Preliminary Study On The Effect Of Antibacterial Peptide Ll-37on Acinetobacter Baumannii Biofilm

BackgroundThe acinetobacter baumannii belongs to the fermentation gram negative bacilli, widely exists in nature, grows well in soil and edema.It was very wide and long-term survival distribution in the hospital environment, medical environment in oxygen humidification bottle, ventilator, transfusion equipment, air conditioning system and the patient and the medical staff of the skin can be caused by pollution of the bacteria infection. The main cause of respiratory tract infections, it can also cause bacteremia, urinary tract infection, secondary meningitis, operation site infections, ventilator-associated pneumonia, and so on. For all have a great threat to the patientsand medical staff, especially for ICU and burn patients, because of its low resistance or large areas of their skin depigmentation, has become a major factor in bacterial invasion of the body.In recent years, as the infection, the detection and the number of strains separated of the acinetobacter baumannii in hospital were increased year by year, the resistance rate increased. Then it appeared extensively drug-resistant acinetobacter baumannii and total resistance of acinetobacter baumannii, so it give more challenges and difficult to our clinical treatment. Research shows that the acinetobacter baumannii genomic make its ability of fasting access and dissemination of antibiotic resistance, multidrug resistance, extensively drug-resistant, all resistant acinetobacter baumannii is widely popular dissemination. The clinical treatment for acinetobacter baumannii often is at a loss what to do. Because the rate of resistance ofacinetobacter baumannii to the third generation and four generation cephalosporins can be as high as70%, the rate of resistant to the common Amikacin, gentamicin, tobramycin and ciprofloxacin are above95%.The majority of strains at present is to imipenem, meropenem, cefoperazone-Shubatan and polymyxin remain sensitive, but the situation is not optimistic.Bauman Acinetobacter resistance on the rate of antimicrobial drugsincreased year by year.More and more researchers believe that the main pathogenic factors of associated with the ability of creating biofilm.The bacterial biofilm possesses the property of structural, coordinative and functional high grade organized community, so as with its correlative antibiotic resistance to infection, becoming one of the factors for clinical refractory infections.Every kinds of the bacteria can exist as biofilms, the United States centers for disease control and prevention estimates that at least65%of the human bacterial infections associated with biofilm. Biofilm has been demonstrated to be associated with chronic otitis media, middle ear cholesteatoma, chronicadenoiditis diseases.The bacterial biofilm is the bacterial community formed with multiple bacteria attaching on the surface of the body or object irreversibly and packaged by the secretion of matrix itself. The bacterial within the biofilm sensitivity to antibiotics compared with the freestate was significantly decreased.The physiological structures of the reasons include biofilm prevented transmission or biofilm drug in the bacterial changes.It had been thought that the reason of antimicrobial resistance of biofilm is mediated antimicrobial drugs to penetrate into the biological membrane. But some studies deny this hypothesis.Research shows deep quinolones can quickly penetrate into the Pseudomonas aeruginosa and Klebsiella pneumoniae biofilm, tetracyclinecan quickly penetrate into Escherichia coli biofilm, vancomycin can quickly penetrate into the Staphylococcus epidermidis biofilm.At present, the only confirmed that aminoglycoside, because the matrix of the biofilm with negative charge, and aminoglycosides with a positive charge, so the aminoglycoside is difficult to penetrate deep into the biological membrane. Because the action of many antibiotics kill on replicating bacteria more powerful, such as penicillins, cephalosporins and carbapenem etc. Regulation is affected by oxygen, the lack of nutrients in the deep of biofilm bacteria and possible quorum sensing system, the bacterial growth, reproduction rate, effect of antibacterial effect of drug.Therefore in the antibacterial drugs in biological membranes, relatively sensitive bacteria can be killed, but the resistant bacteria will continue to exist, but once the antibacterial drugs to stop using, these bacteria will reproduction, growth.Then the bacteria could become drug-resistant associated with having a very high resistance and immune escape ability, escaping antibiotics kill and immune attack in addition. so the bacterial biofilm may be the important reasons which lead chronic persistent infection and refractory wounds. In conclusion, it is very important for each clinical physician to solve and control the problem about infection and resistance.With the development of molecular biology and gene engineering technology, more and more people paid attention to explore a broad spectrum new antibacterial drug with no resistance or low-resistance. As a small molecular active peptide in immune defending system, Antibacterial peptide is an essential part of the innate immune system of organism abounding in bacteria, virus and various animal,most of which have a thermal stability,PH adaptability and broad-spectrum resistant bacteria, fungi, viruses, parasites and tumor cells, and so on. Newly, A slew of scientific research shows,the LL-37is the only known member of the Cathelicidin family of peptides expressed in humans,mainly produced by neutrrophils, various epithelial cells,reported activities of LL-37include broad-spectrum resistant bacteria,immuno-modulatory chemoattractant function,stimulation of angiogenesis and tissue regeneration, and cytokine release. Because the seldom-reported the effect of antibacterial peptide LL-37on acinetobacter baumannii biofilm,this study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37about the effect on bacterioplankton and the bacterial biofilm. To provide the scientific basis for the future research in clinical infection control and new antimicrobial agents.Objectives 1、To observe and study the ability of the formation of acinetobacter baumannii biofilms and explore a method of constructing acinetobacter baumannii biofilms model in vitro.2、To exam the minimal inhibitory concentration of LL-37by broth dilution.3、Preliminary study on the effect of antibacterial peptide LL-37on acinetobacter baumannii biofilm.Methods1、Acinetobacter baumannii biofilm prediction model was established on24-well plate.Picking AB colonies to LB broth of5ml, oscillating culture at37℃. After8hours, adjusting the concentration of bacteria about1.0×108CFU/ml by turbidimetric, adding200μl bacteria into24-well plate,every hole have a cover glass with12x12mm and broth of2ml changed once every24hours,culturing at37℃,after5days, the cover glass were washed3times with PBS and fixed by crystal violet staining,as bacterioplankton does.2、The minimal inhibitory concentration of LL-37was examined by Clinical and Laboratory Standards Institute.3、(1) We putted the sterile sputum suction tube into24-well plate containing the bacteria solution, with1.0cm after autoclaving,and broth was changed once every24hours at37℃in5days.According to statistical requirements,the different wells were randomly divided into experimental group dropped in20μ g/ml LL-37and control group with bacteria free water.After24hours,they were washed with PBS gently3times, fixed in2.5%glutaraldehyde, stored in4℃refrigerator.The sample were observe for Surface Structure under Scanning Electron Microscope when its were sprayed metal after dehydrated by gradient ethanol and dried in high vacuum evaporator.(2)96-well plate containing the bacteria solution of200μl were was changed once every24hours at37℃. After5days,each well was put into different concentrations of LL-37at20p.g/ml,10μg/ml,5μg/ml,2.5μg/ml,1.25μg/ml,0μg/ml. Then it was cultured in24hours at37℃, washed with PBS gently3times, put1% crystal violet staining of2ml when dried. Dyed20min in normal temperature.washed with bacteria free water gently, put95%alcohol when dried. The absorbance value was measured by ELISA at570nm comparing control group with alcohol.Results1、The mature of acinetobacter baumannii biofilm can be found on the cover glass without conditions after5days,The macroscopical of purple dense matter can be observed on the surface of the glass by crystal violet staining,and wo can observe membranous colony by containing a lot of the growth of bacteria under powerful microscope.But the floating bacteria in medium is not.2、Successfully measuring The minimal inhibitory concentration of LL-37was64μg/ml.3、(1) Acinetobacter baumannii attaching on the surface of the sterile sputum suction tube irreversibly were observed definitely and form the mature of bacterial biofilm on scanning electron microscope image,which can proved Acinetobacter baumannii have a way to existence.The mature and dens of acinetobacter baumannii biofilm could be form in5days consistent with previous reported,and much more density compared with the control group.(2) The absorbance decreased significantly when the concentration of LL-37at2.5μg/ml,namely the quantity of biofilm decreased significantly compared the0μg/ml and1.25μg/ml group. Showing this concentration of LL-37can inhibit the formation of acinetobacter baumannii biofilm, and the absorbance decreased gradually with the concentration of LL-37increasing. There was no statistically significant difference between two groups,When the concentration of LL-37increased to10μg/ml,20μg/ml.Conclusions1、The mature and dens of acinetobacter baumannii biofilm could be form in5days consistent with previous reported,proving the acinetobacter baumannii biofilm model in vitro was established successfully. 2、The minimal inhibitory concentration of LL-37was64μg/ml consistent with previous reported.3、LL-37could destroy the structure of Acinetobacter baumannii biofilm, and this occurred at low concentration of2.5μg/ml when far less than MIC.