Study On Dectection Of Sulfonamides Resistance And Florfeniol Resistance Genes And Pfge Of E.coli From Yak And Herdsman

Because of adverse weather condition, distant far-flung and poor condition in Serta County with high altitude and extremely cold climate, there is no study on E. coli of Yak and herdsman in Serta County.Yaks are the most important to production material and living material for Tibetans. Yak E. coli disease is widespread in the pastoral area. As antibacterial is used widely, the problem of drug-resistance increased seriously. Because of the transmission of resistance, it was difficult to cure E. coli disease clinically. In order to get current state of resistance for yak and herdsman in Serta County Tibetans, and the mechanism of resistance transmission, the works had been launched as follows:1. E.coli isolated and serotype identification and 16S rRNA analysis210 samples of yaks faces and 90 human nasal swab were collected from 9 pastures in Serta County Plateau. The conditions were selected for inoculum and conservation of stains. The isolates were identified by biologic character,serotype and pathogenicity, which of 40 stains were identified by 16sRNA. The results showed that survival rate could be raised from 10% to 80% by taking 80% Glycerin and 10% skimmed milk as protective agent.114 E.coli stains of yak and 70 E.coli stains of herdsman were isolated.The O serotype was identified by 165 kind's single factor E.coli O antisera. Results showed that 98 stains were identified of yak in 114. The E.coli strains of yak fell into 24 serotype groups of which the O101 (12.3%),O2 (9.6%),O68 (7.0%). O132 (7.0%),O78 (5.3%),O171 (5.3%),O22 (4.4%),O142 (4.4%),O165 (4.4%) were predominant, accounted for 60%.58 stains were identified of herdsman in 70. E.coli strains of herdsman fell into 12 serotype groups of which the O81 (14.3%), 030 (11.4%).05(11.4%),0165(8.6%),0132(8.6%),O101(8.6%)were predominant, accounted for 62.8%. Serotype of yak was more than herdsman. And there was 7 same serotype between yak and herdsman, including 0132,O101,O15,08,078,0171,017, accounted for 37.5%(13/70). The pathogenicity of stains was checked by challenge assays for mice. Results showed that 22 strains were pathogenic to mice identified.20 strains isolated were identified by O serotype, but 2 strains were not. O78,O26,O111 were accounted for 18.2%,13.6%,13.6%, respectively. The serotype of 078,O26,0111 were predominant of pathogenic E.coli, accounted for 50%. The serotype of pathogenic E.coli of yak was identified firstly, which was important to produce vaccine for pathogenic yak E. coli.40 stains from herdsman and yak were amplified for 16S rRNA and sequenced. All sequences of 40 stains were blasted in GenBank. Results showed that homology of the isolates were 98-99% with other E.coli. And phylogenetic tree was constructed. Results showed that stains between herdsmen clustered in the same group and that stains between yaks clustered in the same group, too. The results showed the genetic relationship of E.coli from Tibetan Area and others.2. MIC testing and comparation of E.coli of yak and herdsman to 15 antibiotic in common use.The MIC of 114 strains of yak and 70 strains of herdsman from 9 pastoral areas were examined for their susceptibility to 15 antimicrobial agents by drugs dilution-plate method. The results of antimicrobial susceptibility test were analyzed according to the standard procedure advised by NCCLS. The results showed that sufisomezole, sulfadiazine and florfeniol may be the most resistant antimicrobial agents tested in this study of yak, accounted for 44%,40.4%,11.4%, respectively. The resistant rate of stains from yak was low to other antibiotics. The resistant rate from high to low was amoxicillin (8.78%),ampicillin(6.14%),gentamicin(3.5%),streptomycin(1.75%).All stains from yak were 100% sensitive to neomycin, doxycycline, terramycin, norfloxacin, ciprofloxacin, nalidixic acid, spectinomycin, ceftiofur sodium. The resistant rate of stains from herdsman was 57.1% and 51.4% to sulfadiazine and SMZ, respectively. The followed antimicrobial agents were ampicillin(22.9%), amoxicillin(25.7%), streptomycin(20%), spectinomycin(14.3%), terramycin(11.4%), ceftiofur sodium(11.4%), norfloxacin(8.57%), doxycycline(5.71%), florfeniol(4.3%), ciprofloxacin(2.86%), Neomycin(0%). In summary, by comparing resistance of yak with herdsman, we can draw the conclusions:1. the resistance of herdsman was more serious than yak's in this study; 2. the sulfonamide-resistance was highest in all the antimicrobial agents; 3. the florfeniol-resistance was detected in 3 strains of herdsman, while florfeniol was prohibited in human to treatment of the diseases.The results of drug sensitivity test of E.coli from Serta County were help to cure E. coli disease. At the same time, the results were useful to parallel examination and data analysis for yak and herdsman, understand the problem of resistance overall.3. Detection of PCR for sulfonamide-resistance genes and florfeniol-resistance gene of E.coli of yak and herdsmanIn order to detected resistance gene of E.coli in Sertar County in Tibetan pastoral area, the multi-PCR for Sul1, Sul2 and Sul3 gene and simple PCR for flor gene were established. By the multi-PCR method,50 strains of yak and 40 strains of herdsman sulfonamide-resistant isolates were checked. Results showed that sulfonamide-resistance was different from yak and herdsman. The Sul2 (>50%) gene of sulfonamide-resistant isolates from yak was predominant, and sul2> sul1> sul3. The sul3 gene was no identified in isolates of E. coli from yak. In 50 isolates, more than one gene coding for sulfonamide resistance was present:sul1 and sul2 in three.The Sul1 (>40%) gene of sulfonamide-resistant isolates from herdsman was predominant, and sul1> sul2> sul3. The sul3 gene was also identified in 3 isolates of E. coli from herdsman. In 40 isolates, more than one gene coding for sulfonamide resistance was present:sul1 and sul2 in five; sul1 and sul3 in one. Sulfonamide-resistant genes of herdsman were more complicated than yak's.16 stains were detected by the simple PCR method of flor gene. Results showed that 14 (87.5%) flor genes was detected in 16 florfeniol-resistant isolates from yak,3(100%) flor gene was detected in 3 florfeniol-resistant isolates from herdsman.4. The study on genetic relation of E. coli of yak and herdsman with the same resistance gene by PFGETo investigate the dissemination of resistance between yak and herdsman in the same Serta County ranch.26 resistant E.coli isolated from two ranch and different host were divided into two groups, and each group had 13 stains with the same similar resistance gene(Sull or flor) profiles were characterized by pulsed-field gel electrophoresis (PFGE) after XbaI digestion by using the CHEF system of Bio-Lab. Software Qantity One was used to analyze genetic relationship study between stains. The results showed that 13 E.coli strains with sul1 gene were classified into 12 different subtypes in the first group. An E.coli clone was detected, and similarity index was up to 0.9 between C2 and C4 from herdsman. In the second group,13 E.coli strains with flor gene were classified into 8 different subtypes. Three E.coli clones were detected, and similarity index was up to 0.88 and 0.93 between B3 and B12,B13 from yak,respectively. The PFGE patterns of strains suggested that transmission of resistant E. coli clones between herdsman and herdsman, as well as yak and herdsman.The resistance relationship between yak and herdsman in Serta County ranch was analyzed for the first time. And the results of transmission of resistant E.coli clones between yak and herdsman were important evidence on managing antibiotics of animal strictly, and preventing resistance widespread.5. Transformation test and conjugation test of E.coli of yak and herdsman with the same sulfonamide-resistance genes or florfeniol-resistanceThe 19 resistant strains were divided into two groups. A group:the 7 strains from yak with flor gene were susceptive to Sulfamethoxazole. B group:the 12 strains from herdsman with Sull or Sul2 gene were susceptive to Florfeniol. Plasmid DNA was purified by Plasmid MiniPrep Kit of TianGen Company. The plasmid profile was analyzed by agarose gel electrophoresis. Gel electrophoresis analysis demonstrated the presence of many visible plasmid bands from every isolate. Plasmids were discovered in all E.coli strains. Each profile contained more than one kind of plasmid. E.coli strains contained maximum plasmid was 10, and minimum plasmid was 2. The molecular weight of plasmids was from 0.4×103 to 90.0×103bp.19 kind plasmids were transformed into E.coli JM109 as recipient by Ca2 transformation. Then transformants was selected, plasmid DNA was extracted, MIC was measured and resistant genes were amplified by PCR. Results showed that the 7 kinds of plasmids with florfeniol-resistance gene from yak were transformed into E.coli JM109 separately.3 kinds of them were transformed successfully. While, The 12 kinds of plasmids with sulfonamide-resistance gene from herdsman were transformed into E.coli JM109 separately.6 kinds of them were transformed successfully. Transformation rate was 52.6% generally。The strains which could be transformed as donor were conjugated into E.coli C00 as recipient. Generated time of transconjugants was measured. Results showed that 2 strains in 4 with florfeniol-resistance from yak were conjugated into E.coli C00 successfully. And plasmid with flor gene was extracted from donor strain A12 and A172.5 strains in 6 with sulfanilamide-resistance from herdsman were conjugated into E.coli C00 successfully. And plasmid with sul1 gene was extracted from donor strain B1,B7 and H1,while sul2 gene from donor strain D 3and H5. The time of conjugation was from 3 to 6 hour. Transconjugants rate was 70% generally。On the one hand, the study revealed transmission of resistance by plasmid transformation and plasmid conjugal transfer, the transmission ratio and speed, which was important to provide the information on controlling transmission of resistance. On the other hand, plasmids with flor gene were identified from donor strain A12 and A172, and plasmids with sul1 or sul2 gene were identified from donor strain B1, B7, H1, D3 and H5,which would help to study on transmission of resistance between wildtype E.coli. The study on transmission of resistance plasmid systematically had not been reported in china Base on the special Tibetan pastureland, pathogenicity, serotype and MIC of E.coli isolates were indentified, which were beneficial to supply evidence to cure E.coli disease and guide the local civil and industrial needs. In the study, the resistance gene was checked from phenotypic resistance strains; genetic relationship was analyzed between yak and herdsman; and the character of transmission of resistance by plasmid was checked. The results were useful to manage antibiotics for animals strictly,E. coli of yak and herdsman collected from Sertar County in Tibetan closure pastoral area in this study. Transmission of resistant E.coli clones between yak and herdsman was proved by PFGE. The character of transfer of resistance to medicinal was studied by plasmid conjugation. The results were useful to manage antibiotics for animals strictly and protect human health and sanitation.