Extraction of biofilm produced by Escherichia coli that are isolated from animals infected with diarrhea and study its protective role

Abstract

This study was conducted to detect the ability of E.coli isolated from diarrhea to produce biofilm and protection against diseases cause by these bacteria compared with whole cell sonicated antigen. One hundred two fecal samples (52 fecal samples from cows, calves and 50 fecal samples from sheep,goat) were collected from College of veterinary medicine-university of Baghdad, College of Agriculture-university of Baghdad, Dora zone and Abu-Ghraib zone. Samples were cultured on MacConkey and Eosin Methylene Blue agar and after purification of cultured bacteria, biochemical tests, API 20 E System and RapIDTM ONE System kit were done. Results showed that 91 out of 102 fecal samples have the characteristics belong to E.coli ,the ability of these isolates to produce biofilm were detected and the results showed that 38 out of 49 E.coli isolates from fecal samples of cow produce biofilm (77.55%)and 39 out of 42 E.coli isolates from fecal samples of sheep produce biofilm (92.85%) with different thickness ranged between (0.2-2)mm, while 11 isolates from 49 fecal samples of cow and calves and 39 isolates from 42 fecal samples of sheep had not produce biofilm. The results showed that protein concentration of biofilm was 92 mg/ml for one isolate and 70 mg/ ml for the whole sonicated antigen of the bacteria that produce biofilm. Three types of bacterial antigens were prepared as follow; Whole cell sonicated antigen (WCA) of biofilm producer E.coli, biofilm extract antigen (biA) with protein concentration 3.5 mg/ml and biofilm extract antigen (biA) with protein concentration 14 mg/ml, then these antigens were injected in 50 healthy White BALB mice. Results showed that survival time of animals in the immunized group with biofilm antigen of high protein concentration (14 mg/ml) was longer (652.8 hrs) than animals immunized with whole sonicated antigen of protein concentration 3.5 mg/ml antigen (378 hrs ) and than the group with biofilm antigen of protein concentration 3.5 mg/ml (513.6 hrs) , heavy bacterial isolation were recorded in the internal organs of the immunized infected animals at (12-48) hours post infection, while moderate bacterial isolation at day 30 post infection. Histopathological examination showed large abscess which caused adhesion of liver with stomach and spleen with stomach and pancreas surround by dense cellular fibrous tissue, the result showed that all animals of control positive group,5 animals of group injected with(WCA) produce biofilm,3 animals of group injected with (biA) 3.5 mg/ml and 1 animal of group injected with (biA) 14 mg/ml were died during 12-48 hours post infection, acute suppurative reaction were seen in internal organs of animals died at 12-24 hours post infection, granulomatous lesions were seen in most internal organs of animals in group 2 , 3 and 4 at day 30 post challenge, while mild inflammatory reaction was recorded in most internal organs of group 4 at day 30 post challenge.