Molecular Characterizations Of The Drug-resistant Pathogenic Bacteria From Bovine Mastitis

Bovine mastitis is one of the most common diseases in the world which can cause significant economic loss in the cattle farm.The common pathogens of bovine mastitis are Staphylococcus,Streptococcus,and Escherichia coli.Severe resistances are brought by using and overusing of the antimicrobial.In order to explore the resistance characteristics and provide the theoretical basis for the prevention and control bovine mastitis,the study on antimicrobial susceptibility testing and molecular epidemiology that were performed for the clinical isolates.Kirby-Bauer method was carried out to study the susceptibility of Staphylococcus.The results showed that most of the 19 isolates of Staphylococcus aureus were resistant to penicillin,erythromycin,ciprofloxacin,and the rate of resistance were 94.74%、84.21%、84.21% respectively,but it showed low resistant to cephalosporins 、 clindamycin 、oxacillin and doxycycline.The Coagulase negative staphylococcus were sensitive to the two drugs cefoxitin,oxacillin,and resistant to the following 7 kinds of drugs like the penicillin and erythromycin.The erm A,erm B and erm C of erythromycin-resistant genes and the mec A of penicillin-resistant gene were detected in 38 staphylococcus strains by PCR.The result showed that the percentage of erm A,erm B,erm C and mec A were 2.67%,7.89 %,13.16 %,7.89 % respectively.The erm C and mec A were detected in two staphylococcus epidermis strains,while the erm B and mec A were detected in the staphylococcus hemolytic strain.By the agar dilution method,only one Staphylococcus epidermidis strain isolated from 38 milk samplesn but it was resistant to florfenicol,the resistance genes fex A and mec A were detected by PCR.The Streptococci isolated from the mastitis milk was detected by antimicrobial susceptibility testing and the detection of resistance genes.12 strains of Streptococci were collected out,including 5 strains of Streptococcus uberis,6 strains of Streptococcus agalactiae and 1 Streptococcus dysgalactiae.The result exhibited that Streptococcus were sensitive to florfenicol,ceftiofur,and resistant to clindamycin,erythromycin.The erythromycin-resistant gene erm B was detected by PCR.Antimicrobial susceptibility testing was performed by broth dilution method.It was serious that the E.coli isolated from cow was resistant to many antibiotics,but the resistance of the strains isolated from the mastitis milk were higher than the strains isolated from the healthy milk.The results showed that 11 strains isolated from the mastitis milkexhibited a high level of resistance to β-lactam-antibiotics（ampicillin,ceftiofur and cefotaxime except cefoxitin）.10 strains of 36 strains of E.Coli carried bla_CTX-M and one carried bla_cmy-2 by PCR and sequencing analysis.The result of conjugation experiments indicated that the resistant plasmid could be transferred to the recipient strain E.coli C600.It is worth noting that bla_CTX-M can be horizontal transmitted between strains by transconjugations.Using PCR for three special genes,we determined which the E.colii strain belongs to according to the results.Multilocus sequence typing technique（MLST）was used by researching sequences of 7 housekeeping genes from 10 E.coli strains.The sequence types（STs）were confirmed by sequences comparing.Then,genetics evolution relationship could obtain by making use of e BURST v3 and MEGA 6 software.The results showed that 2 of the 10 E.coli strains belong to group D,others belong to A or B1.At the same time,the 10 E.coli strains divided into 7 STs,which shows a high otherness.Compared with 3000 ST sequences,the closest genetic distance are M10,M11,M8,E09,G2.Phylogenetic grouping and MLST was used to detect the strains isolated from the healthy milk,the results showed that the 8 strains all belong to group B1 which has nonpathogenic and the 8 E.coli strains divided into 5 STs,2 new ST types were detected in this study which needs to be named.In summary,Staphylococcus,Streptococcus,and Escherichia coli were the main detected bacteria from the mastitis milk.the resistance of Staphylococus was not serious,the percentage of erm A,erm B,erm C and mec A were 2.67%,7.89%,13.16%,7.89%respectively,the erm C and mec A were detected in Staphylococcus epidermis strain.It was also not serious that the Streptococci isolated from cow were resistant to many antibiotics,there were 7 strains who detected the erythromycin-resistant gene in 12 strains of Streptococci.It was serious that the E.coli strains isolated from cow were resistant to many antibiotics,10 strains carrying bla_CTX-M and one carrying bla_cmy-2 were sieved,10 kinds of E.coli strains were divided into 7 STs.Conjugation experiments and plasmid analysis showed that the antibiotic-resistant plasmid could be transferred to the recipient strain C600.We should use sensitivity medicine reasonably to avoid the bovine mastitis to promote the healthy development of dairy farming.