T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens

Abstract

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens.

Fig 1

Morphology and reproduction process of phage Bp7 examined with a JEM-1200EX microscope (JEOL). (a to c) Different stages of phage reproduction. (a) E. coli cell and phage Bp7 in the adsorption phase (bar = 50 nm; the arrow indicates the adsorption of phage Bp7). (b) Phage Bp7 in the maturity phase (the arrows indicate the elongated head and tail; bar = 50 nm). (c) Phage Bp7 in the release period (the arrow indicates phage released by host cells; bar = 200 nm). (d) Transmission electron micrograph of a phage Bp7 virion negatively stained with 2% uranyl acetate (bar = 50 nm; the arrow indicates the phage with the elongated head and tail).

Fig 2

Stability of Bp7 under different temperature conditions. Bp7 could keep its basic infectivity at 40°C or 50°C. With the increased temperature and longer time, phage titers decreased.

Fig 3

Stability of Bp7 under different pH conditions. In the range of pH 2.0 to 13.0, approximately neutral conditions had little or no effect on phage Bp7, while extreme pH conditions affected the stability of phage Bp7.

Fig 4

Comparison of the Bp7 genome with those of JS98, IME08, and T4. Regions with similarity are highlighted by connecting red or blue lines between the genomes; red lines indicate homologous blocks of sequence, and blue lines indicate inversions. The gray bars represent the forward and reverse strands. Gene arrangements and orientations were highly conserved among phages JS98, IME08, and T4, but the genome of Bp7 showed a large-scale genome translocation and can be divided into two parts that are the reverse of the arrangement of phages JS98, IME08, and T4. As seen by the large red blocks connecting both genomes, there is a smaller degree of similarity between Bp7 and T4.

Fig 5

Annotated genome map of bacteriophage Bp7. The genome can be read from left to right and from top to bottom. ORFs with right arrows or left arrows are genes on the positive or negative strand, respectively. The predicted genes were classified into three groups based on their different functions, as shown by different patterns: solid, phage structure and packaging gene; hatched, DNA replication-, transcription-, and translation-related gene; crosshatched, host lysis gene; open, genes with unknown functions. The tRNA coding region was not found. The numbers (1 to 263) show predicted ORFs listed in Table SA1 in the supplemental material.