STUDY OF MOLECULAR COMPOSITION OF VIRULANCE BACTERIA ISOLATED FROM BOVINE MASTITIS WITH BIOFILM PRODUCTION

Abstract

This study was carried out in order to isolate and diagnosis of common bacteria from bovine mastitis in Baghdad city which have the ability to produce biofilm and detect its molecular composition. Twenty five milk samples were collected from different regions in Baghdad city from udders of cows suffering from clinical and subclinical mastitis.Then cultured on blood agar. Gram staining was done to differentiate between bacteria gram positive which cultured on Mannitol Salt Agar and Nutrient Agar while gram negative bacteria were cultured on MacConkey agar and Eosin Methylene Blue agar. All bacterial isolates were subjected to different biochemical tests, API 20 E System, API Staph System and RapID^TMONE System kit to confirm the diagnosis.Christensen tube method was used to detect the ability of the diagnostic bacterial isolates to produce biofilm. Specific forward and reverse primers were designed according to a program from NCBI-Genbank  for Staphylococcus aureus (Genbank code: gb |KR265472.1|), for Escherichia coli : gb|JQ781567.1| and for Klebsiella pneumoniae gb|KT944736.1|, To study  the sequence of these genes  after amplification of 16srRNA genes  by using PCR was appeared because it is sensitive and highly specific assay serve as suitable molecular diagnostic tool for detection and compare these genes sequencing with references strains.Results showed that 8 (32%) out of 25 milk samples were positive for Staphylococcus , 6 (24%) out of  25  samples were positive for Klebsiella pneumonia . and 11(44%) were positive for E.coli . The results showed that 22 (88%) isolates out of 25 milk have the ability to produce biofilm . Genetic identities results showed that Staphylococcus aureus and Klebsiella pneumonia isolates gave 99% matching and resembling the reference strains while Escherichia coli isolates identity showed resemble match of 100% with the reference strain