Isolation,Identification And Drug Resistance Analysis Of A Duck Escherichia Coli Strain O60 Serotype

Avian Colibacillosis caused by avian pathogenic Escherichia coli(APEC), which is characterized with coli-granuloma, peritonitis, salpingitis, pericarditis etc. E. coli is a flagellated, motile and medium-sized Gram-negative bacterium. In recent years, there have been more and more reports of avian colibacillosis with high morbidity and wide epidemic area, which has strongly impacted poultry industry in our country, though the mortality rate is still low. The high diversity of serotype and wide distribution area of APEC as well as the large population of poultry and neglect of management make avian conlibacillosis be a common disease in the poultry industry of our country. Furthermore,because of the abuse of antibiotics,drug-resistant spectrum has grown much wider,making itself a great obstacle to prevent the disease. This study mainly includes isolation, identification and drug resistance analysis of an Escherichia coli strain isolated from a duckery in Tong Guan County of Shaanxi Province, which provides important theoretical basis for the prevention and control of duck colibacillosis.1. Lesion tissues were acquired from a duckery in Tong Guan County of Shaanxi Province for isolation and identification. The tissues were applied to MacConkey agar and broth agar medium in sterile situation. After bacterium cultured under 37 ℃ for 24 h,translucent, moist, smooth, circular-protrusion, and light gray colonies were aquired in the broth agar. While on the surface of MacConkey agar medium, moist, smooth,circular-projection and pink colonies were observed. Colonies picked were Gram stained and medium-sized, nonsporulating and gram-negative bacterium were observed under the microscope. The purified isolate was inoculated in agar slant, Deng Heinz peptone water and glucose peptone water, cultured under 37 ℃ for 24 h. Some colonies were picked and inoculated in biochemical test tube, cultured under 37 ℃ for 24 h, of which the result showed that the isolate was positive for Ferment glucose, lactose, indole and MR, negative for VP. Bacterial universal primers were applied for PCR to amplify 16 S r RNA gene of the isolate. After the target band cut and recovered, the PCR product was gel purified,connected to pMD19-T cloning vector and transformed into DH5α competent cells.Monoclones were picked for PCR amplification and the positive clones were sent for sequencing. Sequence analysis showed 16 S rRNA gene fragments of the isolate was 1534 bp and the similarity with the standard strain of Escherichia coli gene sequence was 99%.The overall judgment showed the isolate labelled as TG-2015 was Escherichia coli strain.Phylogenetic analysis showed that the isolate was positioned in a relatively independent branch.2. The purified isolate was inoculated in agar slant, cultured under 37 ℃ for 24 h. A small amount of 5% phenol saline was used to wash the slant. The suspension acquired was centrifuged for 5 minutes at 2000 r/min. Then the supernatant was placed in another centrifuge tube, centrifuged for 10 minutes at 8000 r/min. With the supernatant discarded,the precipitate was made into bacterial suspension. After sterilization at 121 ℃ for 2 h, O antigen was prepared. The different types of anti- "O" serum were diluted 8-fold with sterile saline and dropped by 25 μL on glass respectively with 25μL O antigen mixed with them.Positive agglutination appeared in 30 s, which indicated the serotype of TG-2015 was O60.3. The purified isolate was inoculated in agar slant, cultured under 37 ℃ for 24 h.Then it was made into the suspension of 1×109 CFU / mL with normal saline. Six mice and six ducklings were injected intraperitoneally with 0.2 mL suspension respectively. Three mice and three ducklings were inoculated with sterile saline as negative control. Within 24 h after injection, all experimental animals in test group died while those in control group were still alive. The results showed that TG-2015 was highly pathogenic.4. The purified isolate was inoculated in MacConkey agar, cultured under37 ℃ for 24 h,Then typical single colony was picked in sterile situation and was formed was made into the suspension of 1×108~1×109. A small amount of the suspension was coated on MH agar medium, then the medium was placed about 5 min following every five susceptibility slips(total 33) attached to the surface of the medium. Then the MH agar medium was placed in an incubator, cultured under 37 ℃ for 16 h. In antimicrobial susceptibility test, there are33 kinds of susceptibility slips, the results showed that TG-2015 was resistant to 25 slips,and sensitive to 8 slips. The sick ducks were recovered 7 days later post treatment with Amikacin, which was sensitive to the isolated strain, and the egg production was also recovered.In conclusion, the serotype O60 field isolated duck E.coli TG-2015, which was high pathogenic, had wide drug-resistant spectrum and in a separated branch, was uncommonly in China.