Antibiotic Sensitivity Test Of Porcine Pathogenic E. Coli And Study On Apramycin & Doxycycline Antibiotic Compound

Stool Samples of suspected cases of Porcine Colibacillosis were collected from pig farms in 21 provinces in China,such as Sichuan,Chongqing,Hubei and so on. 737 strains of E coli.were isolated according to their growth characteristics and results of Gram staining and microscopic examination.After biochemical identification and pathogenicity test with isolates randomly selected,105 isolates were judged as pathogenetic E coli.15 antibiotics of 5 categories were used to test antibiotic susceptivity of pathogenetic E coli.Apramycin and Polymyxin B turned out to be antibiotics with the highest sensitivity(81.9%and 80%respectively),followed by Florfenicol(70.5%) and Spectinomycin(63.8%).Antibiotics with sensitivity lower than 20%included Tetracycline(0%),Ampicillin(3.8%),Enrofloxacin(5.7 %),Arnoxicillin and Potasium Clavulanate(6.7%),Doxycycline(8.6 %),Streptomycin(9.5%),Ciprofloxacin(18.1%).Isolates resistant to at least 6 antibiotics accounted for 85.71%of the total 105 pathogenetic isolates.5 isolates were found resistant to all the 15 antibiotics.None isolate was resistant to only one antibiotic.No isolate tested from Sichuan,Chongqing,Henan,Fujian,Anhui,Gansu, Liaoning and Guangdong was resistant to less than 6 antibiotics.Apramycin was chosen as the major component of antibiotic compound,based on the high sensitivity and its property.6 antibiotics and synergists were selected to combine with Apramycin,according to their sensitivity and theoretical combination effect.Among 6 combination pairs,Apramycin and Doxycycline showed the best synergistic effect.A orthogonal experimrnt of 3 factors,each having 4 levels was carried out to determine the best drug prescription.Result showed the prescription was the best when Apramycin,Doxycycline and Antibiotic Factor were in the ratio of 2:1:2.E coli.O149 standard strain,5 pathogenetic isolates with high resistibility and 10 pathogenetic isolates randomly selected were used to confirm the antibiotic effect of the drug prescription.Result showed the prescription displayed MIC 2～4 times lower than that of Apramycin;2～16 times lower than that of Doxycycline; 1～4 times lower than that of Apramycin and Doxycycline combination in the ratio of 2:1.Modified.Karber's method was used to calculate mice Median Lethal Dose of the compound drug.Result showed LD₅₀ was 2.16 g/kg,and 95%Confidence Interval of LD₅₀ was 1.70～2.74 g/kg,which indicated the compound drug was hypotoxic.F test was applied to compare mice weight of treatment and control group.Result showed only 777.6 mg/kg group showed significant higher weight than the control 3 days after the administration.Content measurement method of the antibiotic compound was studied and it's found that Apramycin and Doxycycline can be measured respectively by microbiological and HPLC method after treated with methanol.This method showed high specificity,precision,recovery rate and repeatability.In order to optimize the adjuvant and the package of antibiotic compound, orthogonal experimrnt of 3 factors,each having 2 levels was carried out in plastic bag, Aluminum foil bag and Penicillin bottle respectively.The factors were drug,bases (soluble amylum,glucose as the control) and anticaking agent(silicon dioxide).After one month's accelerated test,all glucose groups and soluble amylum groups packaging with plastic bag were discolored and caking;other groups didn't show significant difference among levels.Stability was examined in 3 conditions,i.e.10 days in strong light;6 months under condition of 75±5%relative humidity and temperature of 40±2℃;12 months under condition of 60±5%relative humidity and temperature of 25±2℃.Results showed all indexes were consistent with quality stardard,indicating the compound is stable.In conclusion,this research has developed a Apramycin & Doxycycline antibiotic compound soluble powder,and carried out corresponding experiments according to the requirements of new veterinary drug application.This antibiotic compound has provided a new safe and efficient drug for prevention and therapy of Porcine Colibacillosis.