关键词: 前噬菌体, 大肠杆菌, 诱导, 分布特征, 耐药基因

Abstract:

【Objective】 This study investigated the distribution characteristics of prophage in multi-drug resistant Escherichia coli, induction and isolation, as well as the prevalence of drug resistance and virulence genes in prophage, so as to provide a scientific basis for the study of prophage-mediated resistance genes in the spread of bacteria. 【Method】 131 multi-drug resistant E. coli isolating from poultry origin in Guangdong Province from 2018 to 2019 were selected in the laboratory for nucleic acid extraction and whole-genome sequencing. The results of second-generation sequencing were assembled and spliced into a whole-genome sequence and uploaded to the phage. The PHASTER network database was compared and analyzed with the existing phage genome sequences in the database. Drug resistance genes and virulence genes were compared on the CGE database, and then the distribution of drug resistance genes and virulence genes on the prophage were obtained. The mild phage was induced by mitomycin C, separated and purified by using the double-layer plate method. 【Result】 The results of the drug sensitivity test of 131 strains of Escherichia coli showed that the drug resistance rates of ampicillin, tetracycline, florfenicol and compound trimethoprim were all more than 90%, followed by cephalosporin antibiotics, gentamicin, ciprofloxacin, meropenem and colistin with all around 50%, and the resistance rate of tigecycline reached 0.2%. All strains showed multi-drug resistance, and they were all multi-drug resistant Escherichia coli. A total of 736 prophage fragments were detected in 131 strains of multi-drug resistant E. coli, including 329 complete prophage, 66 suspicious phages and 341 incomplete phage, which matched with 40, 20 and 52 known database phage species in different percentages, respectively. The gene sequence of the complete prophage showed that it matched the known phage species better, and the sequence similarity was the highest, with an average of 58.53%. The average number of prophages in 131 strains of E. coli was 5.6, and the average total content was 152.4 kb. Prophage genome accounted for 0.58% to 5.87% of its host genome, with 3.0% being the dominant. The length of the prophage genome ranged from 2.8 to 107.9 kb, and the 13.0 kb prophage had the highest frequency, accounting for 9.1% of all prophages. CGE comparison results showed that the genomes of 131 strains of multi-drug-resistant E. coli detected resistance genes mdf (A), lnu (G) and mcr-1 in 18 prophage sequences. The detected numbers of mdf (A), lnu (G) and mcr-1 were 16, 1, and 1, respectively. 71 strains of multi-drug resistant E. coli prophage carried 6 different virulence genes, and some strains carried 2 or 3 virulence genes. There were 62 prophages carrying the telomerase RNA gene terC, 16 prophages carrying the serum survival increasing gene iss, and the outer membrane protease ompT, among which the adhesin gene iha, the cvaC gene and the ABC transporter gene mchF were at 2, 2, 1, and 1, respectively. Mcr-a gene were detected in prophage of 1 strain multi-drug resistant Escherichia coli. The mdf (A) gene and terC gene were the most common resistance genes and virulence genes in prophage, respectively. The results of mild phage induction experiments showed that the success rate of prophage induction was 84.0%, but the probability of plaque appearance was still relatively low. 【Conclusion】Prophages were widely distributed in multi-drug resistant E. coli and carried a variety of resistance genes and virulence genes. Mild phages had a high induction rate, and have the risk of horizontal transmission of resistance genes and virulence genes, and need to be strengthened and sustained monitor.

Key words: prophage, Escherichia coli, induction, distribution characteristics, resistance genes