WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages.

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

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

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.

Isolation and Characterization of a Virulent Bacteriophage for Controlling Salmonella Enteritidis Growth in Ready-to-Eat Mixed-Ingredient Salads.

ABSTRACT: Ready-to-eat vegetable salads have gained popularity worldwide. However, the microbial safety of these salads is a health concern, primarily due to Salmonella Enteritidis contamination during the growing, harvesting, processing, and handling of produce. In this study, a bacteriophage-based strategy was developed to control Salmonella Enteritidis growth in mixed-ingredient salads. The lytic Salmonella-specific phage SapYZU01 was isolated from a soil sample from a suburban vegetable field in Yangzhou (People's Republic of China). SapYZU01 has a short latent period, a large burst size, and a lytic effect against 13 Salmonella Enteritidis strains isolated from various sources (human samples, pork, deli foods, chickens, and chicken meat). The SapYZU01 genome did not contain virulence or antibiotic resistance genes. SapYZU01 significantly decreased the viability of Salmonella Enteritidis cells in iceberg lettuce, chicken meat, and mixed-ingredient (lettuce plus chicken) salads at 37 and 25°C. Bacterial levels in the salad decreased significantly (by 4.0 log CFU/g) at 25°C after treatment of contaminated lettuce before salad preparation with SapYZU01 at a multiplicity of infection (MOI) of 100. Bacterial levels were decreased by 3.8 log CFU/g at 25°C in lettuce plus chicken salads treated after the salad preparation with SapYZU01 at an MOI of 100. In contrast, treating cooked chicken meat with SapYZU01 at an MOI of 100 before mixing it with contaminated lettuce decreased the bacterial level of the salad by 1.2 log CFU/g at 25°C. These findings indicate the potential application of SapYZU01 as a natural biocontrol agent against Salmonella Enteritidis in mixed-ingredient salads. However, both the treatment method and the bacteriophage MOI must be considered when using this lytic bacteriophage in mixed-ingredient salads.

Isolation and genomic characterization of P.A-5, a novel virulent bacteriophage against Enterobacter hormaechei.

Enterobacter hormaechei is a foodborne pathogen responsible for neonatal sepsis in humans and respiratory disease in animals. In this work, a new virulent phage (P.A-5) infecting E. hormaechei was isolated from domestic sewage samples and characterized. Transmission electron microscopy revealed that P.A-5 belonged to the family Myoviridae having a head size of 77.53 nm and a tail length of 72.24 nm. The burst size was 262 PFU/cell after a latent period of 20 min. Phage P.A-5 was able to survive in a pH range of 4-9 and resist temperatures up to 55 °C for 60 min. The genome sequence of P.A-5 had homology most similar to that of Shigellae phage MK-13 (GenBank: MK509462.1). Pork artificially contaminated with E. hormaechei was used as a model to evaluate the potential of P.A-5. The results clearly showed that P.A-5 treatment can completely inhibit E. hormaechei growth in pork within 8 h, indicating the potential use of P.A-5 as a biocontrol agent for E. hormaechei.

Physicochemical and antibacterial properties of fabricated ovalbumin-carvacrol gel nanoparticles.

The applications of carvacrol are limited due to its poor stability and water solubility, and high volatility; however, ovalbumin can be used to encapsulate hydrophobic molecules, improve their aqueous solubility, and reduce their volatility. In this study, we fabricated ovalbumin-carvacrol nanoparticles (OCGns) under different pH (2, 5, 7, and 9) conditions using a gel embedding method and investigated their physicochemical and antibacterial properties. Rheological experiments revealed that the G' of ovalbumin gels (OGs) prepared under different pH conditions were OG-2 > OG-7 > OG-9 > OG-5. Carvacrol addition reduced the tight structure of ovalbumin and carvacrol under pH 5 and 7 conditions, with hardness first decreasing and then increasing, but increasing under pH 2 and 9 conditions. Fluorescence and infrared spectroscopy indicated complex formation, with carvacrol increasing the average diameter of nanoparticles prepared at pH 2, 5, 7, and 9. Encapsulation reached 89.34 and 91.86% at pH 2 and 9, respectively; however, inhibition experiments revealed that the minimum inhibitory concentration of OCGn-2 against Gram-positive Bacillus cereus and Salmonella (0.08 and 0.16 mg mL-1, respectively) was lower than that of OCGn-9 (both 0.28 mg mL-1). Moreover, OCGn-2 possessed a better dense gel structure and a higher stability, encapsulation rate, and antibacterial activity, suggesting that pH affects gel microstructure and thus the encapsulation efficiency and bacteriostatic properties of the prepared nanoparticles. These results contribute to our knowledge of the design and fabrication of polymeric nanoparticle delivery systems for bioactive compounds with beneficial properties.

Isolation of virulent phages infecting dominant mesophilic aerobic bacteria in cucumber pickle fermentation.

Pickle is a type of mildly lactic acid fermented vegetable and is a traditional dish favored in China, Japan, and Korea. Corruption of spoilage bacteria and accumulation of nitrite during vegetable fermentation are common problems that affect the pickle industry and consumer health. In this work, cucumber juice was used as a vegetable model to study the dominant mesophilic aerobic bacteria (MAB) producing nitrite during pickle fermentation. Virulent phages infecting the dominant MABs combined with Lactobacillus plantarum M6 were used to control these bacteria. Enterobacter cloacae and Pseudomonas fluorescens are the dominant MABs in the fermentation of cucumber juice containing 4% or 8% NaCl, with isolation percentages reaching 30.6% and 23.1%, respectively. Virulent phages PspYZU5415 and EcpYZU01 were isolated using P. fluorescens J5415 and E. cloacae J01 as the host bacteria, respectively. These two phages show a broad host range and strong lytic activity, and their genomes contain no toxins and antibiotic resistance genes. PspYZU5415 and EcpYZU01 were combined into a cocktail (designated as Phage MIX) that effectively inhibits the growth of E. cloacae and P. fluorescens in cucumber juice with different salt concentrations. PhageMIX combined with L. plantarum M6 decreased the counts of P. mendocina and E. cloacae to undetectable levels at 48 h during the fermentation of cucumber juice artificially contaminated with P. mendocina and E. cloacae. In addition, nitrite content increased to 11.3 mg/L at 20 h and then degraded completely at 36 h. By contrast, P. mendocina and E. cloacae remained in the groups without PhageMIX during fermentation (0-48 h). Nitrite content rapidly increased to 65.7 mg/L at 12 h and then decreased to 21.6 mg/L at 48 h in the control group. This study suggests that PhageMIX combined with lactic acid bacterial strains can be used as an ecological starter for controlling the dominant MABs P. mendocina and E. cloacae and for reducing nitrate production during the early stage of pickle fermentation.

Down-regulation of BnDA1, whose gene locus is associated with the seeds weight, improves the seeds weight and organ size in Brassica napus.

Brassica napus L. is an important oil crop worldwide and is the main raw material for biofuel. Seed weight and seed size are the main contributors to seed yield. DA1 (DA means big in Chinese) is an ubiquitin receptor and negatively regulates seed size. Down-regulation of AtDA1 in Arabidopsis leads to larger seeds and organs by increasing cell proliferation in integuments. In this study, BnDA1 was down-regulated in B. napus by over expressed of AtDA1R358K , which is a functional deficiency of DA1 with an arginine-to-lysine mutation at the 358th amino acid. The results showed that the biomass and size of the seeds, cotyledons, leaves, flowers and siliques of transgenic plants all increased significantly. In particular, the 1000 seed weight increased 21.23% and the seed yield per plant increased 13.22% in field condition. The transgenic plants had no negative traits related to yield. The candidate gene association analysis demonstrated that the BnDA1 locus was contributed to the seeds weight. Therefore, our study showed that regulation of DA1 in B. napus can increase the seed yield and biomass, and DA1 is a promising target for crop improvement.