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. 2022 Apr 7:13:830692.
doi: 10.3389/fmicb.2022.830692. eCollection 2022.

Isolation and Characterization of a Lytic Vibriophage OY1 and Its Biocontrol Effects Against Vibrio spp

Lu Gao  1   2 , Min Ouyang  1 , Yi Li  1 , Hui Zhang  3 , Xiang-Feng Zheng  1 , Hua-Xiang Li  1 , Sheng-Qi Rao  1 , Zhen-Quan Yang  1   2 , Song Gao  4   5
Affiliations

Isolation and Characterization of a Lytic Vibriophage OY1 and Its Biocontrol Effects Against Vibrio spp

Lu Gao et al. Front Microbiol. .

Abstract

Vibrio species are important pathogens of marine animals and aquaculture populations and some of them can cause serious infections in humans through consumption of contaminated seafood and aquaculture products. Lytic bacteriophages can potentially alleviate Vibrio contamination in the aquaculture organisms and in the processing of aquatic products and have gained significant scientific attention in recent years. In the present study, bacteriophages were isolated from sewage of local aquatic products markets and grown using Vibrio mimicus CICC 21613 as host cells. The lytic vibriophage OY1 belonging to the newly proposed family Autographiviridae and the genus Maculvirus was identified by observation under electron microscope and comparative genomic analysis. The phage OY1 showed lytic activity against 24 among 32 tested strains belonging to eight Vibrio species. The complete phage OY1 genome consists of a single circular double-stranded DNA of 43,479 bp with a total GC content of 49.27% and was predicted to encode 40 open reading frames (ORFs). To evaluate its potential against vibrios, the one-step growth curve, thermal and pH stability, host range, and lytic activity of the OY1 phage against Vibrio species were evaluated. The results showed that phage OY1 had a range of thermal and pH tolerance, and exhibited a significant inhibitory effect on the growth of tested Vibrio species. Bacterial growth in the fish muscle extract juice (FMEJ) inoculated with Vibrio mimicus CICC 21613, Vibrio parahaemolyticus CICC 21617, Vibrio alginolyticus VJ14, and the mixed bacterial culture was reduced by 2.65 log CFU/ml, 2.42 log CFU/ml, 1.93 log CFU/ml, and 2.01 log CFU/ml, respectively, by incubation with phage OY1 at 25°C for 36 h. Phage OY1 also showed a strong ability to prevent biofilm formation and destroy formed Vibrio species biofilms. These results indicate that phage OY1 is a potential biocontrol agent against Vibrio species in the aquaculture industry and in food safety control.

Keywords: Vibrio species; biocontrol; biofilm; lytic activity; vibriophage.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Morphology of the phage plaque and negative stained phage virions. (A) The plaques formed by phage OY1 on lawn of strain Vibrio mimicus CICC 21613. (B) Electron micrographs of phage OY1, the broad arrow indicate a tail and the narrow arrows indicate tail fibers.
Figure 2
Figure 2
Biological properties of phage OY1. (A) Lytic activity of phage OY1 LB broth supplemented with 3% (w/v) NaCl. (B) One-step growth curve of phage OY1 in V. mimicus CICC 21613 at 37°C L, latent period; R, rise period; P, plateau period. Curves were determined in 3% LB broth at 37°C at multiplicity of infection (MOI) ratio of 0.01. (C) Temperature tolerance from 50°C to 80°C. (D) pH stability of phage OY1. Assays were performed in triplicate. Data are reported as the mean ± SD.
Figure 3
Figure 3
The genome structure of phage OY1. Arrows in clockwise indicate predicted protein-coding genes encoded on the Watson strand with gene names labeled on the external side (black: hypothetical protein; green: DNA metabolism related; yellow: RNA polymerase; blue: structural protein; purple: lysis protein; red: predicted proteins with additional functions). The second cycle (from outside) indicate GC skew (G − C/G + C in a 1-kb window and 0.1-kb incremental shift), value for the outer most line is 0.4 and −0.4 for the inner most. The third cycle indicates the sequencing coverage, the outer most line represents 9,500× and inner most represents 5,500×. The forth cycle is the physical map scaled in kbp.
Figure 4
Figure 4
Comparison of the phage OY1 genome with three Vibrio parahaemolyticus phages, i.e., phages vB_VpaP_KF1 (accession no. MF754111), vB_VpaP_KF2 (accession no. MF754112) and VP93 (accession no. FJ896200). The predicted open reading frames (ORFs) are indicated by arrows. ORFs of unknown function are in black, predicted DNA metabolism ORFs in green, RNA polymerase in yellow, structural protein ORFs in blue, lysis protein ORFs in purple and ORFs with additional functions in red, respectively. Genes named in gray indicate the eight proteins that were predicted (with E-value <10−5) by a separate Standard Protein BLAST (blastp) in GenBank using translated amino acid sequence as the query.
Figure 5
Figure 5
Inactivation of V. mimicus CICC 21613 (A), V. parahaemolyticus CICC 21617 (B), V. alginolyticus VJ14 (C), and mixed bacteria (D) in FMEJ at 25°C. The test was performed in triplicates. Data are reported as the mean ± SD.
Figure 6
Figure 6
Biofilm prevention and destruction using phage OY1 against V. mimicus CICC 21613, V. parahaemolyticus CICC 21617, V. alginolyticus VJ14, and bacterial mixture. *indicates a significant difference (p < 0.01) compared with the blank control.
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