Characterization of a Peptidoglycan-Degrading Protein: Biochemical and Antimicrobial Characteristics, Antibiotic Synergism, and Delivery System Innovation.

The global health threat posed by antibiotic resistance has led to new research involving bacteriophage-encoded enzymes. This study characterized a new peptidoglycan-degrading protein and evaluated its synergism with colistin and its antimicrobial efficacy when conjugated with polycationic-polymer nanoparticles. The gene that codes for endolysin in the vB_PaeM_USP2, a Pseudomonas aeruginosa bacteriophage, was cloned and expressed in Escherichia coli. The recombinant endolysin (rEnd2) was purified and its biochemical properties were determined using peptidoglycan substrate. The enzymatic activity was measured through peptidoglycan layer degradation and a decrease in turbidity of permeabilized Gram-negative bacteria. The antimicrobial activity of rEnd2, alone and in combination with colistin, was evaluated by checkerboard assay. The antibacterial activity of the cationic lipid oleylamine (OAM) conjugated with rEnd2 (OAM-rEnd2) was evaluated by time killing assay. The rEnd2 is structurally analogue with other endolysins and showed muramidase activity. The rEnd2 maintained higher activity between pH 6.0 to 7.5, had maximum activity at 35 °C, and was not affected by chaotropic and reducing reagents. It was sensitive to an increase in surfactant concentration, being inactivated by sodium dodecyl sulfate and cetyltrimethylammonium bromide. Ions exhibited neither a positive nor a negative effect on enzyme activity. The rEnd2 showed clear muralytic activity and decreased turbidity of permeabilized Gram-negative bacteria. However, it did not control bacterial growth despite the combination with an antibiotic and its complexation with polycation (OAM-rEnd2 nanoparticle conjugate). The rEnd2 did not show clear antimicrobial activity suggesting further optimization of conditions for its activity or engineering and modification.

SalmoFree® Phage Additive Proves Its Safety for Laying Hens.

Background: The avian pathogen Salmonella Gallinarum causes avian typhosis in laying hens, leading to high mortality rates among adult birds, which poses a significant problem in the poultry industry. Various products, such as vaccines, antibiotics, probiotics, and disinfectants, are commonly used to prevent and control the disease on farms. An alternative to these products is the use of bacteriophages, which may effectively prevent the colonization of S. Gallinarum. Materials and Methods: This study evaluated the safety of SalmoFree®, a bacteriophage cocktail, administered to 276 laying hens from the first week of age until the 28th week. The hens were divided into two groups: a control group (138 birds) and a treatment group (138 birds). Over the 28-week period, eight doses of SalmoFree® (∼1010 UFP per bird) were administered via drinking water in a controlled environment. Results: The results indicate that the consumption of SalmoFree® has no adverse effects on bird health or zootechnical parameters. Additionally, there is a trend toward improving weight homogeneity (up to 19%), feed conversion (up to 68%), and egg weight (up to 2.7%). The detection of phages by PCR in cloacal swabs suggests that they persist in birds for 2 to 8 weeks post-ingestion. Furthermore, phages were detected in organs and eggshells, indicating that they provide protection beyond the gut. Conclusion: The study demonstrates that SalmoFree® is safe for use in laying hens and may offer additional benefits, such as improved zootechnical parameters and extended protection against S. Gallinarum through the persistence of bacteriophages in the birds.

Bacteriophages Against Bacterial Infections in Poultry Systems: A Scientometric Review.

Poultry production faces challenges from bacterial infections, aggravated by antibiotic resistance, affecting bird welfare and the industry's economy. Bacteriophages show promise as a solution, but their use in poultry systems is still limited. This study uses scientometric analysis to investigate the incidence of bacterial infections in poultry systems and bacteriophage application trends. The Web of Science database was used, and the articles were refined by searching for keywords that included the most rep orted bacteria in the different phases of poultry farming and the application of phages. The articles were analyzed using the CiteSpace and Excel software, allowing the evaluation of publication trends, influential countries, and correlations with antimicrobial resistance and the use of bacteriophages. Results highlight Escherichia coli prevalence in poultry systems and reveal a correlation between the number of publications and poultry productivity, with the United States and China leading both aspects. Findings offer insights into bacterial control gaps in poultry systems, underscoring the need for further research and practical strategies.

Two marine sulfur-reducing bacteria co-culture is essential for productive infection by a T4-like Escherichia coli-infecting phage.

The control of microbiologically influenced corrosion (MIC) challenges the oil exploration sector. The MIC results from electrochemical reactions facilitated by microorganisms such as sulfate-reducing bacteria (SRB), which adhere to the surface of the ducts forming biofilms. SRB uses sulfate as the final electron acceptor, resulting in hydrogen sulfide as the final product, a highly reactive corrosive, and toxic compound. Due to the high diversity of the SRB group, this study evaluated the effect of an Escherichia coli phage, with biofilm degrading enzymes, in preventing biofilm formation by microbial consortium P48SEP and reducing H2S production in a complex SRB community. Three phage concentrations were evaluated (104, 108 and 1012 UFP/ml). High and medium phage concentrations prevented biofilm development, as evidenced by scanning electron microscopy, chemical analysis, and cell counts. In addition, the virus altered the expression pattern of some bacterial genes and the relative abundance of proteins related to biofilm formation and cell stress response. Using a complex culture formed mainly by SRB, it was possible to observe the bacterial growth, H2S, and metabolic activity reduction after the phage was added. This study shows for the first time the ability of an E. coli-infecting phage to prevent the biofilm formation of an SRB consortium and infect and replicate at high concentrations on the non-specific host. This new finding turns the use of non-specific phages a promising alternative for the control of biocorrosion in oil and gas installations, on the other side, alert to the use of large concentration of phages and the influence on bacterial groups with geological importance, opening a research field in phage biology.

Host population structure and species resolution reveal prophage transmission dynamics.

Much knowledge about bacteriophages has been obtained via genomics and metagenomics over the last decades. However, most studies dealing with prophage diversity have rarely conducted phage species delimitation (aspect 1) and have hardly integrated the population structure of the host (aspect 2). Yet, these two aspects are essential in assessing phage diversity. Here, we implemented an operational definition of phage species (clustering at 95% identity, 90% coverage) and integrated the host's population structure to understand prophage diversity better. Gathering the most extensive data set of Acinetobacter baumannii phages (4,152 prophages + 122 virulent phages, distributed in 46 countries in the world), we show that 91% (875 out of 963) of the prophage species have four or fewer prophages per species, and just five prophage species have more than 100 prophages. Most prophage species have a narrow host range and are geographically restricted; yet, very few have a broad host range being well spread in distant lineages of A. baumannii. These few broad host range prophage species are not only cosmopolitan but also the most abundant species. We also noted that polylysogens had very divergent prophages, belonging to different prophage species, and prophages can easily be gained and lost within the bacterial lineages. Finally, even with this extensive data set, the prophage diversity has not been fully grasped. Our study highlights how integrating the host population structure and a solid operational definition of phage species allows us to better appreciate phage diversity and its transmission dynamics. IMPORTANCE: Much knowledge about bacteriophages has been obtained via genomics and metagenomics over the last decades. However, most studies dealing with prophage diversity have rarely conducted phage species delimitation (aspect 1) and have hardly integrated the population structure of the host (aspect 2). Yet, these two aspects are essential in assessing phage diversity. Here, we implemented an operational definition of phage species (clustering at 95% identity, 90% coverage) and integrated the host's population structure to understand prophage diversity better. Gathering the most extensive data set of Acinetobacter baumannii phages, we show that most prophage species have four or fewer prophages per species, and just five prophage species have more than 100 prophages. Most prophage species have a narrow host range and are geographically restricted; yet, very few have a broad host range being well spread in distant lineages of A. baumannii. These few broad host range prophage species are cosmopolitan and the most abundant species. Prophages in the same bacterial genome are very divergent, and prophages can easily be gained and lost within the bacterial lineages. Finally, even with this extensive data set, the prophage diversity has not been fully grasped. This study shows how integrating the host population structure and clustering at the species level allows us to better appreciate phage diversity and its transmission dynamics.

Genome sequences of Mycobacterium sp. Elmwood and accompanying phage, isolated from a public swimming pool in Nebraska.

Genome sequencing of a non-tuberculosis Mycobacterium species, isolated from a public pool, shows that the genome contains several genes for antibiotic resistance and anti-phage defense, which are absent from other related Mycobacteria. Metagenomic binning also provided the genome of the accompanying phage, which is distinct from other mycobacterial phages.

Tools and methodology to in silico phage discovery in freshwater environments.

Freshwater availability is essential, and its maintenance has become an enormous challenge. Due to population growth and climate changes, freshwater sources are becoming scarce, imposing the need for strategies for its reuse. Currently, the constant discharge of waste into water bodies from human activities leads to the dissemination of pathogenic bacteria, negatively impacting water quality from the source to the infrastructure required for treatment, such as the accumulation of biofilms. Current water treatment methods cannot keep pace with bacterial evolution, which increasingly exhibits a profile of multidrug resistance to antibiotics. Furthermore, using more powerful disinfectants may affect the balance of aquatic ecosystems. Therefore, there is a need to explore sustainable ways to control the spreading of pathogenic bacteria. Bacteriophages can infect bacteria and archaea, hijacking their host machinery to favor their replication. They are widely abundant globally and provide a biological alternative to bacterial treatment with antibiotics. In contrast to common disinfectants and antibiotics, bacteriophages are highly specific, minimizing adverse effects on aquatic microbial communities and offering a lower cost-benefit ratio in production compared to antibiotics. However, due to the difficulty involving cultivating and identifying environmental bacteriophages, alternative approaches using NGS metagenomics in combination with some bioinformatic tools can help identify new bacteriophages that can be useful as an alternative treatment against resistant bacteria. In this review, we discuss advances in exploring the virome of freshwater, as well as current applications of bacteriophages in freshwater treatment, along with current challenges and future perspectives.

Characterization and efficacy of Salmonella phage cocktail PHA46 in the control of Salmonella Newport and Typhimurium internalized into cherry tomatoes.

Non-typhoid Salmonella enterica causes salmonellosis illness, and this bacterium can contaminate food throughout the production chain, including those that are consumed as raw products. Salmonella enterica can adhere to and internalize into fresh produce such as cherry tomatoes. It has been reported that lytic bacteriophages (phages) can be used as a biocontrol agent in the agricultural field, being an alternative for the control of Salmonella in red meat, fish, lettuce, and cabbage. The aim of this study was to characterize the two phages present in the PHA46 cocktail to determine their morphology, genome, host range, and resistance to different temperatures and pHs values; and later evaluate their lytic activity to reduce the adherence to and internalization of Salmonella enterica serovars Newport and Typhimurium into cherry tomatoes. In addition, in this work, we also explored the effect of the PHA46 cocktail on the virulence of S. Newport-45 and S. Typhimurium SL1344, recovered from the interior of cherry tomatoes, on the lifespan of the animal model Caenorhabditis elegans. The nematode C. elegans, recently has been used to test the virulence of Salmonella and it is easy to maintain and work with in the laboratory. The results revealed that the morphology obtained by Transmission Electron Microscopy of two phages from the PHA46 cocktail correspond to a myovirus, the analyses of their genomes sequences did not report virulence or antimicrobial resistance genes. The PHA46 sample is specific for 33 different serovars from different Salmonella strains and shows stability at 7 °C and pH 6. Also, the PHA46 cocktail was effective in reducing the adherence of S. Newport-45 and S. Typhimurium SL1344 to cherry tomatoes, at an average of 0.9 log10, respectively. Regarding internalized bacteria, the reduction was at an average of 1.2 log10, of the serovars mentioned above. The lifespan experiments in C. elegans showed by itself, that the PHA46 cocktail was harmless to the nematode, and the virulence from both Salmonella strains grown in vitro is diminished in the presence of the PHA46 cocktail. In conclusion, these results showed that the PHA46 cocktail could be a good candidate to be used as a biocontrol agent against Salmonella enterica.

Valp1, a Newly Identified Temperate Phage Facilitating Coexistence of Lysogenic and Non-Lysogenic Populations of Vibrio anguillarum.

Vibrio anguillarum is a pathogen for several fish and shellfish species. Its ecology is influenced by diverse factors, including bacteriophages. Here, we identify and characterize a new temperate bacteriophage (Valp1) of V. anguillarum. Valp1 is a myovirus with a 60 nm head and a 90 nm contractile tail. Its double-stranded DNA genome of 42,988 bp contains 68 genes, including a protelomerase gene, typical of telomeric phages. Valp1 inhibits the growth of the virulent strain of V. anguillarum PF4, while the derived lysogenic strain P1.1 presents a slight reduction in its growth but is not affected by the presence of Valp1. Both strains present similar virulence in a larval zebrafish (Danio rerio) model, and only slight differences have been observed in their biochemical profile. Co-culture assays reveal that PF4 and P1.1 can coexist for 10 h in the presence of naturally induced Valp1, with the proportion of PF4 ranging between 28% and 1.6%. By the end of the assay, the phage reached a concentration of ~108 PFU/mL, and all the non-lysogenic PF4 strains were resistant to Valp1. This equilibrium was maintained even after five successive subcultures, suggesting the existence of a coexistence mechanism between the lysogenic and non-lysogenic populations of V. anguillarum in conjunction with the phage Valp1.

An Edible Antibacterial Coating Integrating Lytic Bacteriophage Particles for the Potential Biocontrol of Salmonella enterica in Ripened Cheese.

The goal of this research was to create an antibacterial biopolymeric coating integrating lytic bacteriophages against Salmonella enterica for use in ripened cheese. Salmonella enterica is the main pathogen that contaminates food products and the food industry. The food sector still uses costly and non-selective decontamination and disease control methods. Therefore, it is necessary to look for novel pathogen biocontrol technologies. Bacteriophage-based biocontrol seems like a viable option in this situation. The results obtained show promise for food applications since the edible packaging developed (EdiPhage) was successful in maintaining lytic phage viability while preventing the contamination of foodstuff with the aforementioned bacterial pathogen.

Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

Complete genome sequence of the Microbacterium foliorum bacteriophage Garey24.

In this work, we report the discovery and characterization of Garey24, a bacteriophage that forms medium-size plaques with halo rings isolated from a soil sample in Funes, Argentina. Its 41,522 bp circularly permuted genome contains 63 putative protein-coding genes. Based on gene content similarity, Garey24 was assigned to subcluster EA1.

Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

Isolation, characterization, and application of bacteriophage cocktails for the biocontrol of Pseudomonas fluorescens group strains in whole and skimmed milk.

Pseudomonas fluorescens group strains can lead to spoilage of milk as well as loss of quality in dairy products through their heat-resistant enzymes. Phages are important alternatives for combating spoilage bacteria in food industry and used successfully in many applications. The aim of this study was the isolation and characterization of phages and to assess the efficiency of a phage cocktail in whole and skimmed milk. For this purpose, phages effective against Pseudomonas fluorescens (L23.2), Pseudomonas tolaasii (P22.1), and Pseudomonas rhodesiae (A11.1) were isolated. Their host range was found to be highly specific, and the transmission electron micrographs indicates that they belonged to Tectiviridae family. Their genome sizes were found to be vary between 38.3 and 53.5 kb. The latent periods and burst sizes were determined as 15, 10, 15 min and 91, 20, 80 PFU/infected cell for L23.2, P22.1, and A11.1, respectively. All three phages were found to be sensitive to low pH and high temperature. The effect of the phage cocktail was monitored in milk with different fat contents during storage at 4 °C for 5 days. As a result, bacterial reductions up to 4.09 and 5.29 log-units were observed for the whole and skimmed milk, respectively. Thus, the efficacy of a phage cocktail against a bacterial mixture of different P. fluorescens strains was tested in milk samples with different fat contents in accordance with real-life scenarios for the first time.

Chemical-Biology and Metabolomics Studies in Phage-Host Interactions.

For many years, several studies have explored the molecular mechanisms involved in the infection of bacteria by their specific phages to understand the main infection strategies and the host defense strategies. The modulation of the mechanisms involved in the infection, as well as the expression of key substances in the development of the different life cycles of phages, function as a natural source of strategies capable of promoting the control of different pathogens that are harmful to human and animal health. Therefore, this chapter aims to provide an overview of the mechanisms involved in virus-bacteria interaction to explore the main compounds produced or altered as a chemical survival strategy and the metabolism modulation when occurring a host-phage interaction. In this context, emphasis will be given to the chemistry of peptides/proteins and enzymes encoded by bacteriophages in the control of pathogenic bacteria and the use of secondary metabolites recently reported as active participants in the mechanisms of phage-bacteria interaction. Finally, metabolomics strategies developed to gain new insights into the metabolism involved in the phage-host interaction and the metabolomics workflow in host-phage interaction will be presented.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Newly isolated phages preying on Pseudomonas syringae pv. garcae: In vitro and ex vivo inactivation studies in coffee plant leafs.

Coffee canker, or bacterial halo blight (BHB) of coffee, is a disease caused by the phytopathogenic bacterium Pseudomonas syringae pv. garcae (Psg), having been found for the first time in 1955, in the Garça region (State of São Paulo), and which has stood out in the Brazilian coffee plantations in recent years, leading to severe economic losses that seriously affect coffee trade. The treatments available are still scarce, involving frequent spraying of coffee plantations with either copper derivatives or the antibiotic kasugamycin. However, these compounds should be avoided due to environmental toxicity and the development of bacterial resistances. Herein we report the isolation and physical/biological characterisation of two novel lytic phages and their efficacy in the control of Psg. Phages ph002F and ph004F were isolated from coffee plant leaves in Brazil (Sorocaba/SP and Itu/SP cities), using Psg IBSBF-158 as the host. According to the transmission electron microscopy analyses, both phages belong to the class Caudoviricetes and present myovirus-like morphotypes. Phages ph002F and ph004F showed eclipse times of 5 min and 20 min, respectively, and a burst size of 123 PFU/host cell and 12 PFU/host cell, respectively, allowing to conclude they replicate well in Psg IBSBF-158 with latency periods of 50 min. Phage ph002F (reduction of 4.59 log CFU/mL, compared to uninfected culture) was more effective in inactivating Psg than phage ph004F (reduction of 3.85 log CFU/mL) after 10 h of incubation at a MOI of 10. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.26 log CFU/mL at a MOI of 0.1 or reduction of 5.03 log CFU/mL at a MOI of 10, relative to untreated culture), after 12 h of treatment. This study provides evidence that the isolated phages are promising candidates against the causative agent of BHB in coffee plants.

An Edible Biopolymeric Microcapsular Wrapping Integrating Lytic Bacteriophage Particles for Salmonella enterica: Potential for Integration into Poultry Feed.

This research work aimed at developing an edible biopolymeric microcapsular wrapping (EBMW) integrating lytic bacteriophage particles for Salmonella enterica, with potential application in poultry feed for biocontrol of that pathogen. This pathogen is known as one of the main microorganisms responsible for contamination in the food industry and in foodstuff. The current techniques for decontamination and pathogen control in the food industry can be very expensive, not very selective, and even outdated, such as the use of broad-spectrum antibiotics that end up selecting resistant bacteria. Hence, there is a need for new technologies for pathogen biocontrol. In this context, bacteriophage-based biocontrol appears as a potential alternative. As a cocktail, both phages were able to significantly reduce the bacterial load after 12 h of treatment, at either multiplicity of infection (MOI) 1 and 10, by 84.3% and 87.6%, respectively. Entrapment of the phage virions within the EBMW matrix did not exert any deleterious effect upon their lytic activity. The results obtained showed high promise for integration in poultry feed aiming at controlling Salmonella enterica, since the edible biopolymeric microcapsular wrapping integrating lytic bacteriophage particles developed was successful in maintaining lytic phage viability while fully stabilizing the phage particles.

Myriad applications of bacteriophages beyond phage therapy.

Bacteriophages are the most abundant biological entity on the planet, having pivotal roles in bacterial ecology, animal and plant health, and in the biogeochemical cycles. Although, in principle, phages are simple entities that replicate at the expense of their bacterial hosts, due the importance of bacteria in all aspects of nature, they have the potential to influence and modify diverse processes, either in subtle or profound ways. Traditionally, the main application of bacteriophages is phage therapy, which is their utilization to combat and help to clear bacterial infections, from enteric diseases, to skin infections, chronic infections, sepsis, etc. Nevertheless, phages can also be potentially used for several other tasks, including food preservation, disinfection of surfaces, treatment of several dysbioses, and modulation of microbiomes. Phages may also be used as tools for the treatment of non-bacterial infections and pest control in agriculture; moreover, they can be used to decrease bacterial virulence and antibiotic resistance and even to combat global warming. In this review manuscript we discuss these possible applications and promote their implementation.

Tracing ΦX174 bacteriophage spreading during aerosol-generating procedures in a dental clinic.

OBJECTIVE: The aim of this study was to test the plausibility of using the ΦX174 bacteriophage as a tracer of viral aerosols spreading in a dental aerosol-generating procedure (AGP) model. METHODS: ΦX174 bacteriophage (~ 108 plaque-forming units (PFU)/mL) was added into instrument irrigation reservoirs and aerosolized during class-IV cavity preparations followed by composite fillings on natural upper-anterior teeth (n = 3) in a phantom head. Droplets/aerosols were sampled through a passive approach that consisted of Escherichia coli strain C600 cultures immersed in a LB top agar layer in Petri dishes (PDs) in a double-layer technique. In addition, an active approach consisted of E coli C600 on PDs sets mounted in a six-stage cascade Andersen impactor (AI) (simulating human inhalation). The AI was located at 30 cm from the mannequin during AGP and afterwards at 1.5 m. After collection PDs were incubated overnight (18 h at 37 °C) and bacterial lysis was quantified. RESULTS: The passive approach disclosed PFUs mainly concentrated over the dental practitioner, on the mannequin's chest and shoulder and up to 90 cm apart, facing the opposite side of the AGP's source (around the spittoon). The maximum aerosol spreading distance was 1.5 m in front of the mannequin's mouth. The active approach disclosed collection of PFUs corresponding to stages (and aerodynamic diameters) 5 (1.1-2.1 µm) and 6 (0.65-1.1 µm), mimicking access to the lower respiratory airways. CONCLUSION: The ΦX174 bacteriophage can be used as a traceable viral surrogate in simulated studies contributing to understand dental bioaerosol's behavior, its spreading, and its potential threat for upper and lower respiratory tract. CLINICAL RELEVANCE: The probability to find infectious virus during AGPs is high. This suggests the need to continue characterizing the spreading viral agents in different clinical settings through combination of passive and active approaches. In addition, subsequent identification and implementation of virus-related mitigation strategies is relevant to avoid occupational virus infections.