| Shandong Province,which is a major province of broiler industry in China,has an annual output of 1.648 billion broilers and a chicken output of 2.328 million tons,ranking first in China.With the rapid development of broiler industry,the problems of animal disease,waste pollution,food safety and quality of livestock and poultry products also arise.There are many factors that cause these difficult problems in the breeding industry,and Escherichia coli（E.coli）,as a widespread conditional pathogen,is a typical representative of them.Pathogenic E.coli pollutes the environment and food through the discharge of feces,causing food safety and public health issues,and causing severe economic losses.At the same time,the resistance of bacteria to antibiotics,as a potential factor that has a profound impact on the economy,has been valued by countries around the world.Thus,the drug resistance of coli has become a research hotspot.It is found that the drug resistance of E.coli tends to be complicated and serious year by year.So far,there are few studies on the change of E.coli in a fattening cycle in Shandong Province.Based on this,an investigation was conducted on the drug resistance of E.coli in broiler Farms in parts of Shandong Province.In this study,a broiler fattening Farm was selected in Tai’an（Farm 1）,Linyi（Farm 2）,Liaocheng（Farm 3）,Weifang（Farm 4）,Binzhou（Farm 5）and Heze（Farm 6）of Shandong Province.More than 700 samples were collected at three different times within one fattening period.168 strains of E.coli were selected for drug resistance analysis after isolation and purification of bacteria and identification by microbial mass spectrometer.The resistance phenotype of E.coli was analyzed by K-B drug sensitive disk method,and the resistance genes of E.coli polymyxin,β-lactam,aminoglycoside,tetracycline and sulfanilamide were detected by PCR method.The resistance status of E.coli was monitored at the molecular level.The test results are as follows:A total of 707 samples were collected from six broiler Farms in Shandong Province,and375 strains of E.coli were isolated.The isolation rate was 53.04%.There was no statistical difference in the three sampling rates（P>0.05）.The highest isolation rate was 70.83%in Heze（Farm 6）,and 57.81%in Taian（Farm 1）,43.65%in Liaocheng（Farm 3）.According to the sampling time,the separation rate of the first（1-2 days old）sample is 51.28%,the second（17-20 days old）is 52.97%,the third（35-40 days old）is 54.85%,which increases with time.According to source analysis of E.coli,the highest isolation rate was 81.59%（288/353）in animals,in which the isolation rate of Heze（Farm 6）and Tai’an（Farm 1）was over 90%,96.67%and 90.00%respectively;the highest isolation rate of feces was 60.00%（60/100）,and the lowest isolation rate was 9.09%（6/66）in water.Similarly,the environmental samples of Tai’an（Farm1）and Heze（Farm 6）were isolated,and the rate was 29.41%and 27.78%,respectively.For further analysis of drug resistance,168 strains were selected for follow-up analysis.The results of drug resistance tests against 168 strains of E.coli showed that they have different degrees of resistance to 9 common antibiotics.There was a significant difference in the drug resistance between different antibiotics（P<0.001）.The highest drug resistance rate was AMP（92.86%）,and the lowest was FOX（10.12%）.In addition to FOX,AK（20.83%）and PIP（22.02%）,the drug resistance rate of the other six drugs was high,and all of them reached more than 60%.By observing the heat map prepared from the drug resistance rate of each field,it can be seen that the resistance to 9 types in the 6 regions is basically the same.The highest drug resistance rate is to AMP,and the lowest is to FOX.Except for FOX,E.coli in the Binzhou area（Farm 5）had lower resistance to other drugs than other areas,and the resistance rate to FOX was low at 4.17%.The samples came from three different sampling time points（1-2 days old,17-20 days old,and 35-40 days old）were resistant to 9 common antibiotics.Overall,the average drug resistance rate of the strains collected at 1-2 days old was lower than that at the middle and later stages.In addition to 24.07%of the third time of PIP resistance rate of the strains isolated at the middle stage（17-20 days old）of broiler growth was slightly higher than 23.63%of the second time,the other strains isolated at the middle stage had the highest drug resistance rate.The resistance rate of E.coli to CIP,GEN,OFX,AK and ENX was significantly lower than that of the last two times（P<0.001,P<0.001,P=0.012,P<0.001）,while the resistance rate of the other four antibiotics had no significant difference between the three sampling points（P>0.05）.The multi-drug rate of E.coli was 91.07%（153/168）,and the proportion of resistanct to6-drugs was the largest,29.17%（49/168）.Among the multi-drug resistance strains,the multi-drug resistance was mainly between 5-7 drugs,accounting for 75.82%（116/153）of the multidrug resistance strains,and 11 strains（6.55%）were resistant to 8 drugs.The multidrug resistance rates in six areas were:90.00%in Taian（Farm 1）,96.67%in Linyi（Farm 2）,100%in Liaocheng（Farm 3）,93.33%in Weifang（Farm 4）,62.50%in Binzhou（Farm 5）and 100%in Heze（Farm 6）.The multidrug resistance rate of E.coli was 100.00%in the middle（17-20days old）period,and 79.66%in the strains from 1-2 days old,which was lower than that in the middle（17-20 days old）and later（35-40 days old）period.A total of 40 drug resistance profiles were detected in 6 chicken Farms in Shandong Province,including 16 major drug resistance profiles（number of drug-resistant bacteria≥3）,including 9 dominant drug resistance profiles（number of drug-resistant bacteria≥5）.The drug resistance spectrum of 85.12%（143/168）of E.coli tested was the main drug resistance spectrum,and 79.72%（114/143）strains in the main drug resistance spectrum was the dominant drug resistance spectrum.In 168 strains of E.coli,12 drug-resistant genes were detected,including aminoglycosides,β-lactams,tetracyclines,sulfa and polymyxins.The highest detection rate was aadA（80.36%）of aminoglycosides,the lowest was blaSHV（1.19%）ofβ-lactams,and 41strains（24.40%）detected mcr-1.135 strains of aadA,50 strains of aacC2,34 strains of aacC4,42 strains of aphA3,49 strains of bla CTX-M,2 strains of bla SHV,121 and 10 strains of tetA and tetB,48,108 and 20 strains of sul1,sul2 and sul3,respectively,were detected.The detection rate of E.coli resistance gene in the first sampling（1-2 days old）was generally lower than that in the last two samplings（17-20 days old and 35-40 days old）,especially the detection rates of sul1,sul3,aacC4,apha3 and mcr-1 were significantly lower than those in the last two samplings（P<0.05）.However,the detection rates of bla SHV,blaCTX-M,tetB,tetA,aacC2,aadA and sul2 were not significantly different.Among the 41 strains carrying mcr-1,92.68%（38/41）simultaneously carried aminoglycoside resistance gene,36.59%（15/41）simultaneously carriedβ-lactam resistance gene,90.24%（37/41）simultaneously carried tetracycline resistance gene,92.68%（38/41）simultaneously carried sulfanilamide resistance gene.There are 12 strains containing five kinds of drug resistance genes,which indicates that E.coli with myxomycin resistance gene mcr-1 often carries other drug resistance genes.In this study,the resistance of E.coli in broiler Farms in some areas of Shandong Province was studied.The isolation of E.coli confirmed the positive significance of environmental control on the prevention and control of E.coli.The results showed that the problems of E.coli drug resistance and multiple drug resistance are very serious in Shandong Province.It has accumulated experience for the prevention and treatment of Chicken Colibacillosis and has important guiding significance.In addition,the results also showed that the drug resistance increased with the increasing use of antibiotics.During the identification of E.coli,Klebsiella pneumoniae,Proteus mirabilis and other bacteria that can cause zoonotic diseases were also discovered.The transfer of drug resistance between these bacteria has been confirmed,which has a great threat to food safety and human health.Therefore,the monitoring of these pathogens in the breeding process must be highly valued. |
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