Isolation and identification of specific Enterococcus faecalis phage C-3 and G21-7 against Avian pathogenic Escherichia coli and its application to one-day-old geese

Abstract

Colibacillosis caused by Avian pathogenic Escherichia coli (APEC), including peritonitis, respiratory tract inflammation and ovaritis, is recognized as one of the most common and economically destructive bacterial diseases in poultry worldwide. In this study, the characteristics and inhibitory potential of phages were investigated by double-layer plate method, transmission electron microscopy, whole genome sequencing, bioinformatics analysis and animal experiments. The results showed that phages C-3 and G21-7 isolated from sewage around goose farms infected multiple O serogroups (O1, O2, O18, O78, O157, O26, O145, O178, O103 and O104) Escherichia coli (E.coli) with a multiplicity of infection (MOI) of 10 and 1, respectively. According to the one-step growth curve, the incubation time of both bacteriophage C-3 and G21-7 was 10 min. Sensitivity tests confirmed that C-3 and G21-6 are stable at 4 to 50 °C and pH in the range of 4 to 11. Based on morphological and phylogenetic analysis, phages C-3 and G21-7 belong to Enterococcus faecalis (E. faecalis) phage species of the genus Saphexavirus of Herelleviridae family. According to genomic analysis, phage C-3 and G21-7 were 58,097 bp and 57,339 bp in size, respectively, with G+C content of 39.91% and 39.99%, encoding proteins of 97 CDS (105 to 3,993 bp) and 96 CDS (105 to 3,993 bp), and both contained 2 tRNAs. Both phages contained two tail proteins and holin-endolysin system coding genes, and neither carried resistance genes nor virulence factors. Phage mixture has a good safety profile and has shown good survival probability and feed efficiency in both treatment and prophylaxis experiments with one-day-old goslings. These results suggest that phage C-3 and G21-7 can be used as potential antimicrobials for the prevention and treatment of APEC.

Keywords: Avian pathogenic Escherichia coli; Enterococcus faecalis phage; genus Saphexavirus; one-day-old geese; virulent phage.

Figure 1

The host range of the phage. (A) Host spectrum of 8 bacteriophages against different serum groups of APEC; (B) Host profile of phage C-3 and C-5 against 61 strains of E. coli with different serogroups (different sources); Transmission electron micrographs (The bar indicates 100 nm); (C) C-3 was estimated approximately 75 (±1) nm between opposite apices and the head was connected to a long 135 (±1) nm tail; (D) G21-7 was estimated approximately 115 (±1) nm between opposite apices and the head was connected to a long 160 (±1) nm tail.

Figure 2

Growth characteristics: the optimal MOI and one-step experiments. (A) The Optimal Multiplicity of Infection (MOI) phage C-3/G21-7 was tested under different MOIs (1,000, 100, 10, 1, 0.1 and 0.01). When at an MOI of 10, phage C-3 produced highest phage titer; when at an MOI of 1, phage G21-7 produced highest phage titer. (B) The one-step growth curve of phage C-3/G21-7. Sensitivity of Temperature and PH. (C) Sensitivity of temperature: phage C-3/G21-7 suspension was incubated in different temperatures (40, 50, 60, 70, 80°C) for 30 min or 1 h. (D) Sensitivity of PH: phage C-3/G21-7 suspension was incubated in different PH conditions for 10 min. Phage survival rate = (titer after incubation)/(initial titer). The standard deviation is indicated by a vertical line.

Figure 3

Genome analysis. (A) Phage C-3 genome map. (B) Phage G21-7 genome map. Both are drawn using Proksee. (C) After obtaining CDS annotations through Bakta, a collinearity analysis graph was drawn. Comparison of C-3 and G21-7 with the highly homologous phage PHB08 (MK570225, 55,244 bp). The genome encoding the prediction protein is shown in green, tRNA is shown in red.

Figure 4

Phylogenetic analysis and ANI clustering. (A) Whole genome sequences of 12 phages from the NCBI website were compared. Purple indicates Kochikohdavirus genus (including separate clades) and green indicates Saphexavirus genus. Stars have been identified for two phages C-3 and G21-7 in this paper. (B) The ANI values between phage C-3 and G21-7 and Saphexavirus genomes were clustered by Ward’s linkage method.

Figure 5

Tail protein sequence analysis. (A) Tail fibers protein of 8 phages from the NCBI website were compared with domain and motif structure analysis. (B) Phage major tail protein 2 of 8 phages from the NCBI website were compared with domain and motif structure analysis.

Figure 6

Safety test of phage mixtures in vivo, (A) feed efficiency (feed efficiency = Increased weight/feed consumption) was significantly different in the oral group compared with the intraperitoneal injection group (p < 0.05) and the control group (p < 0.05); (B) Kaplan–Meier analysis curves and log-rank test showed that p value = 0.24 and Chi-square = 2.02 between oral, intrabitoneal injection and control group showed no difference. Pairwise comparison showed no difference (p > 0.01, Supplementary Table S11). Treatment test in vivo, (C) feed efficiency of Phage (0.31 kg) was similar to that of the negative control group (0.36 kg) and higher than that of the other antibiotic groups; (D) Kaplan–Meier analysis curves and log-rank test showed that p value <0.0001 and Chi-square = 72.29 among all groups; The Log rank test was conducted between phage and other groups, respectively. The phage showed significant differences with TFSP, FF, SHLHSP, PC, and NC, but no differences with CSSSP and DHSP. Prevention test in vivo, (E) the phage group (0.5 kg) had the highest feed efficiency; (F) Kaplan–Meier analysis curves and log-rank test showed that p value <0.0001 and Chi-square = 200.6 among all groups; the Log rank test was conducted between phage and other groups respectively, phage showed significant differences with SHLHSP and PC, but no differences with other groups. Number of risk shows the number of geese that did not die for 21 days. TFSP, Tiamulin Fumarate Soluble Powder (Veterinary Drug Character (VDC) 020033008, Ringpu, China); FF, Fubennikao Fen (VDC 010122539, Aether Centre (Beijing) BIOLOGY, China); CSSSP, Compound Sulfamonomethoxine Sodium Soluble Powder (VDC 040266233, SHANXI YI KANG ANIMAL’S PHARMACEUTCAL, China); DHSP, Doxycycline Hyclate Soluble Powder (VDC 010126011, Aether Centre (Beijing) BIOLOGY, China); SHLHSP, Spectinomycin Hydrochloride and Lincomycin Hydrochloride Soluble Powder (VDC 20031339, Ringpu, China); PC, positive control; NC, negative control. “*” p < 0.05, “**” p < 0.01, “***” p < 0.001, and “****” p < 0.0001.