ANTI-BIOFILM FORMING ACTIVITY OF NATURAL PRODUCTS PUNICA GRANATUM L., MAGNIFERA INDICA L., AND PROBIOTIC AGAINST BIOFILM FORM BY ESCHERICHIA COLI AND PSEUDOMONAS AERUGINOSA AMONG DIFFERENT CLINICAL SPECIMENS.

Abstract

Escherichia coli and Pseudomonas aeruginosa are gram negative rods. The members of Escherichia coli and Pseudomonas aeruginosa are capable of causing infectious diseases in humans and common cause of community acquired infections. Escherichia coli is a type of bacteria normally present in your intestines and some of its strains that cause gastroenteritis in humans can be grouped into six categories: Shiga toxin-producing E. coli (STEC), enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and diffuse adherent (DAEC) can cause serious infections like diarrhea if ingested in any contaminated food or water, or can be transmitted by person-to-person through fecal shedding and accounts for an estimated 11% of infections, most commonly Escherichia coli causes Gastrointestinal illness and outside intestines Urinary tract infections (UTI). An important strain of Escherichia coli, facultative anaerobic bacteria is Shiga toxin strain O157: H has developed immense attention in food and water borne diseases that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) in humans. O157: H infects the alimentary tract and induces abdominal discomfort, cramps and hemorrhagic diarrhea. Pseudomonas aeruginosa classified as strict aerobes and is present on the surface of water, soil and other moist environment in hospitals cause pneumonia, urinary tract infections (UTIs), and bacteremia. In addition to biofilm formation there are various virulence factors that causes chronic infections in humans like elastase, phospholipase C, protease A, exotoxins and cytotoxins, flagella and pili, pigment production, and QS regulatory system proteins, which regulate both virulence factor transcription and biofilm formation. Escherichia coli and Pseudomonas aeruginosa display a characteristic biofilm forming activity, biofilm consist of colonies of bacteria embedded on extracellular xi polymeric substances (EPS), cell to cell communications within these biofilm are major cause and it further enables bacteria to display infections and intensify its pathogenicity. The objective of this study were to identify the biofilm forming bacteria i.e. Escherichia coli and pseudomonas aeruginosa. To determine the anti-biofilm activity of natural product extracts i.e. pomegranate Punica granatum L. and Mango leafs Mangifera Indica L. To determine the anti-biofilm activity of probiotics having Lactobacillus acidophilus. To evaluate the synergistic activity of Natural product extracts and probiotics. The study design is cross-sectional study. The samples were collected from patients at PNS Shifa hospital Karachi. The sample size is 150. The age groups of the individuals include 15 to 50 years. Ethical permission was taken from hospital review committee informed consent for the study was taken from patients. The data collection was designed to collect the demographic data of patients. The specimens received in lab will be inoculated on Blood agar and MacConkey’s agar culture plates. Culture plate will be inoculated at 37 ℃ in incubator for 24 to 48 hours. Identification of Escherichia coli and Pseudomonas aeruginosa is to be done by colony morphology, gram staining, TSI, Different biochemical test. After identification, 96 well micro titer plate will be used to detect biofilm forming activity of Escherichia coli and Pseudomonas aeruginosa. Then use of natural product extracts and probiotics to evaluate there, anti-biofilm forming activity using 96 well micro titer plate. Results among 150 patients 64% were males and 36% were females. Over all mean age was (33.79±9.94) and (34.02±10.59) years. Urine was the most common specimen (66.7%). (68.7%) patients were found with Escherichia coli and (31.3%) with Pseudomonas aeruginosa. While mean biofilm formation was (1.68±0.85) biofilm formation was achieved among (89.3%) of the patients. We found significant relationship between culture and examination (p-value 0.000) while no significant relationship was found between gender, age and biofilm formation (p-value 0.69) (p-value 0.44) and (p-value 0.57) respectively. Anti-bioiflm forming activity of pomegranate peels extract against Escherichia coli and pseudomonas aeruginosa is (24.46±19.09) and (29.26±19.09) respectively. Anti-biofilm forming activity of Mango leafs extract against Escherichia coli and pseudomonas aeruginosa is (14.90±9.56) and (11.45±9.52) respectively. Anti-biofilm forming activity of probiotic having Lactobacillus acidophilus against Escherichia coli and pseudomonas aeruginosa is (0.5x 10^6 CFU/ml) and (0.5x10^5 CFU/ml) respectively. It was concluded that novel combination of natural products extract and probiotics has shown higher effectiveness against these rapid emergence of biofilm forming pathogens and has tremendously xii decreased the mortality and morbidity and significantly reduced the biofilm production. Therefore, natural products and probiotics represent as an alternative treatment option in this era of multi-drug resistances gram negative bacteria. Hence, it was worth mentioning these new alternatives and providing beneficial aspects in reducing different health care associated infections.