Detection of Mechanism of Invasive Activity of Burn Wound Pathogens and Their Susceptibility to Antibiotic and Natural Plant Products

Abstract

In emergency units burn injuries are considered to be the most agonizing injuries, ranging from minor to lethal. Developing countries such as Pakistan have a high incidence of burn injuries. Burn wound provides an ideal environment for the growth of pathogens. Burn wound serves as portal of invasion by microorganisms. Infections developed in a consequence of burn wound are a leading cause of death. Pseudomonas aeruginosa a non-spore forming, non-fermentative Gram negative bacillus, remains serious wound infection in burn patients and give rise to burn related morbidity and mortality worldwide. This study presents an overview about the mechanism of invasive activity of burn wound pathogens as well as ponders their alternative treatment options with natural plant products. To determine the spectrum of burn wound pathogens, a total of 350 pus samples were collected from patients having burn wound infections with total body surface area (TBSA) ranging from 1-90%. P. aeruginosa was observed in 26% cases as the main culprit of burn wound infection, irrespective of the type of thermal injuries. In addition, the incidence of bacteremia in burn patients was also determined in 150 patients and we observed bacteremia in 43(29%) of cases. It was important to note that in 15 cases the causative agent of bacteremia was P. aeruginosa which confirmed the well defined role of P. aeruginosa in burn patients. It was also of interest to evaluate time related changes in microbial colonization in burn wound infection. Fifty patients were followed up for four weeks at weekly interval. Results revealed that S. aureus was found to be the most frequently isolated pathogen during the 1st week while it was suppressed by Pseudomonas species from 2nd week. As for Candida spp., there was a gradual increase in the frequency of its recovery as time elapsed from admission. The risk regarding the acquisition of P. aeruginosa infection by burn patient is highly confronting because this organism is widely distributed in the hospital setting and can easily be transmitted to burn patients. To determine the exact role of the environment in the acquisition of P. aeruginosa by the burn patient, a total of 130 samples from different hospital sites including wall, floor, patient’s bed, Washing tubs and OT equipments was collected. It was interesting to find that in our results, distribution of P. aeruginosa varies with site of sample collection. Multidrug resistant bacteria have commonly been reported as colonizers of the wounds of burn patients. In order to select an effective antibiotic regimen to overcome the incidence of burn wound infection, the drug sensitivity pattern of burn wound pathogens were determined. Testing of 15 selected antibiotics yielded significant results, as much as 39% of the strains of S. aureus were found to be methicillin resistant. Vancomycin was the only “anti-staph” drug that inhibited the growth of all strains of S. aureus tested while linezolid acted on 95% of strains challenged. Additionally, polymyxin B and piperacillin/tazobactum were the drugs that found decidedly effective against Gram negative isolates of burn wound infection. The emergence of antimicrobial resistance among burn wound pathogens worldwide confine the possible therapeutic alternatives, used to treat burn wound infections. To ponder the alternative treatment options with natural plant products several plants were screened. Our finding reveled, extracts of Brassica rapa sub-spp. Campestris showed inhibitory activity against P. aeruginosa. On the basis of our observations extracts of Brassica rapa (SM2-PE and SM2-EA) were further investigated via gas chromatography-mass spectrometry (GC-MS) technique, for determination of bioactive components. 29 compounds were identified from SM2-PE and 35 compounds were identified from SM2-EA. The major classes of the identified compounds belonged to the monounsaturated fatty acid, methyl and ethyl esters. Both of the extracts were rich in fatty acid esters thus can be used as potential natural therapeutics. It is the first authenticated report on antibacterial activity and chemical composition of Brassica rapa sub-spp. campestris from Pakistan. P. aeruginosa possessed several virulence factors such as, biofilm formation, exotoxin A and type III secretion system. These factors play important role in invasion, survival against the immunological responses of host and spread of infections. Biofilm formation ability of P. aeruginosa was detected by three methods. Among all tested P. aeruginosa isolates 57% were found to be biofilm producers. Additionally, genetic analysis of Exotoxin A gene sequences revealed the little difference in virulence gene distribution in P. aeruginosa isolated from different origins. 92% of P. aeruginosa from burn wound contained Exotoxin A genes while this gene was found in 89% of environmental strains of P. aeruginsa. Furthermore, prevalence of type III secretion system gens (ExoS, ExoT, ExoU, ExoY) of P. aeruginosa from burn wound as well as environmental isolates were determined. Results revealed that all examined burn wound and environmental isolates harboured Exo T gene. While ExoU was found in 58% and 33% P. aeruginosa isolated from burn wound and environmental samples respectively. ExoS-was found in a minority of isolates. The prevalence of ExoSgenes varied from 36% in burn wound isolates and 6 % in environmental isolates. The PCR-based gene detection assay yielded that 90% of burn wound isolates and 10% of environmental isolates contained ExoY gene. To determine the expression of type III secretion exo-enzymes in the P. aeruginosa, we examined P. aeruginosa isolates from burn wound infection among them 5 were invasive strain (ExoS+/ExoU-) and 5 were cytotoxic strains (ExoS-/ExoU+) by (RT–PCR), under inducing condition all invasive strains of P. aeruginosa expressed ExoS while among cytotoxic strains only (40%) expressed ExoU. This is the first study from Pakistan providing evidence of a role for this type III secretion system in burn wound infection. On the basis of our observations, we conclude that burn wound infections and bacteremia are increasing in Pakistan. A continuous monitoring of burn wound pathogens as well as the up to date profiles of resistance to different drugs are necessary to develop better strategies for the treatment of burn patients. Biofilm forming property of P. aeruginosa not only contribute in increasing the severity of infection but it also participate in dilation of wound healing by effecting the defense mechanism of patient. P. aeruginosa that exhibited ETA gene may play an important role in the spread of infection. High prevalence of cytotoxic (ExoU+) strains of P. aeruginosa can induce severe inflammation because of its cytotoxic nature. Induced expression of ExoS proteins under depleted calcium conditions suggested that ExoS+ P. aeruginosa affects the immune system of patients which results in the accumulation of ExoS proteins in burn wound that can contribute in the development of infection. Type III secretion exoenzymes are a major virulence factor of P. aeruginosa which play important role in its pathogenicity, resulting in increased hospital stay and expense of treatment with significant economical burden. We are first time reporting that the fatty acid esters of Brassica rapa sub-spp. campestris Linn. can be use as potential natural therapeutics to treat not only burn wound infections but other infections as well. The results of this study will contribute in the development of new therapeutic agents to conquer the incidence of wound infections in burn patients and will benefit Pakistan economy by saving million of rupees, spend annually on patients who develop wound infection secondary to burn injury.