| Citrobacter freundii is present in the gut microbiota of humans, also known as an opportunistic pathogen, which can cause infection in immunocompromised people. Since its discovery, C.freundii has gained resistance to many common antibiotics, antibiotic treatment only inhibits its growth. In addition, it has ability of resistant to chemical disinfectants, surfactant and UV radiation. Therefore, it is difficult to remove it from medical equipment, and as an important nosocomial pathogens right next to Pseudomonas aeruginosa. Currently the rate of antimicrobial agents developing lags far behind the rate of bacteria acquiring resistance, drug resistant bacteria in particular multi-drug resistant pathogens brought great challenges to clinical therapy. For prevention and treatment of drug-resistant pathogens we need urgent to seek alternatives therapy to antibiotics. The using of bacteriophage to treat bacterial infections has a long history, and has many advantages over antibiotic agent therapy. The recent renaissance of phage therapy offers possible alternative antibacterial treatments and is therefore attracting much attention throughout the world.High-throughput sequencing(HTS), also known as next-generation sequencing(NGS) has many advantages including high throughput, high speed, low cost. It provides solid bases for the development of genomics, also as an important tool in life sciences and medical research. It has a important significance for understanding subjects such as evolution, mutation, gene content and regulation on gene level to complete genome sequences. Nowadays, the main HTS platforms includes: Roche’s 454, Illumina’s Hiseq and Miseq and Life Technology’s Ion Torrent. Among them Illumina’s HTS system dominates the HTS market, for its high throughput and accuracy. Between them Ion Torrent is the fastest, and Roche454 has the longest single Read sequences. Both Ion Torrent and Roche454 have the disadvantages of homopolymers. These sequencers have their own advantages and drawbacks in difference application.In this study, multidrug-Resistant C.freundii strain P10159, isolated from urine samples of a patient with esophageal carcinoma was sequenced using Ion Torrent, also genomics analysis was conducted. Before sequencing antibiotics-resistant characterization and biochemical properties were tested. To solve the fracture problem in genome assembly caused by repeats in the genome, we use both Shotgun sequencing and Mate-Pair sequencing. Assembly was performed by using Newbler v2.8. Because the principle of the Newbler was based on sequences similarity alignment, too much data will not be good for assembling. And we recommend using a multiple of about 30 times the coverage of genome data for assembling. Software Contig Scape and Reference assembly strategy were used for Contigs related information display. Gapfiller was used for filling up the Gaps in the genome. Also we design specific PCR primers to flank the gap and sequence the PCR products, to gain the complete genome. The complete genome sequences of C. freundii P10159 is 5,080,321 bp, encodes 4,768 predicted protein-coding sequences(CDSs), 24 r RNAs and 69 t RNAs, with a GC content of 51.7%. To perform comparative genome analysis, we download the completed genome sequences of other 3 strains of C.freundii in the Gen Bank database. The result shows that the four C.freundii genomes share highly homology. C.freundii P101059 has the highest homology with C.freundii CAV1321. C.freundii P10159 has a large fragment(2.4~3.2 Mbp) reverse mutation when compared with others. Also the protein sequences of the four genome of C.freundii were retreated for subsequent orthologous genes analysis. Those four genomes shared 3,395 CDSs in total. Strain P10159 shared 3,613, 3,606, and 3,488 orthologous CDSs with CAV1321, CAV1741 and CFNIH1, respectively. In addition, 787 CDSs from the P10159 genome were classified as unique, which account the largest amount among the four genomes. Phylogenetic tree based on 3 hourse-keeping genes and one 16 S RNA sequence showed that C.freundii P10159 was clustered with C.freundii CAV1321, CAV1741 and CFNIH1, while some other specises were clustered into different subgroups. In the second chapter we successfully isolated a lytical phage from hospital sewage, which infects C.freundii. Transmission electron microscopy analysis shows that IME-CF2 has an icosahedral head and a contraction tail, displays a morphology resembling Myoviridae family. Whole genome sequences analysis and phylogenetic analysis indicated that IME-CF2 class to T4 bacteriophage. A deeply analysis of its morphological and genomics provides the basis for phage therapy. |
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