Improvement Of Rapid Diagnostic And Antibiotic Sensitivity Tests For Bovine Mastitis-Related Escherichia Coli

Bovine mastitis is one the major diseases of dairy cattle which causes heavy losses to dairy industry. The main pathogenic microorganisms of bovine mastitis include Staphylococcus aureus, Streptococcus and Escherichia coli (E.coli). Treatment of mastitis is based with antibiotics, but the misuse of antibiotics has led to antibiotic residues in milk, widespread drug-resistant strains. There are many methods for diagnosis of mastitis-related E.coli, but the identification procedure is time-consuming and more rapid and simple diagnostic methods are needed.In this study, we improved the previously developed MQQ selective medium by using resazurin sodium (RS) as indicator for more rapid diagnosis and drug sensitive testing of mastitis-related E. coli.On the basis of E. coli selective medium MQQ, we optimized the concentrations of resazurin sodium indicator and the inoculated bacterium. studied systematically cultivation time and selective characteristics, and verified the selection of MQQ-RS with mastitis related bacteria and their combination simulated milk samples, and then diagnosis clinical milk samples of E. coli. The modified medium MQQ-RS remained the strong selective property of the original medium, which supported growth of E. coli but not Staphylococcus and Streptococcus. After inoculation of E. coli pure cultures and cultivation for a few hours, the diagnostic results could be obtained by direct observation of the color change of the medium. The definitive diagnosis could be made in4hours for higher doses of inoculation and in10hours for lower doses of inoculation. The experiments of the simulated milk samples showed that MQQ-RS medium could be used for quick diagnosis of E. coli by direct inoculation of clinical milk samples. For diagnosis79clinical milk samples, the MQQ-RS method had93.6%agreement with the standard method.To establish a more quick antibiotic sensitivity test based on MQQ-RS medium, the antibiotic sensitivities of10different antibiotics against8E. coli isolates were tested by the standard disc diffusion method and the results were used as the reference for adjusting the antibiotic concentrations for MQQ-RS culture. By using adjusted concentrations of antibiotics and the reference E. coli isolates, the similar results were obtained for MQQ-RS quick cultivation method and the standard method. The diagnosis could be made in4-8hours depending on the quantity of the bacterium inoculated. Twenty three E. coli isolates from clinical milk samples and12E. coli-positive milk samples were tested in parallel with MQQ-RS method and standard disk diffusion method. The results showed that, except for streptomycin, the MQQ-RS quick cultivation method had an overall90%agreement with that of the standard method for ampicillin, norfloxacin, tetracycline, polymyxin B, ciprofloxacin, compound sulfamethoxazole, cefoperazone, cephalosporins thiamethoxam cefuroxime and gentamycin.