Characterization and application of a lytic jumbo phage ZPAH34 against multidrug-resistant Aeromonas hydrophila

Abstract

Aeromonas hydrophila is an emerging foodborne pathogen causing human gastroenteritis. Aeromonas species isolated from food such as seafood presented multidrug-resistance (MDR), raising serious concerns regarding food safety and public health. The use of phages to infect bacteria is a defense against drug-resistant pathogens. In this study, phage ZPAH34 isolated from the lake sample exerted lytic activity against MDR A. hydrophila strain ZYAH75 and inhibited the biofilm on different food-contacting surfaces. ZPAH34 has a large dsDNA genome of 234 kb which belongs to a novel jumbo phage. However, its particle size is the smallest of known jumbo phages so far. Based on phylogenetic analysis, ZPAH34 was used to establish a new genus Chaoshanvirus. Biological characterization revealed that ZPAH34 exhibited wide environmental tolerance, and a high rapid adsorb and reproductive capacity. Food biocontrol experiments demonstrated that ZPAH34 reduces the viable count of A. hydrophila on fish fillets (2.31 log) and lettuce (3.28 log) with potential bactericidal effects. This study isolated and characterized jumbo phage ZPAH34 not only enriched the understanding of phage biological entity diversity and evolution because of its minimal virion size with large genome but also was the first usage of jumbo phage in food safety to eliminate A. hydrophila.

Keywords: Aeromonas hydrophila; biofilm; foods; genome; phage ZPAH34.

Figure 1

Lytic curves of phages on Aeromonas hydrophila ZYAH75. Lytic ability of phages ZPAH12 (A), ZPAH21 (B), ZPAH29 (C), ZPAH34 (D), ZPAH71 (E), ZPAH85 (F), ZPAH103 (G), ZPAH106 (H), ZPAH109 (I), and ZPAH118 (J) against A. hydrophila ZYAH75 was determined at various range of infections (MOIs of 0.001, 0.01, 0.1, 1, 10, 100, and 1,000), respectively. The control group was added LB to the bacterial suspension.

Figure 2

Effect of ZPAH34 on A. hydrophila ZYAH75 biofilms. Biofilm inhibition ability of ZPAH34 in 96-well plates and on glass coverslip (A) incubated with bacterial suspension at 28°C for 72 h. Clearance ability of ZPAH34 on mature biofilm in 96-well plates and on glass coverslip (B) after treatment for 12 h. The crystal violet staining method was used to determine the OD₆₀₀ in each well. *p < 0.05; **p < 0.01.

Figure 3

Genomic map of ZPAH34. Circles display (from the outside): (1) the gene coding regions transcribed in the clockwise or the counterclockwise direction. (2) G + C % content. (3) GC skew plot of G-C/G + C are present by green and purple. (4) The inner circle indicates the full length of the ZPAH34 genome. The open reading frames represent genes encoding structural proteins, cell lysis proteins, DNA replication and metabolism proteins, and tRNAs are denoted by the color blue, yellow, oranges, and red, respectively.

Figure 4

Transmission electron microscopy of ZPAH34.

Figure 5

Biological properties of ZPAH34. (A) Adsorption assay of ZPAH34. (B) One-step growth curve of ZPAH34. (C) pH tolerance of ZPAH34. (D) Thermal tolerance of ZPAH34.

Figure 6

Phylogenetic tree based on their major capsid proteins was constructed using IQ-tree. The blue arc is used to indicate phages that are classified as the same subfamily. The square of the same color represents phages classified as the same genus. The roundness indicates the phages of the new proposed genus Chaoshanvirus.

Figure 7

Inhibition of ZPAH34 against A. hydrophila ZYAH75 in lettuce incubated at 4°C (A) and 25°C (B) and fish fillet incubated at 4°C (C) and 25°C (D). Error bars represent the mean ± SD. *p < 0.05; **p < 0.01.