Biological Characterization and Genomic Analysis of Novel Phages DLDT_So2 and BHDT_So9 Against Pseudomonas solanacearum, an Infectious Agent in Tomato in Vietnam.

Tomato (Solanum lycopersicum L.) is an important grown vegetable in Vietnam. Bacterial wilt caused by Pseudomonas solanacearum has been considered to be an important disease resulting in a harvest loss up to 90% and significant economic loss to farmers. In this study, two bacteriophages DLDT_So2 and BHDT_So9 specific to P. solanacearum were isolated. Morphological analysis indicated that DLDT_So2 and BHDT_So9 had podovirus morphology and were classified into Autographiviridae family. The latent period and burst size of DLDT_So2 was found to be approximately 120 min and 20.0 ± 2.4 virions per infected cell. Meanwhile, the latent period of BHDT_So9 was 140 min with a burst size of 11.5 ± 2.8 virions per infected cell. Of the 23 bacterial strains tested, the phages infected 7/11 strains of P. solanacearum and none of the other bacteria tested were susceptible to the phages. Stability of the phages at different temperatures, pHs, solvents was also investigated. The genomes of DLDT_So2 and BHDT_So9 are 41,341 bp and 41,296 bp and long with a total GC content of 63%, contains 48 and 46 predicted protein-encoding CDSs. No virulence or antibiotic resistance genes were found in the genomes, suggesting they would be useful biocontrol agents against P. solanacearum. Classification of the phage using average nucleotide identity, phylogenetic analysis was also carried out. The two phages represented new species when they had overall average nucleotide identity of < 95%. This is first report of the isolation and characterization of P. solanacearum-specific phages from tomato farms in Vietnam. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01090-9.

Complete genome sequence of a novel lytic phage of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen in rice.

Bacteriophage L522, which infects Xanthomonas oryzae pv. oryzae, was isolated from a paddy leaf sample collected in Long An province, Vietnam. The phage shows myovirus morphology based on transmission electron microscopy. It displays a latent period and burst size of approximately 3 h and 63 new virions per infected cell (PFU/infected cell), respectively. The genome of L522 is 44,497 bp in length, with 52% GC content. Of the 63 genes identified, functions were predicted for 26. No virulence or antibiotic-resistance genes were detected. The results of a BLASTn search showed similarity to a previously reported Xanthomonas phage, with 85% average nucleotide sequence identity and 87.15% query coverage. Thus, this L522 is a representative of a new species in the genus Xipdecavirus.

Diverse Bacteriophages Infecting the Bacterial Striped Catfish Pathogen Edwardsiella ictaluri.

Bacteriophages infecting Edwardsiella ictaluri have been less investigated, although the host bacterium is one of the most important fish pathogens causing enteric septicemia of catfish (ESC). We present here two distinctly novel bacteriophages vB_EiM_PVN06 and vB_EiA_PVN09 infecting Edwardsiella ictaluri E1, with their geographical origins from the Mekong Delta, Vietnam. Bacteriophage vB_EiM_PVN06 native to a mud sample reveals complete differences of biological properties with the phage vB_EiA_PVN09 originated from a viscus of a healthy catfish (Pangasianodon hypophthalmus) cultured in the same area. Morphological analyses combined with genomic data indicate that phage vB_EiM_PVN06 is classified to Myoviridae family and shares high similarity with E. ictaluri phage PEi21 genome, while vB_EiA_PVN09 is a member of Teseptimavirus genus, Autographiviridae family, and mostly closes to phage vB_EcoP_IME390. The vB_EiA_PVN09 is a T7-like bacteriophage, which has been firstly found infecting to E. ictaluri, and host range analysis also evidences for the cross-infection of this phage to Escherichia coli K12 and Escherichia coli DH5α. Together, our research highlights the diversity of bacteriophages infecting the pathogen E. ictaluri and suggests further explorations of lytic phages in environmental niches, to be exploited in feasible strategies of phage therapy in ESC disease control.

Protective efficacy of phage PVN02 against haemorrhagic septicaemia in striped catfish Pangasianodon hypophthalmus via oral administration.

Haemorrhagic septicaemia caused by Aeromonas hydrophila in striped catfish (Pangasianodon hypophthalmus) is one of the most important aquatic diseases in the Mekong Delta, Vietnam. However, antibiotic-resistant A. hydrophila strains have become popular and resulted in inadequate control of the disease in striped catfish farms. This study investigates the protective efficacy of bacteriophage PVN02 against haemorrhagic septicaemia in striped catfish via oral administration. The phage-containing pellets were prepared by spraying the phage solution on food pellets at 20 ml/kg. The rate of phage desorption from the food pellets into the water was very low; the phage titres in the water were approximately log 1.0 PFU/ml or undetectable. The in vivo experiment evaluating the protective efficacy of PVN02 against haemorrhagic septicaemia in striped catfish was conducted using 21 groups of 1,260 fish in 50-L plastic tanks in triplicate. The catfish were fed twice daily with phage-sprayed pellets. Different densities of bacterial suspensions were added into the tanks for 24 hr. Without the existence of the phage, the highest mortality rate was 68.3 ± 2.9% at the highest density of bacterial suspension. In contrast, the mortality rate at the highest density of bacterial suspension was significantly reduced to 8.33 ± 2.9% or 16.67 ± 2.9% at the phage dose of log 6.2 ± 0.09 or log 4.2 ± 0.09 PFU/g. This study provides a very practical manner of applying phage therapy to prevent disease in large-scale striped catfish farms.

Antibacterial Activity of Copper Nanoparticles-Chitosan Composite against Vibrio parahaemolyticus.

The Early Mortality Syndrome (EMS) caused by Vibrio parahaemolyticus has recently resulted in a serious loss in shrimp farms in the Mekong delta, Vietnam. Here, antibacterial activity of copper nanoparticles-chitosan composite (CuCS) against V. parahaemolyticus was investigated. Copper nanoparticles were synthesized using L-ascorbic acid as a green reducing agent and chitosan as a biopolymer matrix and stabilizing agent. The physical properties of CuCS were evaluated. Next, antibacterial activity of 2.5, 5.0, 10.0 ppm CuCS against V. parahaemolyticus inoculated in a sterilized shrimp-pond water was examined. CuCS at 2.5 ppm eliminated 91.47% and 95.26% of V. parahaemolyticus after 2 and 4 h of exposure, respectively. Complete elimination was attained following 2 h of 5.0 ppm CuCS exposure. A complete elimination of V. parahaemolyticus in a real EMS-infected shrimp-pond water was also described. This study is the first to report the antibacterial activity of CuCS against V. parahaemolyticus, an important pathogen in shrimp industry in the Mekong delta, Vietnam.

Complete genome sequence of a novel lytic phage infecting Aeromonas hydrophila, an infectious agent in striped catfish (Pangasianodon hypophthalmus).

The bacteriophage vB_AhM_PVN02 (PVN02), infecting Aeromonas hydrophila, was isolated from a striped catfish pond water sample in Can Tho City, Vietnam. The phage had high lytic activity with a latent period and burst size of approximately 20 min and 105 plaque-forming units per cell, respectively. Observation of the phage by transmission electron microscopy indicated that PVN02 belongs to the family Myoviridae. The genome of PVN02 is a double-stranded linear DNA with a length in 51,668 bp and a content of 52% GC. Among the 64 genes, 16 were predicted to encode proteins with predicted functions. No virulence or antibiotic resistance genes were found in the genome, suggesting it would be a useful biocontrol agent. Classification of the phage based on sequence comparisons, phylogenetic analysis, and gene-sharing networks was carried out, and it was found to be the first representative of a new species within a previously undefined genus in the family Myoviridae. This study confirmed that PVN02 is a novel lytic phage that could potentially be used as an agent to control Aeromonas hydrophila in striped catfish in the Mekong Delta, Vietnam.

Virulent bacteriophage of Edwardsiella ictaluri isolated from kidney and liver of striped catfish Pangasianodon hypophthalmus in Vietnam.

Striped catfish Pangasianodon hypophthalmus farmed in the Mekong Delta, Vietnam, represents an important contribution to Vietnamese aquaculture exports. However, these fish are affected by frequent disease outbreaks across the entire region. One of the most common infections involves white spots in the internal organs, caused by the bacterium Edwardsiella ictaluri. In this study, a virulent phage specific to E. ictaluri, designated MK7, was isolated from striped catfish kidney and liver samples and characterized. Morphological analysis indicates probable placement in the family Myoviridae with a 65 nm icosahedral head and a 147 %%CONV_ERR%% 19 nm tail. A double-stranded DNA genome of approximately 34 kb was predicted by restriction fragment analysis following digestion with SmaI. The adsorption affinity (ka) of the MK7 phage was estimated as 1.6 %%CONV_ERR%% 10-8 ml CFU-1 min-1, and according to a 1-step growth curve, its latent period and burst size were ~45 min and ~55 phage particles per infected host cell, respectively. Of the 17 bacterial strains tested, MK7 only infected E. ictaluri, although other species of Edwardsiella were not tested. E. ictaluri was also challenged in vitro, in both broth and water from a striped catfish pond and was inactivated by MK7 for 15 h in broth and 51 h in pond water. Thus, initial characterization of phage MK7 indicates its potential utility as a biotherapeutic agent against E. ictaluri infection in striped catfish. This is the first report of a lytic phage specific to an important striped catfish pathogen.

Development of a bacteriophage-based Method for Detection of Escherichia Coli O157:H7 in Fresh Vegetables.

In this study, a method using a recombinant phage for detection of E. coli O157:H7 in fresh vegetables was investigated. Four kinds of fresh vegetables, i.e. lettuce (Lactuca sativa), mustard greens (Brassica juncea), coriander (Coriandrum sativum), and soybean sprouts were selected since they are commonly used in meals in Vietnam. Firstly, a phage-based method was investigated for detection of E. coli O157:H7 in the four types of vegetables. To support the detection by suppressing growth of background bacteria in vegetables, selective antibiotics, i.e. novobiocin (N) and vancomycin (V) in combination with BHI medium were examined. Secondly, quality of the method was evaluated in terms of sensitivity, specificity, and rapidity. The method enabled the detection of E. coli O157:H7 inoculated at 103, 102, or 101 CFU/ 10 mL of sterile 0.8% NaCl containing 5 g of vegetable and in the presence of several Gram-positive and Gram-negative bacteria inoculated at 107 CFU/10 mL. The time for detection was approximately 16.5 hours for E. coli O157:H7 inoculated at 10 CFU/10 mL of sterile 0.8% NaCl containing 5 g of vegetable. The limit of detection was considered to be 2 CFU g-1 vegetable.

Rapid and simple colorimetric detection of Escherichia coli O157:H7 in apple juice using a novel recombinant bacteriophage-based Method.

In this study, a bacteriophage-based method for the colorimetric detection of E. coli O157:H7 in apple juice was investigated. Firstly, a gene encoding Cytochrome c Peroxidase (CCP) chromogenic enzyme was inserted into a wild type PP01 phage genome to construct the recombinant PP01ccp phage that was used in the production of the chromogenic enzyme through specific infection into E. coli O157:H7. The method was then examined in the colorimetric detection of E. coli O157:H7 in broth, and the appearance of E. coli O157:H7 in broth was confirmed by the color change after a few minutes of the enzyme assay. Secondly, the method was investigated in the colorimetric detection of E. coli O157:H7 in apple juice. A low E. coli O157:H7 concentration as 1 CFU mL(-1) was detected in 15 h that was in a shorter time than in previous bioluminescence phage-based methods. Moreover, the method is much simpler compared to other previous phage-based methods since it enables detection without the need for expensive apparatus.

A novel colorimetric method for the detection of Escherichia coli using cytochrome c peroxidase-encoding bacteriophage.

A new rapid and simple method was developed for the detection of Escherichia coli by constructing a recombinant T4 phage carrying the cytochrome c peroxidase gene derived from Saccharomyces cerevisiae (T4ccp) using which, the colorimetric detection of E. coli K12 was examined. The oxidation activity toward the chromogenic substrate cytochrome c was demonstrated by the cytochrome c peroxidase (CCP) produced from the T4ccp genome. The color change caused by the oxidation of the substrate could be visually perceived. The possibility of interference in the detection by the coexistence of other bacteria was assessed using Pseudomonas aeruginosa as a nontarget bacterium, and it was confirmed that the coexistence of P. aeruginosa caused no interference in the detection of E. coli K12.