The Resistance Of Vibrio Cholerae O1 E1 Tor Strains To The Typing Phage 919TP And Genomic Characterization Of A Mu-like Phage ZP66

Vibrio cholerae is a Gram-negative bacterium and the causative agent of cholera. Seven pandemics of cholera have occurred worldwide over the last two centuries. The ongoing seventh cholera pandemic is caused by El Tor biotype of serogroup 01. A Phage-Biotyping Scheme was developed for the subtyping of 01 El Tor strains and has been used in the cholera surveillance in China . In the biological typing part, El Tor strains can be grouped into 12 biotypes (from a to 1) according to their biological performance in lysogenicity, susceptibility to lysogenic phage 919TP,sorbitol fermentation, and hemolysis. Using the phage-biotyping scheme, El Tor strains isolated from patients in epidemics could be differentiated from those isolated from environmental samples during non-epidemic periods, and the scheme has been used in the surveillance and identification of sources of cholera outbreak. The study of phage typing mechanisms is helpful for revealing genetic differences among the different bacterial strains with the various phage types in the environment and in epidemics.Firstly, the whole genome sequencing and general biological characteristics analysis of the lysogenic phage 919TP used in the Phage-Biotyping Scheme was carried out. Phage 919TP belongs to the family Myoviridae. The optimal multiplicity of infection of 919TP is about 1,One step growth kinetics of the phage 919TP showed that the latent period is approximately 60 rain, the rise period is 60 min or so, and the average burst size is about 4 phage particles per an infected cell. The complete nucleotide sequence of phage 919TP is 33133bp. The G+C content of the 919TP genome is 48.92 %. By gene prediction annotation and whole genome alignment, the phage 919TP was identified as K139 family.Secondly, the resistance of Vibrio cholerae 01 El Tor strains to the typing phage 919TP was studied. A group of 116 01 El Tor V. cholerae strains isolated in different years and regions in China, were selected for the detection of sensitivity to phage 919TP infection, the presence of K139 phage family genes and the wbe gene cluster sequences. Among the test strains resistant to phage 919TP, most contained the lysogenic 919TP phage genome, which facilitated superinfection immunity to 919TP.Our data suggested that this immunity to Vibrio phage 919TP occurred after absorption of the phage onto the bacteria. Other strains contained LPS receptor synthesis gene mutations that disable adsorption of phage 919TP. Several strains resistant to 919TP infection possessed unknown resistance mechanisms,since they did not contain LPS receptor mutations or lysogenic K139 phage genome. Further research is required to elucidate the phage infection steps involved in the resistance of these strains to phage infection.Then the principle of applying lysogenicity in Phage-Biotyping Scheme developed for the subtyping of O1 E1 Tor V. cholerae strains was studied. All 01 Eor V. cholerae strains that produced positive results in lysogenicity, had the lysogenic K139 phage in genome and could both resist to lysogenic phage 919TP and release the K139 family phage. All the 01 Eor V cholerae 22 strains that produced positive results in sensitivity to the lysogenic phage, had no K139 family phage genes and got negative results in lysogenicity. However, the phages of this family were not released from 6 classical strains with positive lysogenicity result. Five serogroup 0139 strains were detected releasing temperate phages K139 without the sensitivity to phage 919TP. Applying the lysogenicity in Phage-Biotyping Scheme for subtyping 01 E1 Tor strains is based on the ability to produce lysogenic bacteriophage K139. The index of "sensitivity to the lysogenic phage" was also associated with this ability.At last,we isolated a novel lysogenic phage ZP66 which is the first Mu-like phage isolated from V. cholerae. Whole genome sequencing and general biological characteristics analysis of the phage ZP66 was carried out in our study. Phage ZP66 belongs to the family Myoviridae. The phage genome is 33499 bp long with an overall GC content of 49.85 %. The genome contains 49 putative open reading frames(ORFs),which constituted 94.78% of its sequence. By gene predictive annotation and protein sequence alignment, the phage ZP66 was determined as Mu-like phage. the results of the phylogenetic tree with the The tail proteins orf homology searches showed that,phage ZP66 were in a branch with prophage sequences in V. cholerae,and clustered into a large clade with other Vibrio and Aeromonas, which coincides with the classification of the bacteria. Phage ZP66 has narrow host range only infected the serogroup 01. The screening of ZP66 genome sequence found that it can't found the genome sequence exception the serogroup 01 biotype classical strains. However, four classical V. cholerae strains which had patical genome sequence were also sensitive to phage ZP66. Moreover, investigation of phage ZP66 receptor on the cell wall showed that LPS O-antigen is the receptor.In this study, we determined principle of applying lysogenicity and susceptibility to lysogenic phage 919TP in Phage-Biotyping Scheme developed for the subtyping of 01 E1 Tor V. cholerae strains. Then analysed the resistance of V. cholerae 01 El Tor strains to the typing phage 919TP. These studies helps us to understand the mechanism of phage typing and the emergence of resistant strains and even find the genetic markers of these resistant strains. At last, we isolated the first Mu-like phage ZP66 from V. cholerae which constribute an advance in our current knowledge of V.cholerae phages.