In Vivo Evaluation of Genetically Engineered Lactobacillus against Uropathogenic E. coli

Abstract

Approximately 80% of the women are suffering from Urinary Tract Infection. Among which 90% is caused by UPEC. There is a condition of Antibiotic resistance due to overuse of antibiotics and hence probiotic can be used as an alternative strategy for this condition. During menstrual cycle, the oestrogen levels decreases due to which there is a decrease in the lactobacillus count, which in turn leads to drop in vaginal pH. UPEC colonization becomes easier in the basic environment. The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocins can be an excellent agent to develop as a novel therapeutic agent and complement conventional antibiotic therapy. Hence genetically transformed lactobacillus containing LL37 and colE2 can be used which will compensate the loss of lactobacillus as well as help in UPEC clearance. In the present study the efficacy of antimicrobial peptide (AMP) produced by genetically transformed lactobacillus (Trivedi et.al 2013) against UPEC was checked in vivo. UPEC binds with the help of virulence factor fim1 to the UPa 1 expressing on the urothellium of the bladder and UPEC internalization takes place with the help of integrins and CD44 genes. This adhesion upregulates the host defence system inducing TLR signalling and help fighting against UPEC. Primers for Genes (UPa 1, CD44, α5β1integrin, TLR 2, TLR 4 and NFĸb) were designed for expression studies. UPEC was intravaginally (107-9 cfu/ml) administered on weekly basis till 45 days and intraperitoneally (103-4 cfu/ml) administered once on the day of induction in murine model. Mice were subsequently treated with L. brevis, intravaginally (107-9 cfu/ml) after the 7th day of infection till the 45th day and in the intraperitoneally induced model, mice were fed orally (107-9 cfu/ml) once on the day of infection. Intraurethrally induced animals were autopsied on 15th and 45th day, Kidney and Vaginal Epithellium were collected for Gene expression studies. Similarly intraperitoneally induced animals were monitored and autopsied on 18th, 32th and 48th hour; Kidney and Small Intestine were collected for Gene expression studies. Blood samples from both the models were collected for Biochemical assays, Antioxidant assays and LPS detection. Urine was collected from both the models for HPLC analysis to check the presence of urinary organic acids i.e. Lactic acid, Acetic acid and Fumaric acid. Samples like Liver, Kidney, Small Intestine and Bladder were collected for Histopathological studies. Page v The levels of antioxidants were significantly less in the group treated with genetically modified L. brevis expressing colE2 as compared to LL37 in both the models. The LPS levels were significantly high in the groups infected with UPEC in both thev models. There was a condition of septic shock in IP infected group, colE2 was more efficient in reducing the LPS levels as compared to LL37. In the control group concentration of lactic acid and acetic acid was observed whereas fumaric acid was absent. In the IU model the levels of Lactic acid increased in the group treated with LL37 and there was a steep decrease in the infected group. In the IP model, the levels of fumaric acid decreased in the group treated with colE2. Acetic acid levels dropped in the infected group while an increase was observed in the group treated with LL37. Morphological changes in the infected group were observed in bladder, liver and small intestine as compared to control group and no major changes was observed in kidney. Gene expression results are still in progress. The results overall indicated that colE2 producing probiotic is better than LL37 and it may be a potential candidate for alternative agent to control Urinary Tract Infection (UTI) and other pathogens.