豬大腸桿菌噬菌體的分離鑒定及受體特性和全基因組序列分析.pdf

1、Porcine post-weaning diarrhea (PWD) occurs sporadically or as major outbreakscause of economic losses in the pig industry.PWD a disease caused by a group of bacteriacalled enterotoxigenic E.coli (ETEC) and Shiga-toxin E.

2、coli (STEC) in piglets.ETEC ofO serogroups 2, 8, 138, 139, 141 and 149 have been associated with porcin PWD.Bacteriophages are diverse forms of organisms and play important role on the balance ofmicrobial world.Antibioti

3、cs have been commonly used in the treatment of infectiousdiseases but their wide spread and improper use has led to antibiotic resistance in porcinecolibacillosis.Recently the potential use of phages as therapeutic agent

4、s in controllinghuman and animal disease was recognized.It were reported that phage treatment issuccessful against animal E.coli infections.The use of bacteriophage to prevent and treatcolibacillosis in chickens and calv

5、es has also been explored. The goal of this study was to detection the distribution of serogroups and virulencegenes of E.coli strains isolated from porcine post weaning diarrhea in Thua Thien Hueprovince of Vietnam.F

6、urther, we isolated phages with lytic activity against enterotoxigenicE.coli (ETEC) to characterize their morphology, genome size, bacterial host range,infectivity, activity at various pH levels, structural protein profi

7、le, genome sequencing,receptor and bacteriophage therapy against E.coli infections. 1.Distribution of serogroups and virulence genes of E.coli strains isolated fromporcine post weaning diarrhea in Thua Thien Hue provi

8、nce of Vietnam Heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic E.coli (ETEC) and Shiga-toxin producing E.coli (STEC) are the important factors associatedwith pathogens to porcine post weani

9、ng diarrhea (PWD).The E.coli strains wereinvestigated serogroups, toxins and adhesion encoding genes.A total of 95 E.coli isolates,collected from feces of porcine PWD.The O-serogroups, toxins and adhesion encodinggenes o

10、f the E.coli strains were detected using polymerase chain reaction (PCR).In theserogrouping with 9 antisera, 76 strains (80.85％) were O-serogroup and belonged to O2,O8, O138, O139, O141, O147, O149 and O157, respectively

11、.While 19 strains (20％) didnot belong to these O serogroup.Predominant serogroup was O8.22.11％ isolates carriedgenes F18, while genes for F4 (K88), F6 (P987) and F5 (K99) were 15.79, 5.26 and 4.21％,respectively.The genes

12、 for STa, STb, LT, Stx2e, Stx2a and Stx21 toxins were identified49.57, 22.14, 13.68, 6.32, 3.16 and 3.16％, respectively.One isolate contained five genesthat is O139/F4/STa/STb/Stx2e, and another isolate contained four ge

13、nes that isO45/F4/Sta/LT/Stx2e.The hemolytic activity on blood agar plates were detected in 62isolates (65.26％) of E.coli.Most of E.coli isolates carried genes for adhesions and toxins.Furthermore, our results suggested

14、that E.coli strains isolated in Thua Thien Hue provinceof Vietnam are mainly ETEC and O8 serotype. 2.Isolation, characterization and classification of bacteriophages for piglet pathogenicE.coli strains Sixteen phag

15、es (JH1 through JH16 designated) were isolated using a mixture of O2,O8, O139 and O141 E.coli strains as hosts.Ten phages (62.5％) were made between Marchand June (JH1, JH4, JH5, JH6, JH8, JH9, JH10, JH12, JH13 and JH15).

16、Six bacteriophages(JH2, JH3, JH7, JH11, JH14 and JH16) were isolated between October and December(37.5％).Nine bacteriophages were isolated from 52 samples of raw sewage (17.3％) andseven bacteriophages were isolated from

17、50 fresh fecal samples (14％).All bacteriophagesproduced clear plaques in double agar.The phage morphology showed that five phages(JH1, JH2, JH9, JH11 and JH14) belong to Myoviridae family, seven phages (JH3, JH4,JH5, JH6

18、, JH8, JH13 and JH15) belong to the Siphoviridae family and four phages (JH7,JH10, JH16 and JH12) belong to Podoviridae family.Unlike typical Podoviridae familymembers' morphologyes with regular hexagonal head, the head

19、of JH16 had an irregularand elongated hexagonal outline.Virion buoyant density in CsCl of JH8 phage is 1.4 g/ml,JH5 is 1.7 g/ml and JH1, JH2, JH3, JH4, JH6, JH7, JH9, JH10, JH11, JH12, JH13, JH14,JH15, JH16 are 1.5 g/ml)

20、.The optimal multiplicity of infection of four phages JH6, JH11,JH13 and JH16 were 0.1, 1, 0.001, 0.01 and 1, respectively.The present study showed thatthese phages have high stability to the physical and chemical factor

21、s.They best grow at thetemperature of 37℃.Phages were incubated at 50℃ for 60mins showed no decline of thetiters.At 60℃ for 10mins, there was no significant difference between experiment andnegative control.However, at 6

22、0℃ for 30 to 60mins there was significant effect on the titerof phages.At 70℃, all bacteriophage were uninfected.All these phages were resistant topH 5-9.All phages' genomes were estimated at ＞20 kb by restriction enzyme

23、sanalysis.Primers designed to amplify the core gene of gp23 which encode major capsid protein ofthe various T4-type phages were used to check two phages JH11 and JH14.Amplicons ofJH11 and JH14 shared high similarity (＞93

24、％) with other T4 like phages, such asECML-134, HX01 and CEV1, thus comfirmed that JH11 and JH14 belong to T4-like phagegenus. 3.Isolation of bacteriophage JH14 receptor from E.coliPolysaccharides by E.coli were no sig

25、nificantly effect on phage adsorption orreplication.Polysaccharides were not significantly affect phage adsorption or replication.Outer membrane protein was extract and purified from VN14 E.coli.It was found thatouter me

26、mbrane protein could block JH14 phage adsorbing to its host strain.Theadsorption of JH14 phage could also be blocked by the antiserum of VN14 E.coli.Thus,we conclude that outer membrane protein of VN14 E.coli.may the maj

27、or receptor ofJH14. 4.Experimenting the protection ability of JH14 phage for mice infecting pathogenicampieillin-resistant E.coli
　　The past few years have shown significant resurgent interest in the old concept of

28、bacteriophage therapy.Some research groups continue to develop whole bacteriophagepreparations as alternatives to antibiotic antibacterial treatment.However, improvements inthe methods of purification of phage preparatio

29、ns opened new opportunities in thesuccessful treatment of antibiotic-resistant bacterial infection.The LD550 of VN4 E.colistrain was 1.548 × 107 cells per 200 microliter.Compared with 0.85％ saline (control), thephage sig

30、nificantly reduced the mutation frequency of E.coli.The minimal dose of theJH14 phage to protective mice was 0.4 × 105 pfu, significantly smaller than that of control.Only with 105 pfu of JH14 phage could protect mice in

31、fected 9 × LD50 (1.4 × 108 cfu) of avirulent strain of E.coll.A single intramuscular dose of phage JH14 was more effectivethan multiple intramuscular doses of Amikacin and Streptomycin.These studies support theview that

32、bacteriophages could be useful in the treatment of animal infections caused byantibiotic-resistant strains of bacteria. 5.Complete genomic sequence of a novel bacteriophage infecting E.coli with highsimilarity to Stap

33、hylococcus phage SA1A novel bacteriophage, JH2, was isolated from fecal samples in Jiangsu pig farm.Phage JH2 has a broad host range and can infect O8 ETEC strains which caused pigletporcine post weaning diarrhea.Phage J

34、H2 showed icosahedral head, neck, contractile andlong tails and belong to Myoviridae family.Microbiological characterization demonstratedthat this phage showed high stability to the physical and chemical factors.JH2 was

35、thermalsensitive and showed acid and alkaline resistance (5-9) and a latent period of 30 min.JH2has a 87.7 kb, circular dsDNA genome with G+C content of 38.82％.The genome of JH2contained 131 putative ORFs.One hundred and

36、 thirty one ORFs were predicted and 121ORFs has putative functions.In database, 22 tRNAs were predicted in JH2 genome.TheJH2 genome was found to similar to the sequences of Staphylococcus phage SA1 completegenome (95％),