TR201808398A2 - New Combined Use for Inhibition and Eradication of Biofilm Production - Google Patents

Abstract

The invention relates to the use of nisin bacteriocin alone and in combination with chlorhexidine (CHX), sodium hypochlorite (NaOCl) and ethylenediamine tetra acetic acid (EDTA) for the inhibition and eradication of biofilm production of Enterococcus faecalis.

Description

DESCRIPTION New Combined Use in Inhibition and Eradication of Biofilm Production The invention is aimed at inhibiting the biofilm production of Enterococcus faecalis bacteria and In the eradication of nisin bacteriocin alone and chlorhexidine (CHX), sodium combined with hypochlorite (NaOCl) and ethylenediamine tetraacetic acid (EDTA) It is related to.

State of the art Various agents are used to combat bacterial biofilm structures, but this The fact that the agents are especially harmful to human health, new and preferably natural agents makes it mandatory. Antibiotic therapy is the most effective treatment for biofilm infections. is the most used method. Antibiotics to combat bacterial infections As a result of uncontrolled and frequent use, resistance to antibiotics has started to develop. And this brought new problems with it. For these reasons, with biofilm structures determination of new agents in the fight or the doses of antibiotics used It is important to determine the auxiliary agents that will ensure the reduction of scientific and As a result of this invention designed in line with technological requirements; clinic all over the world and E. faecalis biofilm structures, which has become a serious problem in the industrial sense. It is a new and effective method in the prevention of the formation of biofilms and the eradication of existing biofilms. A combination of green biotechnological agents was determined.

The existence of numerous patents on combating bacterial biofilm structures, shows its importance. For example; In the patent application numbered CN107108702A; when are you based compounds and their use in the treatment of bacterial infections. has been mentioned. Various modifications on the form of niche available in this patent application As a result, the new forms have a higher level of activity compared to the old forms. shown has not been determined. These agents are used to combat bacterial infections. use has not been determined. However, the Enterococcus used in this invention and especially Nisin+CHX, Nisin+NaOCl and There are no studies on Nisin+EDTA application.

Technical problems that the invention aims to solve In previous techniques, disinfectants are commonly used alone. Resistance may develop over time against disinfectants and also in tissues. may cause irritation. Human health and consumption by FDA with this invention It is accepted as safe for clinical and industrial purposes. Enterococcus faecalis biofilm in combination with commercial disinfectants at varying rates. On the inhibition of the formation of structures and the eradication of mature biofilm structures has not been found to be effective. In this way, the use of these disinfectants together with nisin As a result, disinfectants are effective even at much lower concentrations. has not been determined. In addition, as a result of combining nisin with various disinfectants, biophilin effective disinfectant to be applied in the prevention and eradication of By reducing the concentration of the disinfectants in question, human health and minimizing the negative effects on the environment.

Description of the invention Biofilm structures are the predominant form of microbial growth. only bacteria Many types of microorganisms, such as biofilm structure in response to environmental stress conditions forms. Biofilm structures, which can consist of a single or multiple bacterial species, are classically against sanitation, disinfection and sterilization agents and methods, show a much higher level of resistance than planktonic forms. biofilm These features of their structure make them a major source of clinical and industrial contamination. has not made it. In the fight against microbial infections and contaminations in the prevention of the formation of biofilm structures of the classical agents used and Inadequate eradication of these agents, as well as the applied material, It also has negative effects on health and the environment, in the fight against biofilm structures. It has made it necessary to develop green biotechnological agents and methods. First with pathogenic microorganisms in all areas of life, including medicine and food industry. The struggle continues intensely. The chemical used in this fight (chlorhexidine digluconate, hydrogen peroxide, sodium hypochlorite, formaldehyde, quaternary ammonium compounds, formaldehyde, isopropyl alcohol, ethyl alcohol, benzalkonium chloride and such as sodium dichloro isocyanurate), physical (such as pressure, temperature, sonication and radiation) and biological (enzyme-based detergents, viruses and bacteriocins, antibiotics and other efficacy of agents (such as microbial metabolites) on the basis of planktonic forms. determination and on this basis; disinfection, sanitation and sterilization Due to the development of biofilm methods, biofilm forms are associated with pathogenicity and contamination. factor has gained increasing importance. Therefore, clinical and in industrial microbiology, prevention of microbial contaminations and infections The basic approach in its eradication and eradication is the effects of control agents on biofilm structures. was based on the axis of selection on the basis of activities. of a microorganism the same biofilm form against all of the control agents outlined above. is much more resistant than the planktonic form of the microorganism. known. On the other hand, as a result of intensive and misuse of antibiotics, multiple promoting the development of drug-resistant strains of microorganisms, classical physical and chemical disinfection and sterilization agents as well as human and animal detecting many side effects against health, microbial infection and A new fight against contamination that is environmentally friendly and does not threaten consumer health. has led to the necessity of developing agents and methods.

Detailed description of the invention: Microbial biofilms; individual (planktonic) cells at interfaces, each other or After attaching to the substrates, they are bonded with an extracellular polymeric matrix they produce. occur as a result of their encirclement. The cells that make up the biofilms are very much in these structures. Because they show similar developmental physiology and genetic expression to cellular forms, They acquire structural and functional properties very different from planktonic forms. First Many microorganisms, especially bacteria, are susceptible to environmental stress conditions. It has been determined that they form biofilm structures in response. Mature biofilm structures; organized in such a way as to allow the dispersal of metabolites, nutrients, and waste They are highly organized, monolayer or three-dimensional structures. also above as mentioned, bacterial biofilm that can be composed of a single or multiple bacterial species. structures; against conventional sanitation, disinfection and sterilization agents and methods, show a much higher level of resistance than the planktonic forms of these species. This characteristics, biofilm structures, continuous clinical and industrial has become the main source of contamination. On the other hand, like enterococci Some bacteria, which are considered opportunistic, are pathogenic in their biofilm structures. feature has been found. Enterococcus ciiii (enterococci); Gram-positive, general with sessile, aerobic or facultative anaerobic, catalase negative and cocci morphology. characterized bacterial species. Even in very harsh environmental conditions (high and low temperature, high pressure, high salt concentration) they can continue. enterococci, of which E. faecalis is also a member; natural environments as opportunistic pathogens with cavities, intestines, male and female genital tracts. is defined. Urinary tract infections, endocarditis, intra-abdominal and pelvic infections, catheter-related infections, surgical wound infections, and central nervous nosocomial infections associated with systemic infections (hospital infections) The most common causative agents are members of the Enterococcus genus. There are 12 species of Enterococcus genus. However, E. faecalis is an approximation of the enterococcal infections outlined above. quality gains. This clearly states the clinical importance of E. faecalis. is doing. Because enterococci are common food contaminants, is of great importance in the industry. In food businesses; in production tanks, food by forming a biofilm on transmission lines and other organic and inorganic surfaces. It is a constant source of contamination, metal corrosion, food spoilage. and cause serious economic losses and health problems by causing hygienic problems. is happening. Briefly, biofilm structures; serious in the fields of industry, environment and health. It's obvious that it's causing problems.

In this invention, especially in the world, which has become a serious clinical and industrial problem and 10% of nosocomial infections and 90.1% of human enterococcal infections In the prevention and eradication of the formation of E. faecalis biofilm structures, which are the causative agents of the application method of agents developed with new and effective biotechnological methods. is intended to be developed.

For this purpose, first of all; essential component of cheese starter cultures Lactococcus lactis subsp. of the pure form of the nisin bacteriocin produced by lactis. Faecalis biofilm formation and its effect on eradication were investigated. why all GRAS (Generally Recognised As Safe) It has been described as a bacteriocin and is particularly suitable for Gram-positive bacteria. used in the fight against contamination. However, nisini E. faecalis biophilin No study has been found in which its effectiveness was investigated on its structures. E. faecalis°in It is also found in this invention that nica has a high level of activity against planktonic forms. has not been determined. Based on these data and preliminary studies, against E. faecalis biofilms. Nisin bacteriocin was used as the main agent in the control.

In the second stage of the invention, it is used for commercial and industrial purposes. on enterococcal biofilm structures of the combination of disinfectants and disinfectants at different rates. effect has not been determined. In this way, as a result of combining nisin with disinfectants, effective to be applied in the prevention and eradication of the formation of biofilm structures. by lowering the disinfectant concentration, It has been ensured that the negative effects on health and the environment are minimized. with pure nisin E. faecalis is effective and effective in the prevention and eradication of biofilm structures. a viable concentration and method has been determined.

In this invention, 4 strains of E. faecalis (food-derived E. faecalis strains 73 and 74 and clinical E. faecalis strains 79 and 114) and 2 control strains (E. faecalis ATCC) OGl RF and E. faecalis ATCC 29212) were used. Stock cultures, 15% glycerol at -80°C Added Tryptic Soy Broth (TSB) is stored in liquid nutrient medium. Used Cultures were taken from stock solutions and inoculated into 1/10 TSB liquid nutrient medium. It was developed and tested at 37 °C for 1 night.

Contents of TSB medium: Content g/L Peptone (from Casein5) 17 g Peptone (from Soy) 3 g di-Potassium Hydrogen Phosphate 2.5 g D(+)-Glucose 2.5 mL After the contents of the medium are dissolved in 800 mL of dH20, the volume of the medium is again dH20. made up to 1000 mL with After adjusting the ambient pH to 7.3 ± 0.02, the liquid The medium was dispensed into glass tubes and sterilized in an autoclave at 121 °C for 15 minutes. used.

Contents of M17 Broth medium: Content g/L Polypeptone 5 g Phytopeptone 5 g Yeast extract 2.5 g Meat extract 5 g B-disodium glycerophosphate 19 g Lactose (10%) 50 mL MgSO4.7I-120 l mL Ascorbic acid 0.5 g After the contents of the medium are dissolved in 950 mL of sterile dHzO, the pH of the medium is 7.2 ± It was set to 0.2 and sterilized in an autoclave at 121 °C for 15 minutes. This After the process, the medium is cooled to 45 °C in a water bath and sterilized separately. The medium prepared by adding the obtained lactose solution (50 mL) was used.

Contents of Müller-Hinton Broth medium: Content g/L Meat extract 30 g Casein hydrolyzate 17.5 g Starch 1.5 g After the contents of the medium are dissolved in 800 mL dHzO, the medium volume is again adjusted with dHzO. It is completed to 1000 mL. After adjusting the pH of the medium to 7.3 ± 0.1 °, the broth glass It was distributed into tubes and used after sterilization in autoclave at 121 °C for 15 minutes.

Commercial nisin contains 2.5% of nisin bacteriocin. This bacteriocin in powder form, After dissolving in 0.02 N HC] solution, it is passed through a 0.45 um pore diameter filter, transferred to pre-sterilized microcentrifuge tubes. The nisin solution used, based on the MIC (Minimum Inhibition Concentration) value of each strain, It is freshly prepared before. The chlorhexidine (CHX) concentrations used are very low. First of all, stock CHX solutions with 20% concentration were created, then the concentrations to be used in the study are to dilute the stock solution with water. prepared via

In order to prepare the doses of NaOCl, 10% NaOCl solution should be diluted with water. obtained by dilution of the stock solution with water. It was made into a solution with 1 mL sterile water. EDTA with NaOH buffer solution After dissolving, the solution sterilized in an autoclave for 15 minutes at 121°C. was used in the experiments.

Phosphate Buffered Salt (PBS): Content g/L NazHPO4 1.44 g KH2P04 0.24 g After the contents are dissolved in 800 mL of sterile dHzO, the volume of the solution is again with dHzO. It is complete for 1000 mLs. The prepared solution is autoclaved at 121 °C for 15 minutes. used after sterilization.

Universal Neutralizer Content g/L L-Histidine 1 g L-Cysteine 1 g reduced glutathione 2 g The prepared solution was used after sterilization in an autoclave at 121 °C for 15 minutes.

Investigation of Biofilm Production Characteristics of Enterococcus Strains In the investigation of biofilm forming properties of isolates, Extremina Vd. by recommended method is used. Trypticase Soy Broth (TSB) in liquid medium at 37°C for 18 hours Developed enterococcal isolates were diluted 1:10 using TSB medium. They were then inoculated into 12-well polystyrene microdilution plates and incubated with different incubations. In order to determine the biofilm production levels at 37 °C for 24, 48 and 72 hours incubated throughout. Cells that did not adhere at the end of the incubation Wells were washed three times with phosphate buffered saline (PBS) for removal. washed and the formed biofilm layer was left to dry at 60 °C for 1 hour.

The resulting biofilms were then soaked in 210 µl of 95% methanol for 20 min.

At the end of the period, methanol is removed from the wells and 210 ;11 1% crystalline Viola is poured into the wells. was added, and the dye was dried under fire for 20 minutes to determine the biofilm structure on the plates. painting is done. Then, add 200 µl alcohol ± acetone solution (80% ethanol + It is ensured that the dye kept in it dissolves. The final biofilm structure with the help of a spectrophotometer and Elisa Reader device at a wavelength of 595 nm. has been measured. According to the OD values obtained for bacterial cultures as a result of the measurements rated as. Biofilm trials were 3 parallel and 2 repetitions. has been carried out.

Minimum Inhibitory Concentration (Mic) and Minimum Bactericidal Determination of Concentration (Mbc) In these trials, nisin and disinfectants [Chlorhexidine digluconate (CHX), Ethylenediamine tetra acetic acid (EDTA) and Sodium hypochlorite (NaOCl)], effect against test bacteria, MIC and MBC levels Tong et al. determined on the basis of For this purpose, test bacteria planktonic forms were used. Disinfectants and nitrogen used in the next well It is adjusted to decrease in floor ratio and added to the wells. Then 18 hours of E. It was diluted in the environment and brought to a standard value for the studies. In TSB nutrient medium After the prepared bacterial solutions were diluted at a ratio of 1:10, 96 wells were prepared. They were inoculated into microtiter plates and incubated at 37 °C for 18 hours.

At the end of the incubation period, the disinfectants used prevent the growth of bacterial cultures. the lowest concentration MIC (Minimum Inhibition Concentration) value recorded as.

Calculation of the MIC values of the disinfectants and nisin bacteriocins used. Next, we proceed to the calculation of the minimum bactericidal concentrations (MBC). has been passed. The MIC values determined are the lowest antimicrobial agents that inhibit bacterial growth. While expressing agent concentrations, MBC values represent 99.9% of the bacteria examined. It is defined as the lowest antibiotic concentration (mg/L or ug/mL) that kills.

In the calculation of MBC values, the 96 wells used while determining the MIK values 1007 µL were taken from each of the microtiter plates and added to 900 ”L sterile PBS. dilutions were made. 103 µL of each of the diluted bacterial suspensions were taken and It was dripped onto Müller-Hinton Agar (MHA) plates and kept at 37 °C for 24 hours. left for incubation. At the end of the 24-hour incubation, viable cells from the MHA plates MBCs were determined by counting. When calculating MBC values The following formulas are used; Logarithmic measurement: Log-death = [log]_10(starting cfu/mL)-[log]_10(remaining cfu/mL) Percent death: Percentage of viable cells: Log % viable cells = [log]_10 (%viable cells) Time - Trial of Death In determining the effect times of the disinfectants used, Tong Vd. recommended by method has been used. The main difference of this method from MIK and MBC trials is It allows to determine how long the effect of antimicrobial substances will take place. is recognition. Another difference is that the effect of the disinfectant used is bactericidal or allows the definition of its bacteriostatic character. For this purpose, first 18 hours active E. faecalis cultures were diluted to approximately 107 cfu/mL in TSB nutrient medium. has been made. Then Nisin MIC value, CHX MIC value, EDTA MIC value, NaOCl MIC value in TSB media and incubated for 6, 12 and 24 hours at room temperature. has been made. At the 6th hour of incubation, 100 µl of each tube was taken, containing 900 µl of PBS. transferred to microcentrifuge tubes. With continued serial dilutions to 10'6 level has been reached. MHA by taking 10 pl from each prepared dilution tube (10'1-10'6) The colonies were planted by drop in their medium and after 24 hours of incubation at 37 °C. By counting, the viability levels of microorganisms and therefore disinfectants The effect on cells has not been determined. After 18 and 24 hours incubation, 6 hours as after incubation, 10 µl of each diluted sample Then, drip planting was done in the MHA medium. Incubation for 24 hours at 37 °C Then, colony counts were made and the viability levels of the microorganisms were determined. Each The trials for the group were carried out on different days and consisted of 3 repetitions.

Minimal Biofilm Eradication Concentrations (Mbec) of Antimicrobial Agents Determination Eradication of mature biofilm structures of E. faecalis by antimicrobial agents MBEC (Minimal Biofilm), which enables more sensitive examination Eradication Concentration) method. MBEC values In the determination of Extremina et al. The method suggested by was used. Test bacteria The optical density of the 18-hour active cultures was set to 0.07 in OD5705 and 200 µl of bacterial suspension was transferred to each well of microtiter plates. microtiter In order for biofilm to form on the plates, the plates are shaken in the horizontal plane at 37 °C. It was left to incubate for 24 hours. that does not participate in biofilm formation after incubation In order to remove loosely adherent cells from the medium, the wells were washed 3 times with PBS. has been washed. After the washing step, the microtiter plates formed a biofilm. By adding antimicrobial substances at the following concentrations, the plates were placed in the room. It was incubated for 12, 18 and 24 hours at temperature. At the end of the incubation period The wells were washed again 3 times with PBS solution and with universal neutralization solution for 15 min. incubated at room temperature throughout. Wells treated with universal neutralizer After being processed, it was washed 3 times with PBS and the biofilm structure was kept at 60 °C for 1 hour. left to dry. Fixation of biofilm structures remaining in the wells after incubation 200 µl of 95% methanol was added to the wells for 20 min. incubated at temp. After the fixation process, the biofilm structure For staining, 200 µl of crystal violet was added and the plates were incubated for 20 minutes at room temperature. is pending. Following the dyeing step, in order to remove the dye from the wells, wells were washed with dHzO. Finally, in the biofilm structures remaining in the plaques, The retained dye was dissolved by adding 200 µl of ethanol/acetone (80% / 20%, v/v) to the wells.

In order to determine the remaining biofilm amount, the dissolved dye was 595 nm in the Elisa Reader device. measured at wavelength.

Final concentrations of the antimicrobials tested in the MBEC study; obtained from previous MIK and MBC studies when adjusting the concentration Values are based on and the concentrations to be used are calculated in mM.) NaOCl: Synergetic Effect Trial To determine the synergetic effect between Nisin and other antimicrobial agents In the experiment, the method recommended by Odds was used. 96 in the study nisin in the y-axis of the well microdilution plate, taking into account the MIC value of each strain, It is diluted to be 2 times higher and 4 times lower than the MIC value. If on the X axis dilutions of other antimicrobial agents to be tested were carried out. With this method, The fractional inhibition concentration index (FIC) of nisin and other antimicrobial agents has been defined. The formula given below was used to calculate the FIC index.

FIC index: FIC A + FIC B = (MIC value of A antimicrobial agent in combination/A MIC value of antimicrobial agent alone) + (B antimicrobial in combination) MIC value of agent / MIC value of B antimicrobial agent alone) synergism; If the FIC index is 50.5, positive additive effect; FIC index O.5-4.0 Antagonism; The FIC index is set to 24.0.

Trials were carried out as 3 repetitions on different days.

Determination of Biofilm Production Characteristics of Strains were evaluated as biofilm producing bacteria samples. Based on these values When the results are interpreted, it is seen that the biofilm production characteristics of all strains depend on time. The control strain of the most important differentiation, depending on the incubation period and It was found to occur in E. faecalis ATCC OGlRF strain.

E. faecalis ATCC OGlRF strain OD595= 0.546 after 24 hour incubation period level of biofilm, when the incubation period is 72 hours, biofilm production It increased to OD595=1.336. Considering the isolates used in the experiment, maximum biofilm production was achieved by clinical origin E. faecalis l 14 strain, At the end of the 24 hour incubation period, the biofilm production level was determined as OD595= 1.657. There is no significant difference in the amount of biofilm they produce after incubation periods. While not observed, maximum biofilm production of food-derived E. faecalis 74 strain was observed within 72 hours. at the end of the incubation period and OD595= 1.383. has not been determined.

Minimum Inhibition Concentrations (Mic) and Minimum Bactericidal Determination of Concentrations (Mbc) When the obtained MIK and MBC values are compared, it is seen that the values are very close to each other. is observed. 2 different disinfectants (CHX, EDTA and NaOCl) tried throughout the studies and MIC and MBC values of nisin bacteriocin, for some of the E. faecalis isolates same, MBC values are higher than MIC values as expected for some isolates. found (Table 1).

Table 1. E. of nisin bacteriocins and various disinfectants used in the study. MIK and MBC values determined for inhibition of faecalis isolates MIC CHX EDTA NaOCl CommercialNisin (Nisaplin)* MBC CHX EDTA NaOCl Commercial Nisin (Nisaplin)* * Equivalent to 1 mg/mL Nisine.

Time - Trial of Death In the Time-Death trial study, the eradication of the tested antimicrobial agents It was determined how long the effect took place. Considering the obtained data, MIC values of the disinfectants used (Nisin, CHX, EDTA) with bacterial cells; 6, 12, They have different effects on the growth of bacterial cells after 24 hours of incubation. has been observed. When the Time-Death graph of the 73-coded E. faecalis strain is examined, most effective for every 3 incubation times at different incubation times with disinfectants It is seen that the disinfectant is CHX (0.0002%). When the Time-Death graph of 74 coded E. faecalis strain was examined, EDTA (5 mM) 12 and 24 While it is effective in hours incubation times, it is most effective after 6 hours of incubation. disinfectant nisin ( was determined as ). When the Time-Death graph of 79-coded E. faecalis strain is examined, every 3 incubation times The most effective disinfectant was found to be CHX (0.0002%). 1 When the Time-Death graph of 14 coded E. faecalis strain is examined, 6 and 12 hours incubation Nisin (50 IU/mL) was the most effective disinfectant detected at the end of the day, while incubation for 24 hours Afterwards, the most effective disinfectant detected was NaOCl (050%).

When the Time-Death plot of the E. faecalis OGlRF control strain was examined, 6 hours of incubation Nisin (25 IU/mL) was the most effective agent in inhibiting sus at the end of the study, while incubation for 12 and 24 hours EDTA (5 mM) was determined as the most effective disinfectant at the end of the duration.

When the Time-Death graph of E. faecalis ATCC 29212 control strain is examined, it is seen that 6 hours As a result of incubation with disinfectants, the most effective agent was nisin (25 IU/mL). EDTA (5 mM) was the most effective disinfectant at the end of the 12 and 24 hour incubation periods. has not been determined.

Minimal Biofilm Eradication Concentration (Mbec) of Antimicrobial Agents Determination E. faecalis strains (73, 74, 79, 114 coded test strains and OGlRF) used in the trials disinfectants (Nisin, CHX, NaOCl and EDTA) on the biofilm structures they form. eradication effects Extrainina Vd. It was investigated by the MBEC method proposed by 73 coded E. faecalis strain with disinfectants for 12, 18 and 24 hours. When the data obtained are examined, the most effective concentration of nisin in biofilm eradication is 250. The most effective result is 10% after an hour of incubation and 20% after a 24-hour incubation. EDTA concentration is 10Mm and the most effective CHX after 12, 18 and 24 hours incubation concentration of 08% after 12 and 18 hours of incubation, As a result, it was found as 2.0%. As a result of incubation of 74 coded E. faecalis strain with disinfectants for 12, 18 and 24 hours When the data obtained are examined, the most effective concentration of nisin in biofilm eradication. as 1000 IU/mL after every 3 incubations, the most effective concentration of NaOCl in 12 hours 10%, 18% after incubation and 20% after 24 hours of incubation, the most effective EDTA concentration is 20 Mm after 12 hours incubation, 18 and 24 hours incubation As a result, the most effective CHX concentration was 10 mM after 12 hours of incubation. has been found. As a result of incubation of 79 coded E. faecalis strain with disinfectants for 12, 18 and 24 hours When the data obtained are examined, the most effective concentration of nisin in biofilm eradication. In IU/mL, the most effective concentration of NaOCl 12, 18 and 24 hour incubation times as a result of 20%, the most effective EDTA concentration is 12, 18 and 24 hours incubation. As a result of the incubation times, the most effective CHX concentration was 10 mM, 12 and 18 hours incubation. It was found to be 04% in the result of 24 hours incubation and 1.25% in the result of 24 hours incubation. 1 As a result of incubation of 14 coded E. faecalis strain with disinfectants for 12, 18 and 24 hours When the data obtained are examined, the most effective concentration of nisin in biofilm eradication. concentration of 10% at the end of the 12 hour incubation period, at the end of the 18 hour incubation period In mM, the most effective CHX concentration is 08% after a 12 hour incubation period, 18 hours 06% at the end of the incubation period and 2.0% at the end of the 24 hour incubation period. has been found.

OGlRF coded E. faecalis control water with disinfectants for 12, 18 and 24 hours. When the data obtained as a result of incubations are examined, it is seen that the most effective nisin concentration 750 IU/mL after 12 hours incubation period, 18 hours 500 IU/mL at the end of the incubation period and 1500 IU/mL at the end of the 24 hour incubation period As a result, the most effective concentration of NaOCl was 025, 18% and At the end of the 24 hour incubation period, the most effective EDTA concentrations were 12 and 18, as 050%. 20 mM at the end of the hour incubation period and 10 mM after the 24 hour incubation period As a result, the most effective concentration of CHX was 06% after 12, 18 and 24 hour incubation periods. has been found. When the data obtained as a result of incubations are examined, it is seen that the most effective nisin concentration 750 IU/mL after 12 and 24 hour incubation periods, 18 hours At the end of the incubation period, the most effective concentration of NaOCl is 500 IU/mL, 12 and 18 hours. 10% at the end of the incubation period, 025% at the end of the 24 hour incubation period, effective EDTA concentration 20 mM after 12 hours incubation period, 18 and 24 hours 10 mM at the end of the incubation period, the most effective concentration of CHX every 3 It was found to be 08% at the end of the incubation period.

Synergetic effect (checkerboard) trial In order to determine the synergetic effect between Nisin and other disinfectants, In this trial, Odds Vd. The method suggested by was used. in the study based on mean fractional inhibition concentration (FIC) calculations. This In this method, the lowest drug concentration without growth in all rows and columns wells were included in the evaluation. For each of these wells, drug A and B FIC values were calculated, FIC values were calculated separately for each well, and the obtained values were summed and divided by the number of wells and the average of the FIC values was taken.

FIC index: FIC A + FIC B = (MIC value of A antimicrobial agent in combination/A MIC value of antimicrobial agent alone) + (B antimicrobial in combination) MIC value of the agent / MIC value of the B antimicrobial agent alone) On the plate The MIC values of both disinfectants used were determined simultaneously with the synergetic effect study. has been reworked. The representative view of the plates used in the study are in Tables 2, 3 and 4 are given. The values obtained in the study; synergism; If the FIC index is 50.5, positive additive effect; FIC index 0.5-4.0 Antagonism; The FIC index is rated as 24.0.

Table 2. Nisin+CHX synergetic effect study prepared for E. faecalis strain coded 73 record layout Nisin MIC CHX MIC Table 3. Synigetic effect study of Nisin+NaOCl prepared for E. faecalis strain coded 73 record layout Nisin NaOCI Table 4. Nisin+EDTA synergetic effect study prepared for 73 coded E. faecalis strain record layout Nisin EDTA The data obtained as a result of the study are shown in Table 5. just food Nisin in the inhibition of E. faecalis strain 74 and control strain E. faecalis ATCC 29212 and CHX showed antagonistic effects when used together. 73 code susta best The agent combination showing the inhibitory effect is Nisin+EDTA, 74 coded susta Nisin+NaOCl, 79 coded strain Nisin+EDTA, 114 coded strain Nisin+EDTA, OGIRF control strain Nisin+NaOCl and ATCC 29212 control strain were determined as Nisin+EDTA.

When evaluated in general, Nisin+EDTA is the most effective combination on strains. appears to be. The results of the synergetic effect study carried out with the combination of disinfectants E. faecalis 73 Nisin+CHX 2.58 Positive contribution Nisin+EDTA 1.3228 Positive contribution E. faecalis 74 Nisin+CHX 4.97 Antagonism Nisin+NaOCl 2.3031 Positive contribution E. faecalis 79 Nisin+CHX 3.24 Positive contribution Nisin+NaOCl 2.303 Positive contribution Nisin+EDTA 1.9658 Positive contribution E. faecalis 114 Nisin+CHX 2,784 Positive contribution Nisin+NaOCl 2.27625 Positive contribution Nisin+EDTA 1.5473 Positive contribution E. faecalis OGlRF N-iron+NaOCl 2.2849 Positive contribution Nisin+EDTA 2.3523 Positive contribution E. faecalis ATCC 29212 N is +CHX 5.1339 Antagonism Nisin+NaOCl 2.4708 Positive contribution Nisin+EDTA 2.2152 Positive contribution Enterococci are present worldwide (especially Staphylococcus aureus, group A streptococci or are dangerous pathogenic microorganisms (compared to pneumococci).

In recent years, it is the biggest problem of bacteria-related problems encountered in the field of health and food. are considered as resources. Besides being in the natural flora of foods, Due to their starter culture capacity, they have an important place in the food industry. But virulence genes contained by enterococci strains, antibiotic resistance properties and Due to their biofilm structure, enterococci are used as starter cultures in the food industry. its use creates controversy among researchers. These juices are milk and milk It can be found in meat products, ready-made foods, and even as a probiotic. Even enterococcal strains used can be resistant to antibiotics. enterococcus strains, especially E. faecalis, opportunistic pathogens causing nosocomial infections It has become more common in recent years. This bacterium is particularly infections, bacteremia, meningitis, surgical wound infections, neonatal infections It is the source of many hospital-acquired infections.

Biofilms are formed by a group of bacteria that adhere to surfaces and It is a structure covered with extracellular polymeric matrix produced by cells. biofilm The cells that form it, the developmental physiology and genetic expression similar to multicellular forms structural and functional properties very different from their planktonic forms. they exhibit. Bacterial cells are very well colonized in the biofilm structure in the evolutionary process. have the ability to survive and continue their lives even under the most difficult conditions. has the ability. Today, especially pathogenic microorganisms The main target in studies carried out with biofilms; genetic and biochemical basis prevention and eradication of biofilm structures by determining is to provide. However, the different structures and bacteria that exist in biofilms It enables their structures to gain resistance against antimicrobial agents. And also against conventional sanitation, disinfection and sterilization agents and methods, The strains in biofilms are at much higher levels than their planktonic forms. they show resistance. Therefore, the prevention of microbial infections and basic strategies in its eradication; biofilm, not planktonic forms of pathogenic bacteria are determined taking into account their structures. With microbial infections and contaminations negative effects of the agents and methods used in the fight against the environment and human health. effects, the presence of green biotechnological agents in the fight against biofilm structures and in this direction, it has become necessary to develop methods of struggle.

Bacteriocins are prokaryotic antimicrobial proteins or antimicrobial peptides.

These molecules, which are generally cationic and hydrophobic, are widely used against various pathogens. It can show a broad spectrum antimicrobial effect and have potential biotechnological applications. are green biotechnological agents. Especially today, food-based used in order to have problems caused by pathogens and to prevent food from spoiling. Due to the undesirability of chemical preservatives, the need for new and reliable biopreservatives interest has increased. Therefore, bacteriocins are resistant to various drugs. It has become a very important option in the control of microorganisms. Many bacteriocin to protect human, plant and animal health and to improve food quality and safety. is being researched for improvement.

With this invention made in line with the development of green biotechnological agents and methods; biofilm of E. faecalis strains, which has become a serious clinical and industrial problem. In the prevention of the formation of cheese structures and in the eradication of the formed biofilms, Lactococcus lactis subsp. produced by lactis Formation of E. faecalis biofilm of pure commercial form of nisin bacteriocin and Its effectiveness in eradication has been demonstrated.

Nisin is a GRAS (Generally) Recognized As Safe) has been identified as a bacteriocin. Nisin, especially Gram-positive used to combat bacteria. However, as mentioned earlier, nisin E. There is not yet a study investigating the effect of faecalis on biofilm structures. therefore The study, which is the subject of the invention, is the first in the world.

It has been shown that nix has a high level of activity against planktonic forms of E. faecalis. has not been determined. In the first step of the invention; commercial nisin (Nisaplin), E. faecalis strains with the concentrations used in the inhibition of planktonic forms. of other disinfectants (CI-IX, NaOCl and EDTA) in the planktonic forms of these strains. Concentrations used in inhibition were compared. At this stage, nisin and other The effect of disinfectants against E. faecalis strains is based on MIK and MBC levels. has not been determined. MIC values determined within the scope of the invention are the most important ones that prevent bacterial growth. denotes low antimicrobial agent concentrations. As a result of the trials, Very low concentrations of nisin bacteriocins and other disinfectants used It was determined that even E. faecalis strains prevented the growth. For commercial purposes in this invention The MIK value obtained is generally below the concentration of 50 IU/mL.

Khan et al. Inhibitory effect of nisin bacteriocin on Gram-positive bacteria In their studies to observe the inhibitory effect determined for nisin, concentration ranges from 125-150 IU/mL. Bauttefroy et al. by In the study carried out, nisin at a concentration of 50 IU/mL in general was Gram-positive. It has been found to be effective on bacteria. What has been done about nisin in general? The most important result obtained in the studies is that the nisin is very fast from the pH value of the environment. that it is affected. The effects of nisin on pathogens in environments with high acidity It was observed that the inhibitory effect increased. Nisin in environments with high acidity its antimicrobial effect Tong et al. in the field of endodontology by investigated by studies. Bacteria that cause infection, especially in the oral cavity Since there are Gram-positive bacteria, Streptococcus sanguinis, Streptococcus nisin on bacteria inutans, Lactobacillus fermenti and Lactobacillus acidophilus They investigated its lethal effect and found that nisin was significantly effective. Same At the same time, the fact that the mouth has a low pH value is responsible for some of the enzymes secreted here. not causing harm and especially the presence of Gram-positive bacteria in the environment, nisin stated that they are the most important factors in showing its antimicrobial activity.

When the MIK and MBC values of other disinfectants are examined, they are also very low. were found to be effective on E. faecalis strains at high concentrations. MIC and As a result of MBC studies, the most effective agent on planktonic forms was CHX. has been found. Even at a very low concentration (0.0002%) E. faecalis strains killed almost all of them. Looking at previous studies, CHX to the knowledge that the disinfectant is a very effective disinfectant on pathogenic bacteria. are found. CHX inhibition of E. faecalis pathogens especially in the field of endodontics is used for the purpose. The most effective concentration is stated as 2% (26-28).

Due to the harmful effects of CHXs on human health, it is especially used in the field of endodontics. To reduce the use of CHX, Tong et al. by frequently with E. faecalis ATCC 29212 strain, which is found and causes a wide variety of infections. has been made. In their study with Nisin and CHX, it was found that nisin was also found in E. faecalis strains as well as CHX. They found that it was effective on The study of both antimicrobial agents Since the mechanism is the same, today's studies in this area have replaced CHXs. It supports the use of nisin. Another antimicrobial agent used in the study NaOCl is similar to CHX in its mechanism of action. Earlier As we mentioned, NaOCl has strong antimicrobial effect and organic substances. The most frequently used disinfectants, especially in the field of endodontics, due to their easy dissolving is located between. Besides these advantages of NaOCl, dental plaques are fully not being able to clean, having high toxicity, corrosion in the devices they are used. It has many disadvantages as well. Both agents together There are many studies in which it is used in the literature. One of these studies is Cintra et al. The use of CHX and NaOCl agents together in the cleaning of tooth roots by This is a study of its effects. As a result of their work, the two agents together An orange-colored precipitate with high toxicity is formed. they have determined that he has come. This precipitate called parachloroaniline (PCA), CHX and NaOCl They determined that it was formed as a result of the acid-base reaction occurring between In the continuation of the study, it was determined that the rate of PCA precipitate formed was correct with the NaOCl used. It was stated by the researchers that it is proportional. Cintra et al. this is what they did As a result of the study, it was determined that these two agents, which are used together especially in oral cleaning, It has been stated that it will be very harmful in terms of Dubey et al. by E. faecalis strains In the study, 25% concentration of NaOCF was higher than E. faecalis strains. It was found to have a significant antimicrobial effect. NaOCl and CHX Another study conducted with disinfectants is Jeansonne et al. by has been carried out. Which of the CHX and NaOCl disinfectants are E. faecalis strains? In this study, which was carried out in order to determine which has the most impact on It has been found to be more effective than NaOCl at concentrations. Delany et al. by In the study carried out, it was found that CHX, which has a concentration of 0.2%, is a pathogen in the mouth. It has been found to have a highly lethal effect on bacteria. NaOCl and A study investigating the antimicrobial effects of EDTA disinfectants on E. faecalis. other work Baumgartner et al. made by NaOCl at a concentration of 525% and the use of EDTA at a concentration of 15%, especially in implants. It has been determined as a result of the research that it is very effective in clearing the pathogens. In the study, CHX and NaOCl agents showed on E. faecalis strains. The effect was found very close to each other, in parallel with the literature data.

In the Time-Death trials, how long does the effect of the disinfectants used take? has been found to occur. 6, 12 and 24 hour incubations of disinfectants used MIC values obtained against E. faecalis strains after It has proven that it has activities. When the data is examined, it is seen that the effect of niche is 6 at most. It was determined that it showed at the end of the hour incubation period. General structure of the nisin When examined, it is seen that those who cannot stay in their environment for a long time and deteriorate immediately. It is known that it is a molecule of protein or peptide structure. Therefore, what alone When it is used, it shows its effect at the end of the 6 hour incubation period, compatible with our current knowledge. Even during this incubation period, it has been shown that Its inhibitory effect is quite high compared to other agents. At 12 and 24 hour incubation times, The reason why it cannot be very effective on the waters is that it is very perishable (degradable structure) can be shown. Therefore, increasing the nisin concentration or continuously To achieve more effective results by gradually adding nisin to the environment with systems it will be possible. Compared to other agents, the most effective agent in all 3 incubation periods Found as CHX. Similar results for all 3 incubation times tested Although obtained, the most effective result was achieved at the end of the 6 hour incubation period.

EDTA is the most effective agent on planktonic forms of enterococci after CHXl. has not been determined. The effect they show at the end of all incubation periods is comparable to each other. was found quite close. The lowest effect was shown by the NaOCl agent. out of the way The findings obtained from the agents used are in parallel with previous studies. seen to be carried. While the lethal effect of these 3 disinfectants on all waters is the same, nisin on food-derived E. faecalis 73 strain at the end of the 6 hour incubation period. exhibited a high lethal effect.

Minimal biofilm eradication concentrations (MBEC) of alithymicrobial agents In the study carried out to determine The effects of disinfectants on the biofilm structure they form at the end of their life researched. When the data obtained as a result of the study are examined, it is seen that the biofilms formed The most effective agent in its eradication is NaOCl and the most effective NaOCl concentration is In general, the effective concentration was determined as 5-6% in studies. But a made In this study, 1% reduction in eradication of biofilms formed by E. faecalis strain It was determined that NaOCl was effective in the concentration of Data obtained in the study when subjected to statistical evaluation; All NaOCl used above 0.05% (025%, 0.5%, 1%, 2%) on E. faecalis biofilms. appears to have the same effect. For other agents, mature biofilms The incubation time of the most effective EDTA and CHX concentrations in eradicating and It has been found that the biofilm formed by the strains varies according to its structure.

Nisin concentrations used in the MBEC study, statistical analyzes As a result, it could not be evaluated as significant (p<0.01). In the literature data, only one not successful in eradication of biofilms formed by pathogenic strains per se, It is suggested that it gives more effective results when used with other disinfectants. is coming out. Particularly, it determines the structure of EPS found in the outer part of biofilms. Its use with agents that can spoil it gives very positive results. Nisin and A study in which the effects of the combined use of EDTA on the shelf life of foods were investigated. study Economou et al. carried out by. In their study, chicken meat nisin and EDTA together at certain concentrations in order to prolong the lifespan. use of Pseudonionas sp. and Gram-negative bacteria such as enterobacter and Gram-positive bacteria such as Brochothrix thermosphacta and lactic acid bacteria They determined that they inhibited their reproduction to a large extent and that the shelf life of chicken meat should be at least 4 they determined that the day is longer. Investigation of the combined use of Nisin and EDTAL another study by Kopermsub et al. made by Nisin and EDTA together In this study, in which they aimed to prolong the antimicrobial effect they show, surfactants have been used. In this study, Staphylococcus aureus and E.coli strains were used. In the study, it was determined that the duration of action of encapsulated nisin and EDTA components was prolonged. has been made. When the studies in which it was used as a food preservative are examined, niacin enters the Maillard reaction with the reduced sugars in the food, producing lipid and It was found that it forms non-specific bonds with proteins. Besides that, fresh Nisin, which is used as a preservative in foods, is broken down by proteases in foods. again stated as a result of research. With this study by Kopermsub et al. The disadvantages identified as a result of the free use of the It has been shown on pathogenic bacteria with its use with antimicrobial substances such as EDTA. In addition to prolonging the duration of their antimicrobial action, combinations of agents they are more active.

Data from synergetic effect trials show that nisin is combined with other disinfectants. indicated that the combination made a positive contribution to its antibacterial activities. Especially the combined use of nisin and EDTA agent, E. faecalis suslannin planktonic It gave the most effective results on the forms. The other most effective combination is nisin and It is the combination of NaOCl. Nisin and CHX of the pair with the lowest effect has been detected.

EDTA plays an important role in the eradication of biofilms formed by pathogenic bacteria. has. Especially in the eradication of biofilms formed by Gram-negative bacteria is widely used. Lipopolysaccharides in the structure of the cell walls of bacteria and glycerophospholipids. EDTA, which can chelate with metal ions, divalent cations in the structure of LPSS located on the outer membranes of bacteria (Caü, Mgü) impairs the stabilization of the membrane by binding. As a result, cell wall damage Bacteria that have been exposed to bacteriocins are more easily affected. This One of the studies in which two inhibitor agents were used together was Tu et al. carried out by.

As a result of this study, the combination of nisin and EDTA, Salmonella Typhiinuriuin It has been found to be effective in the pathogen. In the study, the strains of E. faecalis The results obtained by using nisin and EDTA together in the inhibition of both It is considerably higher than the results obtained by using the agent one by one. of the study biofilms formed by the control strain E. faecalis OGlRF, which is the last step. Eradication of the effects of various agents by using them together with SEM. Nisin and EDTA are the most effective inhibitor combinations in imaging the has been observed. Showing the synergetic effects of Nisin and EDTAs with each other. in a study by Branen Vd. by Listeria monocytogenes, E. coli, Salmonella enteriditis and Pseudomoiias fluoresceiis strains. In the study, the silences were 18 and After their 24-hour incubation, the use of EDTA and niche separately, as well as Their combined effects were also investigated using the synergistic effect method. used in the study The concentration of EDTA is between 156-10,000ug/mL for Gram-negative pathogens. nisin concentration 3.9-46.9 ug/mL (between 156th) has changed. The concentration of EDTA used for Gram-positive pathogens is 7.8-500 While the nisin concentration is between tig/ml, 1.95-31.3 ug/mL (7 has changed between When they examine the MIK and MBC values they have obtained, they find 250 ;4ng concentration of EDTAa inhibits the growth of L. monocytogenes pathogen, 1250- At concentrations between 5000 tig/ml, it inhibits the growth of Gram-negative bacteria. found to be inhibited. However, EDTA° did not show any bactericidal effect for any strain. has not been determined. When nisin at a concentration of 312.5 IU/mL is used alone, L. Inonocytogenes strain both inhibits reproduction (bacteriostatic effect) and It has a bactericidal effect, but when used alone, it is against Gram-negative bacteria. They stated that it was not effective. Nisin and EDTA are resistant to L. monocytogenes and E. coli pathogens. showed a synergetic effect when used together against Salmonella and It has been stated that they have little effect against Pseudomonasia when used together. when are you EDTA at low concentrations is sufficient when used together with high when used in concentrations, no change in their activity on pathogens. It is among the findings that did not occur. Branen Vd. In this study by high concentrations of EDTA and nisin on planktonic forms of pathogens. While they have been observed to be effective, in the study conducted by us, they were used at low concentrations. Biofilm formed by E. faecalis pathogens by the combined use of EDTA and nisin It has been determined that it can even eradicate their structures. In the eradication of biofilms, the most One of the agents used is NaOCP. Especially in the field of endodontics, E. faecalis NaOCl is widely used in the eradication of biofilms formed by pathogens. is used. In the studies, it is the most effective method used in the eradication of biofilms. The NaOCl concentration was determined to be between 0.5% and 5.25%. Kozol et al. by In the research, NaOCl, which poses the least danger to human health in clinical use, It was stated that the concentration was 0.025%. In other studies on NaOCl, For NaOCl to exert its cytotoxic and bactericidal effect, it must be at least 0.5%. It was emphasized that it should be used in concentration. carried out in the study As a result of the trials, the most effective NaOCl on planktonic forms of E. faecalis strains It is seen that the concentration is 05%. This finding is consistent with Pashley et al. recommended by compatible with concentration. Time-Death trials were performed at 0.5% concentration. The NaOCl agent shows its highest lethal effect on the strains at the end of the 6 hour incubation period. has shown. In MBEC trials, the most effective agent was determined as NaOCl. NaOCl As a result of the analysis of variance for the data obtained from the MBEC studies of the agent, values were found to be significant at the rate of 1%. Then E. faecalis strains and NaOCl agent E. faecalis biofilms were eradicated in Duncan's analysis for its interaction. that all NaOCl concentrations used for detected. NaOCl at 0.05% concentration has the lowest effect in eradication. has shown. The findings obtained from the trials carried out with NaOCl in the study, above is consistent with the summarized literature data.

In addition to its use as an antimicrobial agent, NaOCF protects human health and the environment. It has many disadvantages as well. High cytotoxic effect, strong odor NaOCF is harmful to have and can be respired, causing metal corrosion. is among the most important disadvantages. The NaOCl agent generally affects the bacteria. It shows by inhibiting enzymes that are important for However, pathogenic bacteria such as E. faecalis microorganisms can show resistance to NaOCl. high in NaOCl It is stated that the use of high concentrations is very harmful for human health. known. Therefore, in our studies, we combined the NaOCl agent with nisin bacteriocin. together, the biofilm structures formed by E. faecalis strains Reducing the effective disinfectant concentration to be applied in eradication is targeted. In order to determine the synergetic effect between NaOCl and nisin, In our study, on the other hand, they generally contributed positively to each other's antimicrobial effects. they have been detected. On Enterococcal biofilm structures of Nisin and NaOCP It is the first findings in which its efficacy was determined and its synergetic interaction was defined. In terms of scientific and technological aspects, our data is important. This event promise different environmental parameters at the concentrations in question are examined and detailed, advanced requires research.

Another of the most used agents in the prevention of the development of E. faecalis strains is CHX, tr. CHX is a disinfectant with cationic bisguanide structure. on bacteria interact with negatively charged phosphate groups on the cell wall, which is its target site. It increases the permeability of the membrane by forming a pore on the cell wall and They cause cell death by disrupting the intracellular balance. CHX at low concentrations When used, small molecules such as potassium and phosphorus leave the cell, while higher when used in high concentrations, it can cause precipitation of the cell cytoplasm and/or causes coagulation. Studies with CHX and nisin show that both agents showed that the mechanism of action was approximately the same. Both agents are bacteria. It causes cell death by forming a pore in the cell wall. Intense in the oral flora In the eradication of planktonic forms of E. faecalis strains, Tong et al. Nisin and CHX agent were used together in the study conducted by. In the study, both the effect of the agent when they are used alone, when they are used together It was found that they could not show. The most important reason for this situation is the effect of both agents. They interpreted it as the same phenomenon of the mechanism. In the study carried out by us, Results obtained when CHX and nisin agents are used alone and together The results obtained when using is similar to its findings. However, it was previously formed by E. faecalis strains. A study in which these two agents were used together in the eradication of biofilms not included in the literature. E. eradication of biofilms formed by E. faecalis strains In the study related to the use of CHX and niche, mature biofilms It was determined that its eradication could not be achieved completely. E. faecalis biofilm structures A study investigating the combined effect of nisin and other disinfectants on its absence in the literature does not provide an opportunity to discuss our results by comparison.

As a result of studies with bacteriocins; Other antimicrobials of bacteriocins When used with agents or a wide variety of phenolic compounds, the target It has been determined that they are more effective in eradicating the microorganism from the environment. Especially A single agent is sufficient for eradication of biofilms formed by pathogenic bacteria. polymeric substances on biofilms, where it does not show a high level of antimicrobial effect. It was determined that it could not clean the matrix and dissolve the organic residues sufficiently. The E. faecalis strains used in the study are resistant to many antibiotics, chemical and mechanical nosocomial resistant to cleaning methods and many antiseptics It is the type of bacteria that constitutes the most important cause of infections. Today, E. faecalis The most used disinfectants against the pathogen are CHX and NaOCl. used for years These disinfectants have many negative effects on both humans and the environment. known. Therefore, with this invention; Biofilms formed by E. faecalis In addition to the chemicals used in its eradication, nisin bacteriocin, which is a green agent, its use reduces the concentration of chemicals and thus human health and It has been determined that it is a very important alternative in terms of environment. With this invention In accordance with the data obtained, when used together with nisin bacteriocin, especially NaOCl and EDTA agents are highly effective even at low concentrations. has not been determined. Because of its previously mentioned properties, it is an anti-nisin It is very difficult to develop resistance, it has side effects against the environment and human health. its absence greatly increases its importance and potential for use. In this respect The absence of any work and patent in the literature indicates the originality of the invention. shows.

Claims (1)

REQUESTS . It is the prevention and eradication of biofilm production of Enterococcus faecalis bacteria, and its feature is; As an antimicrobial, it is related to the use of nisin bacteriocin alone or in combination with chlorhexidine (Cl-IX), sodium hypochlorite (NaOCl) and ethylenediamine tetra acetic acid (EDTA). . It is Enterococcus faecalis bacterium according to claim 1.6, and its feature is to use food origin E. faecalis 73 and 74 coded strains, clinical origin E. faecalis 79 and 114 strains and 2 control strains E. faecalis ATCC OGlRF and E. faecalis ATCC 29212. . It is antimicrobial according to claim 1, its feature is; NaOCl: It is used in concentrations of 2%, 1, 0.5, 0.25.