Evaluation of antibacterial effect by ozone therapy on burn injury

Nội dung text: Evaluation of antibacterial effect by ozone therapy on burn injury

1. Journal of military pharmaco-medicine 7-2013 EVALUATION OF ANTIBACTERIAL EFFECT BY OZONE THERAPY ON BURN INJURY Ho Anh Son*; Nguyen Linh Toan*; Vu Quoc Binh** SUMMARY Accidental and war trauma, burns and all sorts of acute and chronic cutaneous infections can be proficiently treated with ozonated water. In current study, 24 burned rabbits were divided into 4 groups (6 rabbits in each group): topical treatment with saline; topical treatment with ozone; topical treatment with ozone and ozone autohemotherapy (OAHT); and topical treatment with silvirin. The results showed that topical use of ozone in burn wounds present in animal model allow a rapid antibacterial effectiveness during the treatment periods. * Key words: Bacteria; Ozone autohemotherapy; Burn; Rabbit. INTRODUCTION Human skin is the barrier to prevent entering of bacteria to the body, when the skin is burned, these bacteria enter the wound and cause infection site. Bacteria can enter the burn wounds from healthy skin around, waste of patients, from the care people, from linens, from health care workers, and the atmosphere, drugs, the medical instruments Many reports have warned that burns hospital infections are a major cause of death in burn patients (accounting for 50 - 70%). The serious treated problem of drug-resistant bacteria present in the burn is also mentioned. Besides, the cost of using systematic and tropical antibiotics accounts for a large proportion of funding treatment. Therefore, we conducted this study using ozone as an antimicrobial substance with strong effect has been known for a long treated history, with a very low cost to assess their ability to antibacteria on the surface of burn wounds. SUBJECTS AND METHOD 1. Study subjects. Healthy Newzealand rabbits (from a farm in Hungyen province), medium weight 2 kg/each. Rabbits were breed in experimental conditions, individual clean cages during the study period and free access to food, vegetable. Total number of rabbits was 24, including both male and female, were randomized divided into 4 groups of 6 rabbits: - NaCl group: animals were burned and the wounds were cleaned by saline solution. - NaCl + O3 group: animals were burned and the wounds were cleaned by ozonated saline solution. - O3 + O3 group: animals were burned and the wounds were cleaned by saline solution and OAHT. - Silvirin group: animals were burned and the wounds were treated by Silvirin. The experiment were performed in Pathophysiology Department, Vietnam Military Medical University. 2. Method. * Ozone autohemotherapy: The method was descibed somewhere [1], in brief: 5 ml/kg body weigh ear vein blood was collected in heparined plastic container with mixture of ozone-oxygen. The blood is exposed to the gas mixture for 5 minutes and transfered back to the rabbit. The process of OAHT was conducted in total of 6 times, 3 times per week, starting with 50 mcg/ml ozone dose and increased to 50% after every 2 time of treatment [1, 2, 3]. 1
2. Journal of military pharmaco-medicine 7-2013 Figure 1: Transfusion process of ozone exposure to rabbit vein blood. * Experimental burn rabbit model: Rabbits fur on back are removed, then they were mounted on the table. Animals were anesthetised by thiophental, 50 mg/kg dose. A cyclindal container was poured by boiling water and keep perpendicular to the back skin, followed by heavy weights 1 kg set up container for 30 seconds. The wound was done on both spine sides with grade III lesions, and covers about 10% of the total surface area of rabbit skin. * Ozonated saline to rinse the wounds: Ozone dose of saline to wash the wound began 80 mcg/ml. After the first washed day, the ozonated saline doses were reduced 10 mcg/ml per day. When the dose is reduced to 10 mcg/ml, this concentration is maintained until the wounds were heal. * Assess the level of infection in burn wounds: Microbiological tests in burns: rolling sterile swab on the surface burns injury, which is 1 cm2 area, is limited by the plastic paper. The test were performed before, after the of 3, 7 and 14 days of ozone treatment. The sterile cotton swab soaked in 0.9% NaCl sterile, push the swab into a glass tube to reduce water, gently rolling on the wounds, roll across the gaps in the plastic, so that the burn fluid stained on the entire circumference of the swab for 20 seconds. The swab specimens were put into 5 ml tubes available 0.9% NaCl sterile. Shaking slightly the tube for about 1 minute, transfer to lab microorganisms. Specimens were staging on the different cultures agar, incubated at 370 C for 24 hours. Count the number of colonies on the agar culture, then calculate the number of bacteria on the surface of 1 cm2 burn wounds. The technique was conducted on Bitex system in Paraclinical Department, National Institute of Burns. * Data analysis: Data was analyzes by Stata software and One way-ANOVA, SPSS 12.0. RESULT Table 1: The rate of infection in rabbits burns day 3rd. Group Rate of infection (%) (n = 12/group) b (-) b 105 NaCl group (1) 0 0 0 0 100 2
3. Journal of military pharmaco-medicine 7-2013 NaCl + O3 group (2) 33.3 0 0 0 66.7 O3 + O3 group (3) 33.3 0 0 0 66.7 Silvirin group (4) 0 0 0 0 100 p > 0.05 (b: bacteria) Results showed that the rate of microbial infections in burn wounds after 3 days of treatment was quite common in the study groups. With two groups using ozonated saline to clean wounds, rate of burns infection was 33.3%. Other burns in the remaining groups had over 105 bacteria/cm2. Chi-square statical analysis showes no significant diference of rate infection among agoups. Table 2: The rate of infection in rabbits burns day 7th. Group Rate of infection (%) (n = 12/group) b (-) b 105 NaCl group (1) 0 0 0 16.7 83.3 NaCl + O3 group (2) 0 0 0 16.7 83.3 O3 + O3 group (3) 16.7 0 0 50 33.3 Silvirin group (4) 0 0 50 0 50 > 0.05 p3-4 p3-1,2 < 0.05 p p4-1,2,3 < 0.005 0.05 Data showed: infection rates increased significantly on day 7 after the process of burn treatment. Only 2 burns (16.7%) in O3 + O3 treated group, bacteria did not grow in the culture dish. In the other burns, population of bacteria was over 103/cm2. In range 103 - 104/cm2 bacterial infected burns, there were 6 (50%) of 12 wounds in 2 silvirin treated group, it is significanly lower than other groups (X = 8.0; p3-4 < 0.005). The range 104 - 105/cm2 bacterial infected burns, there were 6 (50%) of 12 wounds in 2 silvirin treated group, it is significanly lower than silvirin groups (X = 8.0; p3-4 < 0.005). A mong bacterial infected burns over 105/cm2, there were four (33.3%) of 12 wounds in 2 O3 + O3 treated group, it is significanly lower than NaCl and NaCl+O3 treated group (X = 6.17; p3- 1,2 < 0.05). Table 3: The rate of infection in rabbits burns day 14th. 3
4. Journal of military pharmaco-medicine 7-2013 Group rate of infection (%) (n = 12/group) b (-) b 105 NaCl group (1) 0 0 0 16.7 83.3 NaCl + O3 group (2) 83.3 0 0 16.7 0 O3 + O3 group (3) 66.7 0 0 0 33.3 Silvirin group (4) 25 0 25 0 50 p2-1 < 0.001 p2-4 < 0.005 p p1-2<0.001 p3-1 < 0.005 P1-3<0.05 p3-4 < 0.05 p4-2<0.005 Results showed advantages of using ozone therapy can reduced infected burns significantly. Negative culture bacteria accounted for 66.7% in O3 + O3 treated group and 83.3% 2 in NaCl + O3 treated group, it is significantly higher than NaCl and silvirin treated group (X = 2 2 2 17.1, p2-1 < 0.001; X = 8.22, p2-4 < 0.005; X = 12, p3-1 < 0.005; X = 4.2, p3-4 < 0.05). Meanwhile, the NaCl and silvirin treated group were 100% and 75% burn-positive 2 2 2 bacterial culture, respectively (X = 17.1, p1-2 < 0.001; X = 6.17, p1-3 < 0.05; X = 8, p4-2 < 0.005). Table 4: Speed of narrowing the wound size. Group Wound size (cm2) (n = 12/group) Day 3rd Day 7th Day 14th Day 21th Day 28th NaCl group (1) 15,1 0,86 10,8 0,77 6,6 0,36 2,93 0,18 1,24 0,45 NaCl + O3 group (2) 13,42 0,79 9,26 0,44 5,92 0,36 2,02 1,31 0,28 0,06 O3 + O3 group (3) 14,3 0,86 9,5 1,14 5,37 0,65 1,31 0,32 0,39 0,17 Silvirin group (4) 14,8 0,67 10,7 0,56 6 1,03 2,15 0,89 0,32 0,13 p > 0,05 p1 > 0,05 p1,3 < 0,05 p1,3 < 0,05 p1,2;1,3;1,4 < 0,05 4
5. Journal of military pharmaco-medicine 7-2013 The results showed that, in the first 7 days of treatment, there was no significant difference of the burn area in the groups. The other treatments have not demonstrated a significant improvement in speed of wound narrowing. Figure 2: Speed of narrowing the wound size. th th However, from day 14 to day 21 , the area of the O3 + O3 treated group has narrowed significantly compared to NaCl treated group. By the end of the experiment on day 28 th, the difference in size between the control group (NaCl) compared with othe groups treated with ozone and silvirin was significantly larger (p < 0.05). DISCUSSION Ozone increases the immune response and the role of anti-free radical system when the body suffers serious stress. It could preventing, reducing, normalized the chronic oxidative stress and enhanced release of bone marrow stem cells. In addition, ozone is applied for the treatment of local infections, chronical ulcers. Disinfection probability of ozone is based on the chemical structure of three oxygen atoms. Ozone is strong oxidants, capable of destroying most of the organic compounds, ozone is also capable of killing fungal spores and bacteria. Therefore, ozone is often used as an antiseptic and has the strongest effect. In burn wounds, 80 mcg/ml ozonated saline in the early days had strongly antiseptic effect and rinse the necrosis tissue (Bocci, 2005) [3]. The next days, lower concentration of ozone was still able to kill bacteria but are harmless to tissue in the wound healing process. Local treatment with ozonated saline and/or in combination with OAHT could strongly effect on reducing the burns infections (Giunta et al, 2001; Zamora et al, 2005) [5]. The advantage is not only comparable with normal saline treated group, but also with silvirin treated group. By observation, we found the burns surface treated by ozonated saline, dryer and cleaner than other treated groups. Thus, besides the probability to kill bacteria, ozone has a cleansing effect, dry surface and promote the burn healing (De Souza et al, 2010) [4]. By that way, ozone is directly or indirectly reducing the amount of bacteria in surface burns injury. CONCLUSION This study evaluated the bacterial resistant effectiveness of ozone, were performed on 24 burned rabbits, which were burned with the same area and depth. Rabbits were divided into 4 groups, including group of saline treatment, ozonated saline, OAHT and topical creams silvirin. The results indicated that ozone therapy is a promising treatment method in burn injury, it could significantly reduces burn wound infections and promotes wound healing. REFERENCES 5
6. Journal of military pharmaco-medicine 7-2013 1. Vu Quoc Binh, Ho Anh Son, Nguyen Linh Toan. No effect of ozonenate autohemotherapy on plasma SOD activity, glucose, acid uric, IL-1α, TNF-α and IL-10 levels in healthy rabbits. Journal of 108- clinical Medicine and Pharmacy. 2011, 3 (6), pp.24-29. 2. Bocci B, Valacchi G, Corradeschi F, Fanetti G. Studies on the biological effects of ozone: Effects on the total antioxidant status and on interleukin-8 production. Med Infl. 1998, 7, pp.313-317. 3. Bocci V. Ozone - a new medical drug. Springer. 2005. 4. De Souza Y.M, Fontes B, Martins J.O, Sannomiya P, Brito G.S, Younes R.N, Rasslan S. Evaluation of the effects of ozone therapy in the treatment of intra-abdominal infection in rats. Clinics. 2010, 65, pp.195-202. 5. Giunta R, Coppola A, Luongo C, Sammartino A, Guastafierro S, Grassia A, Giunta L, Mascolo L, Tirelli A, Coppola A. Ozonized autohemotransfusion improves hemorheological parameters and oxygen delivery to tissues in patients with peripheral occlusive arterial disease. Ann Hematol. 2001, 80, pp.745- 748. 6. Zamora ZB, Borrego A, López OY, Delgado R, González R, Menéndez S, Hernández F, Schulz S. Effects of ozone oxidative preconditioning on TNF-alpha release and antioxidant-prooxidant intracellular balance in mice during endotoxic shock. Mediators Inflamm. 2005, 24, pp.16-22. 6