. aureus was greater on pe ( od595 0.315 ) than on sss ( od595 0.190 ) ( p  =  0.024 ) and ti ( od595 0.0386 ) ( p  <  0.001 ) ( fig . 
2a ; table  1 ) and the same trend was seen for bf by s. epidermidis ( pe : od595 0.248 , sss : od595 0.104
[ p  <  0.001 ] ; ti : od595 0.0578 [ p  <  0.001 ] ) ( fig .  2b ; table  2).table  1optical density ( 595  nm ) for a 7-day - old biofilm by s. aureusmaterial7-day - old biofilmbackground absorbancepolyethylene0.3150.167surgical stainless steel0.1900.101titanium0.03860.0801table  2optical density ( 595  nm ) for a 7-day - old biofilm by s. epidermidismaterial7-day - old biofilmbackground absorbancepolyethylene0.2480.162surgical stainless steel0.1040.0800titanium0.05780.0740 optical density ( 595  nm ) for a 7-day - old biofilm by s. aureus optical density ( 595  nm ) for a 7-day - old biofilm by s. epidermidis s. aureus and s. epidermidis were observed for bf during 3 , 5 , 7 , and 9  days . on day 3 , biofilms of both bacteria occurred in insufficient quantity to be measured . on ti ,
pe , and sss , most biofilm was formed by s. aureus on day 7 and day 9 ( day 7 versus day 9 [ all p values    0.198 ] ) ( fig .  3a ) .
the amount of biofilm formed by s. epidermidis on pe was equal at all times of incubation ( day 5 versus day 7 [ p  =  1.000 ] , day 5 versus day 9 [ p  =  0.074 ] ) ( fig . 
the greatest quantity of biofilms was formed by s. epidermidis on sss and ti ( day 7 versus day 9 [ all p values    0.141 ] ) ( fig . 
3a  bthe graphs show the amount of bf after various incubation times and answer the fourth research question , whether quantity of biofilms differed among 3 , 5 , 7 , or 9  days .
( a ) biofilms by s. aureus kept on growing until day 9 on pe , ti , and sss .
( b ) the largest amount of biofilm by s. epidermidis was formed within 7 to 9  days on sss and ti .
* for these comparisons all p values  <  0.001 ; all other comparisons were similar .
the graphs show the amount of bf after various incubation times and answer the fourth research question , whether quantity of biofilms differed among 3 , 5 , 7 , or 9  days .
( a ) biofilms by s. aureus kept on growing until day 9 on pe , ti , and sss .
( b ) the largest amount of biofilm by s. epidermidis was formed within 7 to 9  days on sss and ti .
the addition of es to 7-day - old biofilms of s. aureus and s. epidermidis reduced these biofilms compared with biofilms without addition of es on all tested biomaterials ( figs .  4 , 5 ) .
the biofilm of s. aureus on sss and pe decreased for all concentrations of es to minimums of 40.5% on sss for 0.31  g es per well ( p  <  0.001 ) and 39.2% on pe for 0.93  g es per well ( p  < 
0.001 ) as compared with the control biofilm without es ( fig .  4a  b ) .
on ti , only 8.33  g es per well showed a reduction ( p  =  0.005 ) of biofilm ( fig . 
biofilms of s. epidermidis formed on sss and pe also were reduced for all concentrations of es ( fig . 
biofilms decreased to minimums of 32.3% on sss for 25  g es per well ( p  <  0.001 ) and 7.6% on pe for 8.33  g es per well ( p  <  0.001 ) compared with the control .
on ti , concentrations of 0.31  g es per well ( p  <  0.001 ) and 0.93  g es per well ( p  =  0.021 ) showed less biofilm ( fig . 
4a  cthese graphs compare the mean bf produced by s. aureus with es and the mean control bf after 8  days without es and answer the last research question whether es reduced biofilms produced by s. aureus .
all values lower than the 8-day - old biofilm showed biofilm reduction . ( a )
the biofilm on sss was decreased for all concentrations with a maximum of 59.5% for 0.31  g es per well .
( b ) the biofilm on pe also was reduced for all concentrations with a maximum of 60.8% for 0.93  g es per well .
( c ) on ti , only 8.33  g es per well showed biofilm reduction . * for these comparisons all p values  <  0.001 ; all other comparisons were similar.fig . 
graphs compare the mean bf produced by s. epidermidis with es and the mean control bf after 8  days without es and answer the last research question whether es reduced biofilms produced by s. epidermidis .
( a ) biofilm on sss was reduced for all concentrations with a maximum of 67.7% for 25  g es per well .
( b ) biofilm on pe also was decreased for all concentrations with a maximum of 92.4% for 8.33  g es per well .
( c ) on ti , only 0.31  g es per well and 0.93  g es per well showed biofilm reduction . * for these comparisons all p values  <  0.001 ; all other comparisons were similar .
these graphs compare the mean bf produced by s. aureus with es and the mean control bf after 8  days without es and answer the last research question whether es reduced biofilms produced by s. aureus .
( a ) the biofilm on sss was decreased for all concentrations with a maximum of 59.5% for 0.31  g es per well .
( b ) the biofilm on pe also was reduced for all concentrations with a maximum of 60.8% for 0.93  g es per well .
( c ) on ti , only 8.33  g es per well showed biofilm reduction . * for these comparisons all p values  <  0.001 ; all other comparisons were similar .
these graphs compare the mean bf produced by s. epidermidis with es and the mean control bf after 8  days without es and answer the last research question whether es reduced biofilms produced by s. epidermidis .
( a ) biofilm on sss was reduced for all concentrations with a maximum of 67.7% for 25  g es per well .
( b ) biofilm on pe also was decreased for all concentrations with a maximum of 92.4% for 8.33  g es per well .
( c ) on ti , only 0.31  g es per well and 0.93  g es per well showed biofilm reduction . * for these comparisons
biofilm - associated infections related to implants can not be treated easily and frequently the implanted device must be removed with severe consequences for the patient . therefore , new approaches for these kinds of infections are needed .
previously reported maggot es reduced bf of p. aeruginosa . in the current study , we investigated whether other bacterial species , namely , s. aureus , s. epidermidis , k. oxytoca , e. faecalis , and e. cloacae , were able to form biofilms on pe , ti , and sss , and if so , whether the quantity of bf differed between the bacterial species , whether the quantity of bf depended on the biomaterial surface , and whether the largest amount of bf was formed after 3 , 5 , 7 , or 9  days . finally , we hypothesized sterile maggot es could reduce bf on the biomaterials .
first , we performed only a crystal violet assay as a standard method to measure biofilms , but other methods could be used in vitro   and in vivo .
we modified the crystal violet assay to our design with the combs of pe , ti , and sss , however the assay was based on the commonly used methods of pitts et al .   and stepanovic et al . .
second , every experiment was performed with one pool maggot es , but we did not compare the effectiveness between the various pools . the use of different pools will not likely influence the conclusions of this research , because in an earlier study , the effectiveness of es was compared and similar results of biofilm reduction occurred using various pools of es .
for example , es were collected under standard conditions in darkness , had an acidity of ph 8 , and were incubated at 35c .
furthermore , the protein concentration from each collected es was measured to standardize the various pools .
the biomaterials were produced according to specifications for common implants for patients and all bacterial species were isolated from implant infections of patients in our clinic .
therefore , we believe these results were representative and sterile maggot es reduce not only biofilms of p. aeruginosa   but also existing and mature biofilms of s. aureus and s. epidermidis .
these bacterial species were expected to form biofilms , as they all were isolated from device - related infections .
the possible biofilm - forming capacities of the five tested species on medical devices have been described by others [ 4 , 10 , 20 ] .
bf by k. oxytoca , e. faecalis , and e. cloacae was insufficient to be quantified .
nutrient media other than bhi were used to test bf by these bacteria but did not show bf .
the highest amount of biofilm was formed by s. aureus and s. epidermidis , which are the most frequently isolated pathogens from orthopaedic surgical wounds .
.   described equal bf but a stronger adherence to material surfaces by s. epidermidis .
the quantity of bf depended on the biomaterial surface , however published studies show differing results .
.   found that the kind of metal , ti or sss , plays a minor role in the quantity of bf , whereas mackintosh et al .   reported biomaterial surface characteristics
we found ti had the least amount of biofilm in vitro formed by s. aureus and s. epidermidis . in a previous study ,
therefore , we conclude that the quantity of bf depends on the biomaterial and on the bacterial specie creating the biofilm .
after 7  days of bf , the highest amount of biofilm was formed . in the first 7  days , the nutrient medium in the microtiter plate was not changed , because the biofilm bacteria were still viable as the biofilm kept growing .
new nutrient medium with or without es was added after 7  days of incubation to maintain the viability of the bacteria .
the average time for biofilm maturation is 6 to 8  days [ 10 , 22 ] .
although the 7-day - old biofilms in this study were not examined by confocal microscopy , their maturation process was more advanced than that of the previously tested 24-hour - old pao1-biofilms which were decreased by es .
thus , this research shows more evidence for the therapeutic potential of es for treatment against biofilm - associated infections which involve mature biofilms .
the biofilm - decreasing capacity of maggot es seems optimal in low protein concentrations from 0.31 up to 8.33  g es per well ( 3.1 to 83.3  g es per ml ) . in previous studies ,
protein concentrations of as much as 20  g es per well were reported as the most effective concentrations for biofilm reduction [ 6 , 28 ] .
there are several possible explanations for the fact that the influence of es at higher concentrations than 20  g es per well declined .
we hypothesize a cofactor necessary to regulate the process was depleted   or there is a nonenzymatic interaction . in future studies
concentration ranges less than 0.31  g es per well will be tested to examine the lower limit amount of es causing biofilm reduction .
previous research suggests maggot es do not possess direct bactericidal or bacteriostatic activity against planktonic organisms .
therefore , es are not expected to reduce biofilm by destroying the bacteria in the matrix . however , the influence of es on biofilm bacteria will be investigated in future studies , by examination of total viable counts of bacteria .
maggot es broke down existing biofilms , because all measurements were lower than the control bf after 7  days .
however , they also seem to inhibit additional growth of bf as es reduced bf to less than 100% in the experiments with a greater amount of biofilm on day 8 compared with day 7 ( fig . 
4a  b ) . therefore , potential therapeutic use of es could be prevention or inhibition of bf , eg , flushing surgical wounds before closing and treatment against existing biofilm - associated infections , especially on orthopaedic medical devices .
further research is necessary to clarify the mechanism(s ) of biofilm reduction by maggot es and identify the substance(s ) in es responsible for these reductions .
 backgroundbiofilm - associated infections in trauma surgery are difficult to treat with conventional therapies . therefore , it is important to develop new treatment modalities . 
 maggots in captured bags , which are permeable for larval excretions / secretions , aid in healing severe , infected wounds , suspect for biofilm formation . 
 therefore we presumed maggot excretions / secretions would reduce biofilm formation.questions/purposeswe studied biofilm formation of staphylococcus aureus , staphylococcus epidermidis , klebsiella oxytoca , enterococcus faecalis , and enterobacter cloacae on polyethylene , titanium , and stainless steel . 
 we compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times . 
 maggot excretions / secretions were added to existing biofilms to examine their effect.methodscomb-like models of the biomaterials , made to fit in a 96-well microtiter plate , were incubated with bacterial suspension . 
 the formed biofilms were stained in crystal violet , which was eluted in ethanol . the optical density ( at 595  nm ) of the eluate was determined to quantify biofilm formation . 
 maggot excretions / secretions were pipetted in different concentrations to ( nonstained ) 7-day - old biofilms , incubated 24 hours , and finally measured.resultsthe strongest biofilms were formed by s. aureus and s. epidermidis on polyethylene and the weakest on titanium . 
 the highest quantity of biofilm formation was reached within 7  days for both bacteria . 
 the presence of excretions / secretions reduced biofilm formation on all biomaterials . 
 a maximum of 92% of biofilm reduction was measured.conclusionsour observations suggest maggot excretions / secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials . 
Introduction
Materials and Methods
Results
Discussion