Isolation And Identification Of Pasteurella Multocida From Ducks And The Distribution And Structural Characteristics Of CRISPR Sites

Pasteurella multocida is a classical pathogen which causes hemorrhagic septicemia and infectious pneumonia on livestock and can also be survived in mouth and pharyngeal mucosa of many healthy animals. This pathogen induces a significantly economic influence on duck industry because of its worldwide spreading. Nowadays, due to the abuse of antibiotics, the drug resisrance of Pasteurella multocida becomes more serious. For bacterium, one of the main ways to acquire the drug resistance is through horizontal gene transfer of drug resistance gene. However, the CRISPR system discovered in prokaryote can prevent horizontal gene transfer effectively. In order to do deep research about the relationship between CRISPR-Cas systems and drug resistance of Pasteurella multocid, the study analyzed the CRISPR array structures in Pasteurella multocida, and investigated the relationship between CRISPR loci and drug resistance of Pasteurella multocida initiatively.In this research, samples were gained from four different large-scale duck farms. The ducks suspiciously infected by fowl cholera were all with typical symptoms like phemorrhagic septicemia, multifocal hemorrhage on the epicardium and haemorrhage in coronary fat, petechial haemorrhage on mucosa and serosa, and pin-pointed white necrotic foci in liver. We identified four clinical isolates using MacConkey medium, and other methods such as Gram stain, Biolog Microstation System, polymerase chain reaction (PCR) and Kirby-Bauer. The results showed that all of the four clinical isolates are Pasteurella multocida with multiple drug resistance.To analyze the structures of CRISPR-Cas systems, the genomes were extracted from four clinical isolates and seven strains which were kept in laboratory and then measured by high-throughput sequencing. The other eleven Pasteurella multocida genomes were downloaded from NCBI. The whole CRISPR-Cas systems in these twenty-two P. multocida strains were compared. The analysis of distribution, leaders, repeats and spacers in CRISPR-Cas systems showed that there were three types of CRISPR-Cas systmes including Ⅰ-F,Ⅱ-C and Ⅲ-U in twenty-two P. multocida strains. we also tound that the leader, repeat and Cas proteins interact with each other in every CRISPR-Cas system. In addition, the fourth base in every single repeat has the most significant effect on secondary structure in type Ⅰ-F systems while the ninth base in every single repeat has the most significant effect on secondary structure in type Ⅲ-U systems. There are 1314 spacers in three types of CRISPR-Cas systmes. Only Pasteurella multocida phage F108 matched with them. And the strains derived from different district had different numbers of spacers. Meanwhile, to undercover the relation between CRISPR loci and drug resistance of Pasteurella multocida in production, we focused on the four clinical isolates. It revealed that more serious drug resistance given less numbers of spacers.The results in this study will offer data for further investigating the relationship between CRISPR-Cas systems and drug resistance mechanism of Pasteurella multocida in future, especially in horizontal gene transfer. Simultaneously, it can also provide references for the research of bacteria virulence, detection of epidemics of Pasteurella multocida and application of Cas proteins in production.