Virulence Measurement And Genome Sequencing And Comparative Genomics Analysis Of Two Listeria Monocytogenes Isolates

Listeria monocytogenges (Lm) is pathogenic for humans and animals, and causes listeriosis, such as gastroenteritis, meningitis, septicemia, abortion and et al. Lm is the food-borne pathogen which easily infects neonates, the old, pregnant women and immunocompromised individuals. Lm is a facultative intracellular microorganism which is suitable as a model bacterium for carrying out the studies of intracellular infection and T cell mediated immunity. In addition, Lm can be as carrier system for the development of live vaccines against intracellular infection and tumor.In this study, the virulence of two Lm isolates LM605and LM201which isolated from animal food was measured in vitro and vivo, respectively. The genome sequencing, assembly and annotation were carried out for two isolates, respectively. Comparative genomics analyses for virulence genes were performed. Taken together, these data and the two isolates as parental strains will provide the basis for exploring Lm pathogenic mechanism and developing Lm attenuated live vaccine vehicle.1. Virulence measurement of LM605and LM201in vitroHemolysis test shows that the two isolates just could form a smaller P hemolytic ring. Compared to L. innocua LI56, invasion and adhesion capability of LM201and LM605for RAW264.7cell are significantly higher (P﹤0.01), while invasion and adhesion ability of LM201is sligthtly higher than that of LM605(P>0.05). The result of biofilm formation ability measurement demonstrates that under the condition of37℃cultivating24h, two isolates can present a secondary biofilm formation ability (2ODc﹤OD≤4ODc).2. Virulence measurement of LM605and LM201in vivoTo measure the LD50of LM201and LM605, five week old mices as test animal were infected with two isolates, and the clinical symptoms and pathological changes in autopsy were observed. The datum indicated that the LD50of LM201was6.3×106CFU/mL, while that of LM605was8.5×107CFU/mL. According to the result, we knew that the strain LM201was high virulent strain because of logLD50﹤7. Additionally, observation results of infected mice’s clinical symptom and pathologic anatomy and histopathological changes showed the mices of the test group appeared depressed spirit and losed appetite and conjunctivitis. Autopsy results showed that the lungs of infected mices had hemorrhagic spot while livers had thanatosis. Observed result of pathological section dispalyed many tissues and organs was monocytosis with bleeding and inflammation compared with the control group.3. Genome sequencing of LM605and LM201 LM605was found to be3,001,147bp chromosome with a G+C content of37.8%,2,678,396bp coding area size and contained2948protein-coding genes by high throughput sequencing. The genome of LM201was composed of3,200,729bp chromosome with a G+C content of37.4%,2,836,379bp coding area size and contained3165protein-coding genes. Based on the protein-coding genes, the COG function classification and prediction in LM605and LM201genome was analyzed.4. Comparative genomics analysis of LM605and LM201(1) Based on the genome sequencing, serotyping results showed that two isolates were4b serotype.(2) Comparison of phylogenetic trees based on the16S rRNA sequences, we found that the evolutionary distance between strain LM605and LM201is nearer, besides evolutionary distance among the strain LM201and Lm F2365and Lm07PF0776were nearest.(3) To analyze the nine main virulence genes of LIPI-1and LIPI-2of Lm, Lm F2365and Lm EDG-e as reference strains, conduct homologies comparison in amino acids and nucleotides sequence of LM201and LM605, respectively. The result demonstrated the nine virulence genes were conservative, while different degrees of insertions and deletions or replace were observed in the genes.(4) According to the difference of the phylogenetic tree based on the proteins sequences encoded by the nine virulence genes, we speculated that there was different level of evolution between the LM201and LM605.(5) The test predicted the sixty virulence genes reported of the Lm on two isolates. The result indicated that fifty seven genes of the sisty virulence genes (95.0%) predicted existed in the LM201, and there was no measure auto and inlK and gtcA genes associated to the adhesion and invasion; the result indicated that fifty nine genes of the sisty virulence genes (98.3%) predicted existed in the LM605, lacking of the gene inlK associated to the adhesion and invasion of Lm.