Isolation and characterization of a novel phage belonging to a new genus against Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is a major foodborne pathogen that contaminates aquatic products and causes great economic losses to aquaculture. Because of the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages are considered promising agents for their biocontrol as an alternative or supplement to antibiotics. In this study, a lytic vibriophage, vB_VpaM_R16F (R16F), infecting V. parahaemolyticus 1.1997T was isolated, characterized and evaluated for its biocontrol potential. METHODS: A vibriophage R16F was isolated from sewage from a seafood market with the double-layer agar method. R16F was studied by transmission electron microscopy, host range, sensitivity of phage particles to chloroform, one-step growth curve and lytic activity. The phage genome was sequenced and in-depth characterized, including phylogenetic and taxonomic analysis. RESULTS: R16F belongs to the myovirus morphotype and infects V. parahaemolyticus, but not nine other Vibrio spp. As characterized by determining its host range, one-step growth curve, and lytic activity, phage R16F was found to highly effective in lysing host cells with a short latent period (< 10 min) and a small burst size (13 plaque-forming units). R16F has a linear double-stranded DNA with genome size 139,011 bp and a G + C content of 35.21%. Phylogenetic and intergenomic nucleotide sequence similarity analysis revealed that R16F is distinct from currently known vibriophages and belongs to a novel genus. Several genes (e.g., encoding ultraviolet damage endonuclease and endolysin) that may enhance environmental competitiveness were found in the genome of R16F, while no antibiotic resistance- or virulence factor-related gene was detected. CONCLUSIONS: In consideration of its biological and genetic properties, this newly discovered phage R16F belongs to a novel genus and may be a potential alternate biocontrol agent.

Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi.

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

A newly isolated roseophage represents a distinct member of Siphoviridae family.

BACKGROUND: Members of the Roseobacter lineage are a major group of marine heterotrophic bacteria because of their wide distribution, versatile lifestyles and important biogeochemical roles. Bacteriophages, the most abundant biological entities in the ocean, play important roles in shaping their hosts' population structures and mediating genetic exchange between hosts. However, our knowledge of roseophages (bacteriophages that infect Roseobacter) is far behind that of their host counterparts, partly reflecting the need to isolate and analyze the phages associated with this ecologically important bacterial clade. METHODS: vB_DshS-R4C (R4C), a novel virulent roseophage that infects Dinoroseobacter shibae DFL12T, was isolated with the double-layer agar method. The phage morphology was visualized with transmission electron microscopy. We characterized R4C in-depth with a genomic analysis and investigated the distribution of the R4C genome in different environments with a metagenomic recruitment analysis. RESULTS: The double-stranded DNA genome of R4C consists of 36,291 bp with a high GC content of 66.75%. It has 49 genes with low DNA and protein homologies to those of other known phages. Morphological and phylogenetic analyses suggested that R4C is a novel member of the family Siphoviridae and is most closely related to phages in the genus Cronusvirus. However, unlike the Cronusvirus phages, R4C encodes an integrase, implying its ability to establish a lysogenic life cycle. A terminal analysis shows that, like that of λ phage, the R4C genome utilize the 'cohesive ends' DNA-packaging mechanism. Significantly, homologues of the R4C genes are more prevalent in coastal areas than in the open ocean. CONCLUSIONS: Information about this newly discovered phage extends our understanding of bacteriophage diversity, evolution, and their roles in different environments.

[Effects of global climate change on the ecological characteristics and biogeochemical significance of marine viruses--A review].

As the most abundance biological agents in the oceans, viruses can influence the physiological and ecological characteristics of host cells through viral infections and lysis, and affect the nutrient and energy cycles of the marine food chain. Thus, they are the major players in the ocean biogeochemical processes. The problems caused by global climate changes, such as sea-surface warming, acidification, nutrients availability, and deoxygenation, have the potential effects on marine viruses and subsequently their ecological and biogeochemical function in the ocean. Here, we reviewed the potential impacts of global climate change on the ecological characteristics (e. g. abundance, distribution, life cycle and the host-virus interactions) and biogeochemical significance (e. g. carbon cycling) of marine viruses. We proposed that marine viruses should not be ignored in the global climate change study.

Endolysins: a new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria.

Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.

A broad-host-range lytic phage vB_VhaS-R18L as a candidate against vibriosis.

Vibriosis is one of the most common bacterial diseases that cause high rates of mortality and considerable economic losses in aquaculture. Phage therapy has been considered as a promising alternative method to antibiotics in the biocontrol of infectious diseases. Genome sequencing and characterization of the phage candidates are prerequisites before field applications to ensure environmental safety. In this study, a lytic phage, named vB_VhaS-R18L (R18L), was isolated from the coastal seawater of Dongshan Island, China. The phage was characterized in terms of morphology, genetic content, infection kinetics, lytic profile, and virion stability. Transmission electronic microscopy indicated that R18L is siphovirus-like, comprising an icosahedral head (diameter 88.6 ± 2.2 nm) and a long noncontractile tail (225 × 11 nm). Genome analysis indicated R18L to be a double-stranded DNA virus with a genome size of 80,965 bp and a G + C content of 44.96%. No genes that encode known toxins or genes implicated in lysogeny control were found in R18L. A one-step growth experiment showed that R18L had a latent period of approximately 40 min and a burst size of 54 phage particles per infected cell. R18L showed lytic activity against a wide range of at least five Vibrio species (V. alginolyticus, V. cholerae, V. harveyi, V. parahemolyticus, and V. proteolyticus). R18L was relatively stable at pH 6-11 and at temperatures ranging from 4°C to 50°C. The broad lytic activity across Vibrio species and the stability in the environment make R18L a potential candidate for phage therapy in controlling vibriosis in aquaculture systems.

Virus-Host Interactions Drive Contrasting Bacterial Diel Dynamics in the Ocean.

Marine organisms perform a sea of diel rhythmicity. Planktonic diel dynamics have been shown to be driven by light, energy resources, circadian rhythms, and the coordinated coupling of photoautotrophs and heterotrophic bacterioplankton. Here, we explore the diel fluctuation of viral production and decay and their impact on the total and active bacterial community in the coastal and open seawaters of the South China Sea. The results showed that the night-production diel pattern of lytic viral production was concurrent with the lower viral decay at night, contributing to the accumulation of the viral population size during the night for surface waters. The diel variations in bacterial activity, community composition, and diversity were found highly affected by viral dynamics. This was revealed by the finding that bacterial community diversity was positively correlated to lytic viral production in the euphotic zone of the open ocean but was negatively related to lysogenic viral production in the coastal ocean. Such distinct but contrasting correlations suggest that viral life strategies can not only contribute to diversifying bacterial community but also potentially piggyback their host to dominate bacterial community, suggesting the tightly synchronized depth-dependent and habitat-specific diel patterns of virus-host interactions. It further implies that viruses serve as an ecologically important driver of bacterial diel dynamics across the ocean, highlighting the viral roles in bacterial ecological and biogeochemical processes in the ocean.

Diversity and function of mountain and polar supraglacial DNA viruses.

Mountain and polar glaciers cover 10% of the Earth's surface and are typically extreme environments that challenge life of all forms. Viruses are abundant and active in supraglacial ecosystems and play a crucial role in controlling the supraglacial microbial communities. However, our understanding of virus ecology on glacier surfaces and their potential impacts on downstream ecosystems remains limited. Here, we present the supraglacial virus genome (SgVG) catalog, a 15-fold expanded genomic inventory of 10,840 DNA-virus species from 38 mountain and polar glaciers, spanning habitats such as snow, ice, meltwater, and cryoconite. Supraglacial DNA-viruses were highly specific compared to viruses in other ecosystems yet exhibited low public health risks. Supraglacial viral communities were primarily constrained by habitat, with cryoconite displaying the highest viral activity levels. We observed a prevalence of lytic viruses in all habitats, especially in cryoconite, but a high level of lysogenic viruses in snow and ice. Additionally, we found that supraglacial viruses could be linked to ∼83% of obtained prokaryotic phyla/classes and possessed the genetic potential to promote metabolism and increase cold adaptation, cell mobility, and phenolic carbon use of hosts in hostile environmental conditions using diverse auxiliary metabolic genes. Our results provide the first systematic characterization of the diversity, function, and public health risks evaluation of mountain and polar supraglacial DNA viruses. This understanding of glacial viruses is crucial for function assessments and ecological modeling of glacier ecosystems, especially for the Tibetan Plateau's Mountain glaciers, which support ∼20% of the human populations on Earth.

Isolation and Characterization of a Novel Cyanophage Encoding Multiple Auxiliary Metabolic Genes.

As significant drivers of cyanobacteria mortality, cyanophages have been known to regulate the population dynamics, metabolic activities, and community structure of this most important marine autotrophic picoplankton and, therefore, influence the global primary production and biogeochemical cycle in aquatic ecosystems. In the present study, a lytic Synechococcus phage, namely S-SZBM1, was isolated and identified. Cyanophage S-SZBM1 has a double-stranded DNA genome of 177,834 bp with a G+C content of 43.31% and contains a total of 218 predicted ORFs and six tRNA genes. Phylogenetic analysis and nucleotide-based intergenomic similarity suggested that cyanophage S-SZBM1 belongs to a new genus under the family Kyanoviridae. A variety of auxiliary metabolic genes (AMGs) that have been proved or speculated to relate to photosynthesis, carbon metabolism, nucleotide synthesis and metabolism, cell protection, and other cell metabolism were identified in cyanophage S-SZBM1 genome and may affect host processes during infection. In addition, 24 of 32 predicted structural proteins were identified by a high-throughput proteome analysis which were potentially involved in the assembly processes of virion. The genomic and proteomic analysis features of cyanophage S-SZBM1 offer a valuable insight into the interactions between cyanophages and their hosts during infection.

Insignificant Response of Bacterioplankton Community to Elevated pCO2 During a Short-Term Microcosm Experiment in a Subtropical Eutrophic Coastal Ecosystem.

Ocean acidification, as one of the major consequences of global climate change, markedly affects multiple ecosystem functions in disparate marine environments from coastal habitats to the deep ocean. Evaluation of the responses of marine microbial community to the increasing partial pressure of CO2 (pCO2) is crucial to explore the microbe-driven biogeochemical processes in the future ocean. In this study, a microcosm incubation of eutrophic coastal water from Xiamen Bay under elevated pCO2 (about 1,000 µatm) and control (ambient air, about 380-410 µatm) conditions was conducted to investigate the effect of ocean acidification on the natural bacterioplankton community. During the 5-day incubation period, the chlorophyll a concentration and bacterioplankton abundance were not significantly affected by increased pCO2. Hierarchical clustering and non-metric multidimensional scaling analysis based on Bray-Curtis similarity among the bacterioplankton community derived from the 16S rRNA genes revealed an inconspicuous impact of elevated pCO2 on the bacterial community. During the incubation period, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Epsilonbacteraeota were predominant in all microcosms. Despite the distinct temporal variation in the composition of the bacterioplankton community during the experimental period, statistical analyses showed that no significant difference was found on bacterioplankton taxa between elevated pCO2 and control, indicating that the bacterioplankton at the population-level were also insensitive to elevated pCO2. Our results therefore suggest that the bacterioplankton communities in the fluctuating and eutrophic coastal ecosystems appear to be adaptable to the short-term elevated pCO2.

Viral Regulation on Bacterial Community Impacted by Lysis-Lysogeny Switch: A Microcosm Experiment in Eutrophic Coastal Waters.

Marine viruses are major drivers of global biogeochemical cycles and energy fluxes, yet the importance of viral impacts on the succession and diversity of the bacterial community remains largely unexplored. Here, we explored viral life strategy and its potential effect on the bacterial community by experimental incubations of eutrophic coastal waters under lysogen-induced and non-induced treatments. The lysogen-induced treatment showed relatively constant viral and bacterial abundances, lytic and lysogenic viral production throughout the experimental period, together with the progressive declines in not only the relative abundances for SAR11, Rhodobacteraceae, Alteromonadaceae, and SAR86 but the bacterial community diversity. Conversely, the non-induced treatment observed the marked variation in the abundances of viruses, bacteria and cells with high nucleic acid content over the time course of incubation, which was congruent with the drastic shift in lytic and lysogenic viral production as well as the succession of bacterial community. Our results supported the hypotheses that a high level of lysogeny would occur with the increasing density of bacteria with rapid growth rate, which may contribute to a relatively lower host community diversity, whereas the lysogeny to lysis switching would fuel growth opportunities for less-active or initially rare bacterial taxa and generate a more diverse bacterial community. Altogether, the present study underscored the crucial regulatory role of the viral lysis-lysogeny pattern in bacterial community dynamics, composition and diversity, highlighting the viral impact on the microbial food web and biogeochemical processes.

Erratum: Yang, Y.; et al. A Novel Roseosiphophage Isolated from the Oligotrophic South China Sea. Viruses 2017, 9, 109.

The authors wish to make the following changes to their paper [...].

A Novel Roseosiphophage Isolated from the Oligotrophic South China Sea.

The Roseobacter clade is abundant and widespread in marine environments and plays an important role in oceanic biogeochemical cycling. In this present study, a lytic siphophage (labeled vB_DshS-R5C) infecting the strain type of Dinoroseobacter shibae named DFL12T, which is part of the Roseobacter clade, was isolated from the oligotrophic South China Sea. Phage R5C showed a narrow host range, short latent period and low burst size. The genome length of phage R5C was 77, 874 bp with a G+C content of 61.5%. Genomic comparisons detected no genome matches in the GenBank database and phylogenetic analysis based on DNA polymerase I revealed phylogenetic features that were distinct to other phages, suggesting the novelty of R5C. Several auxiliary metabolic genes (e.g., phoH gene, heat shock protein and queuosine biosynthesis genes) were identified in the R5C genome that may be beneficial to the host and/or offer a competitive advantage for the phage. Among siphophages infecting the Roseobacter clade (roseosiphophages), four gene transfer agent-like genes were commonly located with close proximity to structural genes, suggesting that their function may be related to the tail of siphoviruses. The isolation and characterization of R5C demonstrated the high genomic and physiological diversity of roseophages as well as improved our understanding of host-phage interactions and the ecology of the marine Roseobacter.

Evaluation of Tangential Flow Filtration for the Concentration and Separation of Bacteria and Viruses in Contrasting Marine Environments.

Tangential flow filtration (TFF), which has been widely adopted to concentrate a diverse array of microbes from water, is a promising method of microbial separation or removal. However, it is essential to select an optimal membrane suitable for the specific filtration application. This study evaluated two different scales of TFF systems for concentrating and separating microbes (including bacteria and viruses) from contrasting marine waters. Among bacteria-size membranes, polyvinylidene difluoride (PVDF) membranes showed higher bacterial recovery, but lower viral permeation efficiencies than polyethersulfone (PES) membranes, regardless of environments and scales of TFF. Estuary samples showed significantly higher percentages of bacterial retention than nearshore and ocean samples. For virus-size membranes, a higher viral recovery and lower sorption was observed for regenerated cellulose membrane than PES membranes in the small-scale TFF. Similar viral recoveries were observed between PES membranes in the large-scale TFF, with higher viral concentrations being observed in estuary samples than in nearshore samples. Deep ocean samples showed the lowest recovery of viruses, which was consistent with observations of bacterial recovery. Synthetically, PVDF may be more suitable for the concentration of bacterial cells, while PES would be a better choice for the collection of viruses. When compared with the PES membrane, regenerated cellulose is better for viral concentration, while PES is recommended to obtain bacteria- and virus-free seawater.

Complete genome sequence of vB_DshP-R2C, a N4-like lytic roseophage.

vB_DshP-R2C, a lytic phage that infects the marine bacterium Dinoroseobacter shibae DFL12(T), one of the model organisms of the Roseobacter clade, was isolated. Here we report the overall genome architecture of R2C. Genome analysis revealed that R2C is an N4-like phage with a 74.8 kb genome that contains 85 putative gene products.