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dc.contributor.authorErum, Rakhshanda-
dc.description.abstractSurgical site infections (SSIs) are infections that occur in the wound created by an invasive surgical procedure and are associated with increasing rates of morbidity, prolonged hospital stay, considerable socio-economic costs and death. SSIs are the most widely recognized nosocomial infections which represent 38% of all healthcare-associated infections. As compared to developed countries surgical-site infections are more often reported, 2-5 times higher rate from resource limited countries such as Pakistan. Infection with resistant pathogens including Candida species particularly Candida albicans is associated with an increased treatment episodes of surgical patients. C. albicans is a dimorphic fungus. C. albicans found as normal flora in oral cavities, urogenital and gastrointestinal tracts of healthy people but it can cause an assortment of infections which depends on the immune status of host. This study presents an overview about the spectrum of surgical wound pathogens, their mechanism of virulence as well as ponders their alternative treatment options with natural plant products. The study also highlight the role of Candida species in SSIs which is went unrecognized and was underestimated for long time by clinicians and microbiologists, resulted in unreported and untreated infections actually caused by this ubiquitous yeast. To determine the spectrum of surgical wound pathogens, a total of 450 pus samples were collected from patients having surgical site infections. Candida species were observed in 21% cases irrespective of their age and sex. Additionally, the incidence of bacteremia secondary to SSIs was also determined in 110 patients and we observed bacteremia in 23(21%) of cases. Furthermore, a total of 105 swab samples collected from tong dorsum and jugal mucosa of healthy individuals were also collected to determine the prevalence of C. albicans in healthy individuals as well as to compare their virulence properties with Candida spp. isolated from surgical site infections. 19.04% of samples from healthy individuals yielded C. albicans. Surgical site infections are becoming more important given the current situation of increasing antibiotic resistance in human pathogens. For choosing an efficient empirical chemotherapy to prevent surgical site infection drug susceptibility patterns of surgical wound pathogens were investigated. Testing of 15 selected antibiotics and 4 antifungal agents on surgical wound pathogens yielded significant results, as many as 34% of the S. aureus were noted to be methicillin resistant. Vancomycin was the only “anti-staph” drug that was effective on all the S. aureus isolates challenged; however, Linezolid acted on 92% of strains tested. Also noted to be decidedly capable in effect among the drugs tested were polymyxin B and piperacillin/tazobactum against Gram negative isolates. C. albicans isolates showed 100% susceptibility to voriconazole and amphotericin followed by itraconazole 98% and fluconazole 89%. The emergence of antimicrobial resistance limits the available therapeutic options for effective treatment of surgical wound infections. To develop new alternative antifungal agents, several plants were screened for their potential antifungal activity. Our finding reveled, extracts of Mansoa alliacea Lam. showed inhibitory activity against all Candida species tested. On the basis of these observations, the bioactive components of Mansoa alliacea Lam. were analyzed through gas chromatography-mass spectrometry (GC-MS) technique. Altogether 25 compounds were identified from two different extracts i.e. CGLM-PE and CGLM-EA in which 21 are identified first time from this source. The major classes of identified compounds in both fractions were saturated fatty acid, alkane, terpene and phytosterol. 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 antifungal activity and chemical composition of Mansoa alliacea Lam. from Pakistan. C. albicans has many virulence factors such as dimorphism, phenotypic switching, biofilm formation and hydrolytic enzymes that contribute to infection, penetration, and survival against the host defense systems. Biofilm formation is an important virulence factor of C. albicans. Biofilm formation by certain Candida species strains was verified by using two modalities, and we observed biofilm production in 46% of C. albicans isolates. Phospholipase and proteinase are two putative virulence factors of Candida species which are assumed as enhancer of active penetration and extracellular nutrient acquisition. In this study we compared the virulence potential of Candida species, isolated from surgical site infections and healthy individuals by estimating the production level of their exo-enzymes. Results revealed that phospholipase and proteinase activity was more pronounced in Candida Species from surgical wound in contrast to species isolated from healthy individuals. To date, ten members (SAP 1–10) have been recognized in family of secreted aspartic proteases (SAPs) of C. albicans which were encoded by multigene family. To analyze genetic versatility of C. albicans, prevalence of six virulence genes (SAP1-SAP6) of secretory aspartyl proteinase of C. albicans was determined. Among all 54 C. albicans tested, synchronous presence of all tested genes was detected in 24 isolates. SAP5 and SAP6 genes were synchronously lacking in eight isolates while six isolates were found to be synchronous deficient for SAP4 and SAP5 genes. However, sixteen C. albicans showed synchronous presence of all tested genes except SAP5. To evaluate the difference in the patterns of SAP 1-10 genes expression of C. albicans strains isolated from patients with surgical site infections, we screened strains of C. albicans for SAP gene expression under Sap-inducing and non-Sap-inducing condition by (RT-PCR). C. albicans strains showed marked differences in the expression of SAP1-10 genes in inducing and non inducing conditions in vitro. The expression levels of SAP9 – SAP 10 were more pronounced in both inducing and non inducing conditions, while SAP1 and SAP3 were higher in the inducing condition. SAP2 expression was also more frequent in inducing condition as compared to Sap non-inducing condition. Majority of C. albicans strains express SAP4-SAP6 independent of the inducing and non inducing conditions. One of the important finding of this study was the detection of SAP7 mRNA in strains of C. albicans analyzed. The lack of SAP7 expression under non-Sap-inducing conditions and less frequent expression under Sap-inducing conditions indicated that SAP7 may undergo a different transcriptional regulation. This is the first study from Pakistan demonstrating the role for this proteinase family in surgical site infections. On the basis of our observations, we conclude that surgical site infections and bacteremia are increasing in Pakistan. Development of resistance to fluconazole in Candida species has become a matter of concern for treating Candidal infections in surgical patients. For selection of an efficient empirical chemotherapy to prevent SSIs, a continuous monitoring of microorganisms and their drug susceptibility pattern are needed. Candida species have ability to form biofilm which not only delay wound healing but also enhance severity of infection by compromising the immune system of patient. The more pronounced activity of exo-enzymes in Candida species isolated from surgical wound in contrast to species isolated from healthy individuals highlights the role of these enzymes in surgical wounds. The high frequency and the high rate of association patterns of SAP genes in C. albicans strains suggest that secretory aspartyl proteinase is a major virulence factor of C. albicans which contributes significantly to its pathogenicity. The more frequent expression of SAP2 under inducing condition indicated that this proteinase is involved in colonization, infection as well as tissue damaging in SSIs. Moreover, on the whole, the patterns of SAP1-10 expression clearly demonstrate that multiple SAP expression profiles exist during surgical site infection which might prone C. albicans strains to behave as virulent strains. In order to minimize the risk of surgical site infections there is need to support patient’s immune mechanisms, rapid elimination of reservoirs of infection, suppression of infection transfer and development of alternative therapies to aid patients in recovering from surgical site infection. We are first time reporting that extract of Mansoa alliacea Lam. possessed antifungal activity and are rich in fatty acid esters so these fatty acid esters can be use as potential natural therapeutics to develop more effective and non toxic agents to treat Candida infections. The results of this study will support the development of new therapeutic agents to overcome the incidence of surgical wound infections and will contribute to the economic growth and stability by reducing the rate of infections as well as by giving sustainable reduction in morbidity and mortality of patients with surgical site infection.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.publisherUniversity of Karachi, Karachi.en_US
dc.titleTo Study the Spectrum of Microbial Pathogens and Their Mechanism of Virulence in Patients with Postoperative Wound Infectionen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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