Contribution Of Quorum Sensing To The Development Of Biofilm And Virulence Of Pseudomonas Aeruginosa

Bacterial colonization and infection are important factors in compromised wound healing particularly in chronic wounds. Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals .These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. Bacterial biofilms are aggregations of bacteria that live in a highly structured and organized community. Biofilms typically become established by attachment of planktonic cells to a surface, followed by production of an extracellular polymeric substance (EPS) that may consist of polysaccharide, proteins, and nucleic acids. This interconnecting matrix links the bacterial cells together, and begins the establishment of an organized ommunity. Biofilms represent a locus for infection, which is protected from the effects of antibiotics, and present additional obstacles to phagocytes, Particularly chronic P.aeruginosa infections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Bacterial biofilms are increasingly recognised as a major cause of persistent and destructive infectious diseases. However the biofilm formation in critical colonization and a role of biofilms on wounds are well not understood. Although it has been suggested that bacteria are living within a biofilm in the condition of critical colonization, biofilm formation on wounds are well not understood. The aim of this study was to produce a rat model P.aeruginosa inoculated, and demonstration that biofilm formation on the skin wounds morphologically. The full-thickness and partial-thickness wounds were created on the backs of SD rats, Suspensions of P.aeruginosa carring the gene encoding the green fluorescent protein were applied on wounds, and bacterial suspension was not applied in a control group. Both wounds were kept in closed environment during this experiment. Wounds were harvested at 8 hours, 1, 3, 7 days postwounding, processed for histology and immunohistochemictry. Partial-thickness wounds are proper to observe P.aeruginosa biofilm formation, Fluorescence microscopy showed biofilm from 8 hours, and thickened day by day until 3 days after the inoculation. The rate of wound reduction was increased in the experimental group inoculated bacteria compared to the control group. In conclusions, we have developed a experimental model of wound to observe P.aeruginosa biofilms on damaged skin. Full-thickness wounds hava been used to observe rat wounds usually. Howevere, we used partial thickness wounds in this study because we thought full-thickness wounds may be inappropriate to observe biofilm formation.In recent years, it has been demonstated that expression of a large number of these virulence factors in P.aeruginosa is regulated by quorum sensing. The extracellular virulence factors include proteases, pigments, haemolysins, exoenzymes and exotoxin. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms which naturally develop in wounds. Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. When antimicrobial therapy stops, the biofilm can act as a nidus for recurrence of the infection. Biofilm infections can linger for months, years or even a lifetime and usually persist until the colonized surface is surgically removed from the body.Localized tissue ischemia and bacterial colonization were recognized as important factor in compromised wound healing, particularly in chronic wounds. Wound healing in ischemic tissues such as flap margins due to inadequate blood supply is still a source of considerable morbidity in surgical practice. Therefore, we set out to develop a model of ischemic rat wounds infected with Pseudomonas aeruginosa PA01 for use in biofilm stud. Tissue from the excised wounds visualized by fluorescence microscopy showed a significantly greater biofilm biomass and thickness in control group compared to those ischemic group. During the course of 7-day biofilm was found to progressively wax over time Quorum-sensing in Pseudomonas aeruginosa (PAO1) known to regulate several aspects of pathogenesis, including virulence factor production, biofilm development, and antimicrobial resistance. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells. However, the role of quorum sensing in this skin infection models utilizing rat has not been examined. Furthermore these are little known about the importance of Quorum-sensing on biofilm in vivo. Both quorum sensing and biofilm formation are important in the pathogenesis of P. aeruginosa infection, thus knowledge of how quorum sensing systems of P. aeruginosa operate during infection may help us to find a new approach to the treatment of P. aeruginosa wound infections. The objective To understand the importance of quorum sensing in Pseudomonas aeruginosa wound infection, the in vivo pathogenic effects of the wild-type P. aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signalgenerating parts of the quorum sensing systems are defec were compared. These results suggest that the lasI and rhlI genes of P. aeruginosa play an important role in the horizontal spread of P. aeruginosa within wound skin and development of biofilms. In this study, we have used the skin wound model to examine the contribution of quorum-sensing systems to the pathogenesis of P. aeruginosa infections in skin wounds. Our results suggest that both quorum-sensing systems (lasI and rhlI) contribute to the virulence of P. aeruginosa. In comparison with P. aeruginosa parent strain PAO1, lasI, and rhlI mutants showed a reduction in virulence. In addition, mutants were less efficient than PAO1 in spreading within the tissue, confirmed in this study, the in vivo virulence of the mutant is significantly lower than that of PAO1. It is clear from the present results that mutations in the quorum-sensing systems interfered with the ability of P. aeruginosa to cause general and local damage in the wound infection. Based on these data, the expression of quorum-sensing genes may be a mechanism whereby P. aeruginosa gains a protective advantage, by making a concerted effort as a population to overcome host defenses and establish an infection. In this study, Blank grops and lasI /rhlI mutant showed a greater level of wound epithelization on day 7 and compared to PAO1 group. One possible explanation for these findings is that the epithelization was delayed by infection, or owing to the biofilm, directed to inhibition the epithelization process or other factors that are controlled by either quorum-sensing system. these studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants , the biofilm of PAO1 had significantly greater biomass and thickness, compared to the biomass and thickness of lasI /rhlI mutant, the rhlI mutant formed only slightly less biofilm than the wild type. Our data indicate that the Quorum sensing regulates the expression of several secreted virulence factors and lasI/rhlI mutant strains are attenuated for virulence in rat wound models. Ffurthermore, the Quorum sensing is a potent inducer of biofilm in rat wound. Thus all these data indicate that the lasI and rhlI gene of P. aeruginosa play an important role during the infectious process. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies.Antiseptics have long and widely been used on wounds to prevent infection. It is well known that Povidone–iodine has a broad anti-microbial spectrum and is very effective in the treatment of various types of wounds, and no resistance has been determined . The usefulness of antisepics on intact skin is well established and broadly accepted. However, the use of antiseptics as prophylactic anti-infective agehts for open wounds has been an area of intense controversy for several years. Povidone iodine is available in several forms (solution, cream, ointment, scrub), but without control of dosage, iodine can cause tissue staining and possible local toxicity. The Two Component Gel (TCG) is a new iodine formulation which generates low, safe and effective levels of molecular iodine at the point of delivery to a wound. Ischemic flaps were raised using aseptic technique, two rectangular (18 x 55 mm) random skin flaps were raised superficial to the deep muscular fascia. Full thickness skin wounds were created at 45mm from the flap base. Bacterial suspension (Pseudomonas aeruginosa) was inoculated onto the base of the ulcer . A total of 28 wounds were created. Each wound was randomly assigned to either the antimicrobial gel (two Component Gel [TCG] or the Gel Vehicle (without iodine). The 2 days after operation was designated as day 0. On days 0, 2 and 4 the swabs were taken from the base of the wound for total bacterial count. At the final timepoint, the wounds were removed in their entirity using a scalpel. Each wound was divided into 2 equal parts, one part was used for total bacterial count and the other part was rapidly frozen in liquid nitrogen or embed in paraffin directly. The biopsies of each wound were stained with haematoxylin and eosin or Rhodamine-conjugated Concanavalin A and were then observed by light and fluorescence microscopy. Biopsy samples were observed for evidence of epithelialisation and biofilm. In this study, TCG effectively reduces bacterial counts and wound exudates, in the swab, the TCG treated wounds were less contaminated, TCG showed significant decreases in quantitative bacteriological counts. That show a significant difference in bactericidal activity in vivo between pre-TCG and post-TCG, on day2 gradually fewer microorganisms were cultured from the wounds of the TCG ointment treated group. On day4, the number of colony forming units per swab was significantly more reduced in TCG group. Tissue from the excised wounds visualized by fluorescence microscopy showed a significantly greater biofilm biomass and thickness in wounds treated with the TCG vehicle compared to those treated with the TCG .In rat wounds that were treated with the TCG, reduction in wound exudate and faster epithelialisation was observed relative to the vehicle control. Also, the TCG inhibited biofilm development on the surface of the wound. Thus we suggest that the TCG is a promising treatment to inhibit biofilm development and clear Pseudomonas aeruginosa cells within biofilm from skin lesions of exudative or infectious wounds and to prevent wound exacerbation.