Isolation And Biological Characteristics Of Enterotoxigenic E.coli And Phages From Swine

Enterotoxigenic Escherichia coli(ETEC)is an important pathogen that causes morbidity of humans and young animals(newborn piglets,lambs and calves).Newborn animals often death due to severe watery diarrhea and rapid dehydration after infected with ETEC,It's morbidity and mortality are high,and bring serious economic losses to the aquaculture industry.In this experiment,ETEC was isolated from 150 commercially available pork samples and farm pig manure.The strain was identified by physiological and biochemical markers and molecular biology.The single colony of ETEC were isolated and purified by specific primers.Using the isolated single colonies as host bacteria,screening of lytic phages,and then examine the biological characteristics of phages,and make the foundation for the application of phages.The pork samples were shredded and diluted with a homogenizer.The pig manure samples were pretreated and cultured in Brain Heart Leaching Broth,and then coated on the selective culture medium.The results showed that the colonies of the dominant colonies were pink,surface uplift,neat,smooth and rounded.Then,the total DNA of the mixed bacteria was extracted and the MRT-PCR reaction was carried out by using the five primers and probes designed and synthesized.The positive samples were reserved for the next test.Further phylogenetic analysis of the 16 S rDNA fragment(1436bp)obtained by PCR amplification showed it had 99% homology with Escherichia coli and could be identified as Escherichia coli.Isolated pathogens are athletic and can metabolize metabolized glucose,ornithine,lysine,tryptophan,urea,phenylalanine,mannitol,inositol,honey disaccharide,raffinose,no catabolism Sorbitol,ribitol.The specific PCR was carried out by priming the LT gene and ST gene.The single colony of ETEC was isolated and purified.The results showed that there were 7 strains of ETEC isolated from 150 pig source samples.Three strains contained only LT gene,two strains carrying only ST gene,two strains carrying both LT gene and ST gene.ETECs that carry ST and LT genes have higher pathogenicity than other strains.10 commonly used antibiotics were chosen to detect ETEC strains of drug resistance,according to the American Clinical Laboratory Standardization Institute 2011 version of the KB method of operation and judgment criteria.The results showed that ETECs were extremely sensitive to ciprofloxacin,cotrimoxazole and amoxicillin,highly sensitive to streptomycin and tetracycline,moderate sensitivity to chloramphenicol and doxycycline,to polymyxin B and Gentamicin produces resistance.Using of ultrafiltration centrifugal separation of ETEC produced by the outer membrane vesicles,and observed by transmission electron microscopy.The results showed that ETEC outer membrane vesicles of about 100 nm,was round or oval.Using two ETECs carrying both the LT gene and the ST gene as the host bacteria,collecting the waste water from the farm and the hospital,centrifuging and filtering to obtain the phage solution;and inoculating the phage solution into the isolated logarithmic growth phase Culture,filtration sterilization,get high titer phage proliferation fluid.Two different sizes of plaques were obtained by separation and purification by double plate method.The results of transmission electron microscopy showed that the two phages(PES-1 and PES-2)belonged to Siphoviridae.The biological characteristics of phage were determined by in vitro antibacterial test,one-step growth curve,pH,temperature stability,chloroform sensitivity test and phage particle concentration.Enzyme digestion showed that the genome of PES-1 was linear and the genomic DNA of PES-2 was circular.PES-1 cleavage ability is better than PES-2,the cocktail of PES-1 and PES-2 can significantly inhibit the growth of pathogens.SDS and bile salts were added to the phage and host bacteria according to different concentration gradient.The OD600 of the bacteria was determined.Compared with the control group with only phage and antibiotics alone,the results showed that SDS and bile salt can decline phage lysis ability in different degrees;if the phages were administrated orally,bile salts as mammalian intestinal secretions,phages will be affected by bile salts,phage should be coated orally.The combination of phage and antibiotics is an effective method to control the resistance of resistant bacteria and control the evolution of strain resistance.Different concentrations of amoxicillin,streptomycin and gentamicin and phage co-acting on ETEC,the determination of bacterial OD600 value showed that phages and amoxicillin,streptomycin,gentamicin had positive synergistic effect.