microbial control in the endodontic system represents a challenge to the solution of refractory infections when chemical and surgical procedures do not provide the desired response for control of infection .
the chemomechanical preparation used during endodontic therapy is the main factor responsible for eliminating the microorganisms lodged into the root canal system20 . in most clinical situations ,
however , some cases demand the application of intracanal medication as an adjuvant therapy to resolve the infection .
this occurs because microorganisms may colonize the endodontic system , demanding deeper and more guaranteed therapeutic action . in these cases , intracanal medication not only plays the role of inflammatory reaction moderator , but is also imperative to combat the remaining infection . in most cases ,
this resistance is related to microbial capacity to remain in a latent phase , at a low metabolic rate for a long period of time .
however , environmental changes , such as coronal leakage , may lead to activation of these microorganisms and their consequent proliferation6,14 .
associations of antimicrobial agents have been proposed with the goal of increasing the spectrum of action and combating the persistent infection9,11,17 .
the choice of vehicle associated with intracanal medication is also of paramount importance , as they are responsible for transporting the drug to the interior of the root canal systems and for the speed of its diffusion24 .
the purpose of this study was to investigate the antimicrobial action of ciprofloxacin hydrochloride and metronidazole , and the vehicles polyethylene glycol and natrosol , either alone or associated , supported by the fact that intracanal medication is an important adjuvant of the endodontic treatment protocol .
twenty - three microbial strains were selected from the american type culture collection ( atcc ) and the university of taubat culture collection ( ccut ) ( table 1 ) . the susceptibility of the microorganisms to the antimicrobial agents metronidazole ( henrifarma , china ) and ciprofloxacin hydrochloride ( pharmanostra , usa ) and to the vehicles polyethylene glycol ( pm 1000 ) and natrosol , was assessed , either alone and in the following associations : a ) ciprofloxacin and metronidazole ; b ) ciprofloxacin and natrosol ; c ) ciprofloxacin and polyethylene glycol ; d ) metronidazole and natrosol ; e ) metronidazole and polyethylene glycol ; f ) ciprofloxacin , metronidazole and natrosol ; and , g ) ciprofloxacin , metronidazole and polyethylene glycol .
the antimicrobial agents were submitted to filtration ( 0.22 m , millipore membrane ) and the vehicles were sterilized by gamma radiation ( co , gammacell , atomic energy of canada limited , canada ) .
the mller - hinton agar ( difco , detroit , mi , usa ) dilution method was used to determine the minimum inhibitory concentration ( mic)15 .
culture media were prepared , adding antimicrobial agents in two - fold serial dilutions , obtaining final concentrations of 16 to 0.25 g / ml of medium . for the vehicles ,
culture media containing 50 , 45 , 40 , 35 , 30 and 25% were used . for the associations ,
the substances were added to the culture media at the same concentrations and percentages used for the substances tested separately , in a total of 292 combinations . for each test ,
the selected microorganisms were plated in mller hinton agar and incubated at 37c for 24 hours . after growth and morphologic confirmation in smears stained by gram method ,
suspensions were prepared with isolated colonies and sterile saline in accordance with tube 0.5 ( approximately 1.5  10 cells / ml ) of mcfarland scale , for the bacteria , and tube 2 ( approximately 10 cells / ml ) for yeasts ( recommendation of the national committee for clinical laboratory standards12,13 ) .
next , aliquots of the suspensions were placed into the wells of the steers replicator , and then plated in the culture media .
the plates were incubated at 37c for 24 hours and the occurrence of microbial growth was evaluated .
the mic was defined at the lowest concentration of the drug that inhibited microbial growth .
minimum inhibitory concentration results for the antimicrobial agents ciprofloxacin and metronidazole , and for the vehicles ( natrosol and polyethylene glycol ) are shown in table 2 .
r = resistant at the highest concentration tested ( 16 lg / ml or 50% ) ; s = sensitive at the lowest concentration tested ( 0.25 g/ ml or 25% ) ;  interval of concentration of the tested products 
 table 3 shows the results of the association of ciprofloxacin with the vehicles .
ciprofloxacin / natrosol association partially increased the effects of the products , also presenting effects against candida species .
s = sensitive at the lowest concentration tested ( 0.25 g / ml or 25% ) ;  interval of concentration of the tested products 
 table 4 shows the results of the association of metronidazole with the tested vehicles .
metronidazole/ polyethylene glycol association inhibited all microbial strains , using low concentrations of the antimicrobial agent , as well as low percentages of polyethylene glycol .
resistant at the highest concentration tested ( 16 lag / ml or 50% ) s  sensitive at the lowest concentration tested ( 0.25 g / ml or 25% )  interval of concentration of the tested products the mic values of ciprofloxacin / metronidazole association and these with the vehicles are expressed in table 5 .
the association of ciprofloxacin with metronidazole presented lower mic for the bacteria than ciprofloxacin alone , except for escherichia coli .
polyethylene glycol added to the formulation eliminated all bacteria , at the lowest concentration of antimicrobial agents and vehicles .
the performance of ciprofloxacin / metronidazole / polyethylene glycol association was equivalent or lowest to the association ciprofloxacin / polyethylene glycol .
r = resistant at the highest concentration tested ( 16 lag / ml or 50% ) ; s = sensitive at the lowest concentration tested ( 0.25 g/ ml or 25% ) ;  > interval of concentration of the tested products . in all plates of culture media
in which the antimicrobial agents and vehicles were not added ( controls ) , there was growth of all microbial strains .
these difficulties are generally related to the anatomic characteristics of the root canal system7 , resistance to antimicrobial agents3,6 and the presence of apical biofilm18,25 . for cases of resistance to antimicrobial agents ,
the use of alternative medications , or the combination of antimicrobial agents is suggested to increase the spectrum of action and act particularly on the facultative anaerobic microorganisms . in endodontics ,
antimicrobial agents are used topically , due to the absence of blood irrigation in the root canal system at the time of therapy . in most cases ,
intracanal medication is used in aqueous solution , allowing the system to be well filled . however , the capacity to remain there and to have a prolonged action is limited and it is in contact with the healthy or contaminated periodontal fluid .
the vehicle associated with the antimicrobial agents has a strict relationship with the time of action of the antimicrobial agent19 , penetration of the medication in the root canal system2,24 , in addition to the dissociation of the principle of the drugs5,8 .
the results of the present study demonstrated , although metronidazole had no action against the tested microorganisms , which was expected , the ciprofloxacin/ metronidazole association resulted in a positive interaction , that is , there was synergism in this proposed combination .
slots , et al.21 ( 1990 ) found that the ciprofloxacin/ metronidazole association was more effective against enterobacteria collected from periodontal pockets than ciprofloxacin used alone .
all mic values for this association were lower than those of ciprofloxacin used alone or combined with metronidazole .
another advantage of the association ciprofloxacin / polyethylene glycol was the effective action on yeasts resistant to the other formulations .
all tested bacteria were sensitive to the association of ciprofloxacin and metronidazole with the vehicle polyethylene glycol , while c. albicans and c. tropicalis were eliminated with this vehicle in concentrations equal to or lower than 35% by volume of the solution .
metronidazole at 16 g / ml concentration and lower , associated with polyethylene glycol at a percentage of 30 to 50% , was responsible for eliminating all strains , including the yeasts .
as the volume of polyethylene glycol in the culture media was reduced , the number of resistant strains increased .
the results obtained were expected , since the action spectrum of metronidazole is selective for anaerobic bacteria .
therefore , the importance of the results is based on the knowledge of the interaction of metronidazole with the other studied substances .
the effectiveness of the association of ciprofloxacin , metronidazole and natrosol shows a trend towards 100% sensitivity , as the concentration of the antimicrobial agents increases .
for this combination of substances , the highest mic was 4 g / ml and 50% of natrosol .
similarity of the results with the group without the addition of metronidazole was noted . as metronidazole did not present action against the facultative anaerobic bacteria tested in this experiment , further studies are necessary with other species of microorganisms to assess the feasibility of its application .
the vehicle polyethylene glycol was capable of eliminating 100% of the microorganisms tested with mic 40% . as the volume of polyethylene glycol was diminished ( below 30% ) , some microorganisms were capable of growing , particularly c. albicans and c. tropicalis .
it is interesting to point out that polyethylene glycol ( pm 1000 ) is the vehicle that allowed greater dentinal penetration of the association calcium hydroxide , ciprofloxacin and metronidazole24 .
when studied separately , natrosol prevented the growth of some microorganisms , particularly when administered in higher percentages .
however , at no time was it capable of inhibiting the growth of all the microorganisms .
the susceptible of the microorganisms to the vehicle polyethylene glycol was similar to that of the associations used in the present study .
this suggests that the antimicrobial action demonstrated was exercised by the action of this vehicle . this may be related to the hydrophilic property of polyethylene glycol . removing water from the culture media ,
the vehicle possibly did not allow microbial growth , as a certain amount of water is required to enable them to multiply and develop .
it is suggested that further studies be conducted , to find whether this effect occurs inside the root canal system .
the microbial concentration used for the suspension was determined by using the standard 0.5 on the mcfarland scale for bacteria and 2 for the yeasts , as the latter present a greater cellular volume12,13,15,18 .
it must be emphasized that almost all clinical situations are solved by performing the simple endodontic technique , which complies with the conditional factors of good preparation of the root canal system . in spite of the usual therapeutic protocol ,
the medication here proposed can be indicated when the routine alternatives are not able of producing the desired effects .
analysis of the results obtained by the application of the experimental model allowed the following conclusions :  ciprofloxacin showed antimicrobial action against all tested microbial strains , and its association with metronidazole was synergic . 
 ciprofloxacin associated with polyethylene glycol was the most effective combination for reducing the tested bacteria and yeasts .
 in some situations , endodontic infections do not respond to therapeutic protocol . in these cases , 
 it is suggested the administration of an alternative intracanal medication that presents a wide spectrum of action and has an in - depth effect on the root canal system . 
 the purpose of this study was to assess the antimicrobial action of ciprofloxacin , metronidazole and polyethylene glycol and natrosol vehicles with different associations and concentrations . 
 the minimum inhibitory concentration ( mic ) was determined by using the agar dilution method . the culture media ( mller - hinton agar ) 
 were prepared containing antimicrobial agents at multiple two - fold dilutions of 0.25 to 16 g / ml , and with the vehicles at the concentrations of 50 , 45 , 40 , 35 , 30 and 25% . 
 twenty - three microbial strains were selected for the study . 
 metronidazole was not capable of eliminating any of the tested microorganisms . 
 the association of ciprofloxacin with metronidazole resulted in a reduction of the mic . 
 the vehicle polyethylene glycol inhibited the growth of 100% of the tested strains , while natrosol inhibited 18% of the strains . 
 ciprofloxacin formulations with polyethylene glycol presented better effects than those of formulations to which metronidazole was added . 
 it was possible to conclude that ciprofloxacin presented antimicrobial action against all tested bacterial strains , and its association with metronidazole was synergic . 
 the vehicle polyethylene glycol showed antimicrobial effect and the ciprofloxacin / polyethylene glycol association was the most effective combination for reducing the tested bacteria and yeasts . 