| Background:With the rapid development of global economy, traffic accidents and social conflict occurred more and more frequently, and the incidence of body injury is increasing. The skin is the biggest organ in human being. It has an very important role in the prevention of organisms invading. Pseudomonas aeruginosa (PA), Staphylococcus epidermidis, staphylococcus aureus (SA) and Acinetobacter baumannii (AB) which are the most common pathogens lead to infection and complications when skin is suffered severe trauma (burns, scalds, etc.). Totally 1235 cases of trauma complicated with wound infection from 2005 to 2010 collected by Yeli Dong et al. The isolation rate of AB was 7.70%, and what’s worse, the resistance rate of AB showed increasing trend year after year, especially to carbapenem.Acinetobacter baumannii belongs to the nonfermenters gram negative bacilli. As a kind of opportunistic pathogen, AB is widely existed in nature and belongs to the normal flora of the skin, respiratory tract, gastrointestinal tract of human body. It has a great threat to the patients in ICU and burns, because of the wide distribution and long-term surviving in the hospital environment. Its risk factors include intensive care nursing, duration of hospitalization, use of mechanical ventilation, invasive procedures, wide use of broad-spectrum antibiotics, long time use of immunosupp-ressants and correlated serious original diseases. Broad-spectrum antibiotics, especially the third and fourth cephalosporins and carbapenem were widely use in clinic which created a favorable environment for the quantity of MDR-and XDR-strains increasing, because AB has the ability to get fast and spread resistance and the mechanism is very complicated. Now, Acinetobacter baumannii can resistant to virtually all antibiotics and form many strains which resistant to multiple antimicro-bial drugs, include multidrugresistant Acinetobacter baumannii (MDRAB), extensively drug resistant Acinetobacter baumannii (XDRAB) and pan drug resistant Acinetobacter baumannii (PDRAB). According to CHINET, the isolation rate of AB in clinic was about 16% from 2010 to 2013, and the resistance rate of imipenem of imipenem and meropenem both about 60%. Totally 2748 strains of pathogens were isolated from 478 burn patients hospitalized in Institute of Burn Research of Southwest Hospital, Isolation rate of PA ranked the first (996 strains accounted for 36.24%), followed by SA (495 strains accounted for 18.01%) and AB (395 strains accounted for 14.37%). The resistant rates of AB to most of antibiotics was 57.91% to 100.00%, and it showed a trend of increase year by year. AB might replace PA as the main pathogenic bacterium that cause the death of burn patients with infection.With the increase in number of multi-drug resistant Acinetobacter baumannii and the development of resistance in recent years, we are badly in need of new drug to resistant AB. Antibacterial peptide (ABP) is an important component of innate immune system which widely distributed in plants and animals. It is a active polypep-tides which produced by the organism immune defense system to resistant pathogen. A large study showed that the LL-37 is the only known member of the Cathelicidin family of peptides expressed in humans, mainly produced by neutrophils and various epithelial cells. It exhibits broad spectrum anti-microbial activity and the antibacterial spectrum include gram-positive bacteria, gram-negative bacteria, fungi and part of virus with envelopes. In addition, ABP has other biological function include neutralize endotoxin, immune regulation, inducing angiogenesis, promoting repair of wound and so on. At present, the effect of the topical application of antibacterial peptide LL-37 on Acinetobacter baumannii infection of wound, and the model of Acinetobacter baumannii infection of deep second-degree burn wound have not been both reported. In this research, the model of deep second-degree burn wound with Acinetobacter baumannii infection and traumatic wounds infection of Acinetobacter baumannii were set up firstly, then the preliminary evaluation on the effect of LL-37 on Acinetobacter baumannii infection wound compared with positive control group of mafenideObjectives:1. To determin the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of LL-37 to the Acinetobacter baumannii in vitro.2. To establish the model of Acinetobacter baumannii infection of deep second-degree burn wounds in Wistar rats.3. To preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of deep second-degree burn wounds in rats.4. To preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of traumatic wounds in rats.Methods:1. The MIC and MBC of LL-37 to the Acinetobacter baumannii were determined by mircrobroth dilution assay (it was strictly complied with the Clinical Laboratory standardization committee, CLSI).2. The model of Acinetobacter baumannii infection of deep second-degree burn wound were established in Wistar rats:Forty Wistar rats (by the Experimental Animal Center of Southern Medical University) were removed hairs on back by 80 g/L sodium sulphide the day before starting the experiment. The model of deep second-degree burn wound was designed on the hairless area of 3cm×2cm with 85℃ hot water for 15 seconds after anaesthetized with 1.5% pentobarbital sodium (20 ml/kg). At 10 minutes post-injury, the Acinetobacter baumannii infection model was established by evenly daubed with Acinetobacter baumannii liquid (the bacterial concentration of 1× 108 CFU/ml) of 0.5 ml. All rats were kept under stable conditions, temperature of 24±5℃ after wound dressing. One piece of the infection tissue under the scab was obtained at 12th,24th,48th,72th hour. Finally, plate count method was used to count the quantity of bacteria. Tissue homogenate according to 100 mg of organization to join 0.9 ml isotonic saline, then the liquid were planted on agar medium for bacterial culture after gradient dilution. The quantity of bacteria was counted by plate count method and the infection model would be establish when the the quantity of bacteria up to standard of 1×105 CFU/g.3. Preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of deep second-degree burn wound in rats:Seventy-two Wistar rats were enrolled in this study, and the Acinetobacter baumannii infection model was reproduced according to the above-mentioned measures. Then they were randomly divided into C(control, n=24, with wet compress of isotonic saline), M(n=24, with hydropathic compress of 100 g/L mafenide), L(n=24, with wet compress of 0.2g/LL-37) groups. Dressing change and general observation were performed once a day before the rats were executed at 1st,3rd,5th,7th day after treatment. Two pieces from the infection skin and the muscle tissue under the scab was obtained under aseptic conditions, one used to compare the histology change under HE stain and the other to count the quantity of bacteria by plate count method.4. Preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of Traumatic wounds in rats:The model of thirty Wistar rats were reproduced by excision of the full layer of dorsal skin with an area of 1.5 cm×1.5 cm, then the Acinetobacter baumannii liquid (1×108 CFU/ml) of 0.1 ml was evenly daubed on the wound. Next, they were randomly divided into L(n=10, with hydropathic compress of 0.2g/L LL-37 at 3 hours post-injury), M (n=10, with hydropathic compress of 100 g/L mafenide), C(control, n=10, with wet compress of isotonic saline) groups. General observation were performed once a day before determining the quantification of bacteria in subeschar tissue and comparing the histology change under HE stain at 3 days post-injury.Results:1. Successfully determin the minimal inhibitory concentration of LL-37 to the Acinetobacter baumannii was 64μg/ml and minimal bactericidal concentration was 128μg/ml.2. Acinetobacter baumannii liquid (the bacterial concentration of 1× 108 CFU/ml) of 0.5 ml was planted on the deep second-degree burn wound after 10 minutes, and the quantification of bacteria was detected respectively that is 0.09×105 cfu/g, 0.77×105 cfu/g,2.16×105 cfu/g,7.17×105 cfu/g at 12th,24th,48th,72th hour.3. Preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of deep second-degree burn wounds in rats:All rats had survived in the observational trial. The wounds from all the three groups were pale, exudative and red in cut edge. The exudation and red became increasing in group C and L at 3d and 5d, and there was reddish-brown scab in partial wound and little granulation under the scab at 5d. But those had improved at 7d. The group M became dry and scabby with the development of sepia scab three days later, and the growth of granulation tissue had increasing at 5d,7d. The amount of bacterials of tissue in group C and L more than group M at the every time point(P<0.05) While the amount of bacterials between group C and group L had no significant difference(P>0.05). The amount of bacterials in group C and L was increasing from 1d to 5d, but it was beginning to decrease at 7d, while it was decreasing from Id to 7d in group M. The degree of inflammation reaction had no difference among rats from three groups at 1d, There were more obvious inflammatory cellular infiltration in group C and group L than group M at 5d.4. Preliminary observation on the effect of LL-37 on Acinetobacter baumannii infection of traumatic wounds in rats:The wounds were damp with more exudation in group C, while that in other two groups were dry and scabby. The bacterial count in subeschar tissue demonstrated a difference between group M and the other groups at 3 days after injury [group L:(11.9±6.7)×102 CFU/g; group M:(15.2±11.4)×102 CFU/g; group C:(68.1±36.4)×104 CFU/g] (p<0.01). The exudates coated the wounds in three groups, but there were more inflammatory cellular infiltration in group C than group C and group L.Conclusion:1. The MIC of LL-37 to the Acinetobacter baumannii was 64μg/ml and MBC was 128μg/ml.2. The bacteria isolated from the infection wounds was identified as Acinetobacter baumannii. AB liquid with the bacterial concentration of 1×108 CFU/ml was planted on the deep second-degree burn wounds, and the model of infection wounds was established after 48 hours(the count of bacterials>1×105 CFU/g).3. There was no antibacterial effect of the human antibacterial peptide LL-37 on Acinetobacter baumannii infection of deep second-degree burn wound in rats.4. There was obvious antibacterial effect of the human antibacterial peptide LL-37 on Acinetobacter baumannii infection of traumatic wounds in rats. |
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