Podoviridae Family Research Articles

Phage cocktail amikacin combination as a potential therapy for bacteremia associated with carbapenemase producing colistin resistant Klebsiella pneumoniae

The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types—K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages’ selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Phage-resistant Streptomyces abietis and its telomycin bioactive metabolite as a possible alternative to antibiotics

Multidrug-resistant pathogens are now thought to be the primary global causes of disease and death. Therefore, it is imperative to develop new effective bioactive compounds from microbial sources, such as Streptomyces species. Nevertheless, the pharmaceutical industry suffered financial losses and low-quality end products as a result of Streptomyces bacteriophage contamination. To reduce the likelihood of phage-induced issues in the medical industry, it is crucial to develop a method for finding phage-resistant strains. Hence, we aimed to isolate and characterize Streptomyces spp. and Streptomyces phages from various rhizospheric soil samples in Egypt and to investigate their antibacterial activities. Moreover, we targeted development of a Streptomyces phage-resistant strain to extract its active metabolites and further testing its antibacterial activity. Herein, the antibacterial activities of the isolated 58 Streptomyces isolates showed that 10 (17.2 %) Streptomyces isolates had antibacterial activities against the tested bacteria including Listeria monocytogenes, E. coli O157, Acinetobacter baumannii, methicillin resistant-vancomycin-intermediate Staphylococcus aureus (MRSA-VISA) and Micrococcus luteus. Three lytic bacteriophages (ϕPRSC1, ϕPRSC2, and ϕPRSC4) belonging to the families Siphoviridae and Podoviridae were obtained from the rhizospheric soil samples using the most potent S. abietis isolate as the host strain. The three isolated Streptomyces phages were thermostable, ultraviolet stable, infectious, and had a wide range of hosts against the 10 tested Streptomyces isolates with antibacterial activities. The DNA of the ϕPRSC1 and ϕPRSC4 phages were resistant to digestion by EcoRI and HindIII, but the DNA of ϕPRSC2 was resistant to digestion by EcoRI and sensitive to digestion by HindIII. Of note, we developed a S. abietis strain resistant to the three isolated phages and its antibacterial activities were twice that of the wild strain. Finally, telomycin was recognized as an antibacterial metabolite extracted from phage-resistant S. abietis strain, which was potent against the tested Gram-positive bacteria including L. monocytogenes, MRSA-VISA, and M. luteus. Thus, our findings open new horizons for researching substitute antimicrobial medications for both existing and reemerging illnesses.

Isolation and Biological Characteristics of a Novel Phage and Its Application to Control Vibrio Parahaemolyticus in Shellfish Meat.

Vibrio parahaemolyticus is a common foodborne pathogenic bacterium. With the overuse of antibiotics, an increasing proportion of drug-resistant strains are emerging, which puts enormous pressure on public health. In this study, a V. parahaemolyticus-specific phage, VP41s3, was isolated. The head length, width, and tail length of the phage were 77.7 nm, 72.2 nm, and 17.5 nm, respectively. It remained active in the temperature range of 30-50°C and pH range of 4-11. The lytic curve of phage VP41s3 showed that the host bacteria did not grow until 11 h under phage treatment at MOI of 1000, indicating that the phage had good bacteriostatic ability. When it was added to shellfish contaminated with V. parahaemolyticus (15°C, 48 h), the number of bacteria in the experimental group was 2.11 log10 CFU/mL lower than that in the control group at 24 h. Furthermore, genomic characterization and phylogenetic analysis indicated that phage VP41s3 was a new member of the Podoviridae family. The genome contained 50 open reading frames (ORFs), in which the ORF19 (thymidine kinase) was an enzyme involved in the pyrimidine salvage pathway, which might lead to the accelerated DNA synthesis efficiency after phage entered into host cells. This study not only contributed to the improvement of phage database and the development of beneficial phage resources but also revealed the potential application of phage VP41s3 in food hygiene and safety.

Symbiotic virus-bacteria interactions in biological treatment of coking wastewater manipulating bacterial physiological activities

Biological treatment is commonly used in coking wastewater (CWW) treatment. Prokaryotic microbial communities in CWW treatment have been comprehensively studied. However, viruses, as the critical microorganisms affecting microbial processes and thus engineering parameters, still remain poorly understood in CWW treatment context. Employing viromics sequencing, the composition and function of the viral community in CWW treatment were discovered, revealing novel viral communities and key auxiliary metabolic functions. Caudovirales appeared to be the predominant viral order in the oxic-hydrolytic-oxic (OHO) CWW treatment combination, showing relative abundances of 62.47 %, 56.64 % and 92.20 % in bioreactors O1, H and O2, respectively. At the family level, Myoviridae, Podoviridae and Siphoviridae mainly prevailed in bioreactors O1 and H while Phycodnaviridae dominated in O2. A total of 56.23–92.24% of novel viral contigs defied family-level characterization in this distinct CWW habitat. The virus-host prediction results revealed most viruses infecting the specific functional taxa Pseudomonas, Acidovorax and Thauera in the entire OHO combination, demonstrating the viruses affecting bacterial physiology and pollutants removal from CWW. Viral auxiliary metabolic genes (AMGs) were screened, revealing their involvement in the metabolism of contaminants and toxicity tolerance. In the bioreactor O1, AMGs were enriched in detoxification and phosphorus ingestion, where glutathione S-transferase (GSTs) and beta-ketoadipyl CoA thiolase (fadA) participated in biodegradation of polycyclic aromatic hydrocarbons and phenols, respectively. In the bioreactors H and O2, the AMGs focused on cell division and epicyte formation of the hosts, where GDPmannose 4,6-dehydratase (gmd) related to lipopolysaccharides biosynthesis was considered to play an important role in the growth of nitrifiers. The diversities of viruses and AMGs decreased along the CWW treatment process, pointing to a reinforced virus-host adaptive strategy in stressful operation environments. In this study, the symbiotic virus-bacteria interaction patterns were proposed with a theoretical basis for promoting CWW biological treatment efficiency. The findings filled the gaps in the virus-bacteria interactions at the full-scale CWW treatment and provided great value for understanding the mechanism of biological toxicity and sludge activity in industrial wastewater treatment.

Biological control of Pseudomonas aeruginosa (ATCC-27853) by isolated lytic bacteriophage

Background Pseudomonas aeruginosa is a common bacterial species; it can grow in all fields, even in distilled water. One of the obvious characteristics of P. aeruginosa species is its low antibiotic susceptibility (antibiotic-multi-resistant species); this reflects the urgent need for alternative strategies. Unconventionally, we try to use an alternative destructive antibacterial agent, phage. phage application in our environmental fields (engineering relevance) is a promising safe avenue for replacing chemical biocides. Objective To isolate a lytic phage as a bio-control agent for Pseudomonas aeruginosa (ATCC-27853), evaluate the ability of the isolated phage to inhibit or suppress the growth of the target bacteria. We study phage stability while performing biological and physical characterization, optimizing phage action against bacteria. Materials and methods We use an activated, purified young culture of Pseudomonas aeruginosa (ATCC-27853) as the target bacteria to isolate a specific lytic bacteriophage from a mixture of Enriched sewage and marine water through a sequence of experiments: a Broth clearing assay using nutrient broth and a spot assay test by using nutrient agar medium, both for the detection of phage presence and its lytic action. For the purification of phage, the plaque assay test was performed in many successive subcultures by using the serial dilution method on a semisolid medium. The high-titer lysate obtained from the confluent plates was investigated by TEM to determine its morphology and taxonomy. We performed biological characterization of the isolated phage: one-step growth curve, Phage adsorption rate, host range (using nutrient double layer agar), as well as a bacterial challenge test (using nutrient broth, based on the optical densities). Also, we performed physical characterization of the phage to determine phage Stability, pH, thermal stability, and the influence of UV and organic solvents on the lytic action of the phage. Results and conclusion Pyocyanin and pyoverdin pigments (both fluorescent under UV illumination) are secreted by P. aeruginosa (ATCC-27853), also their ability to thrive at 42°C. After spotting P. aeruginosa plates with enriched sewage filtrate, the results indicated lytic area (+ve). Plaques have a pin tip and are translucent. The lytic profile of bacteria after phage infection revealed that the bacteria was reduced to a MOI of 10 in 4 h. However, the multiplicity of infection(MOI) of 1 and 0.01, respectively, was longer (10, 22/h). The electron microscopy revealed that the phage is a member of the podoviridae family, with a noncontractile short tail (18 nm) and a polyhedral (heptagonal) head (45 nm) in diameter and a phage length of 63 nm. The adsorption rate of the phage was greatest at 20 min. Our goal with the phage application is to replace harmful chemical biocides with lytic bacteriophages in various environmental systems (Phage for Biological Control). Phage bio-control treatment thus holds promise for eliminating the requirement for hazardous chemical biocides.

Characterization of two bacteriophages specific to Acinetobacter baumannii and their effects on catheters biofilm.

Multidrug-resistant strains of Acinetobacter baumannii cause major nosocomial infections. Bacteriophages that are specific to the bacterial species and destroy bacteria can be effectively used for treatment. In this study, we characterized lytic bacteriophages specific to A. baumannii strains. We isolated lytic bacteriophages from environmental water samples and then investigated their morphology, host range, growth characteristics, stability, genome analysis, and biofilm destruction on the catheter surface. Our results showed that the efficacy of the phages varied between 32% and 78%, tested on 78 isolates of A. baumannii; 80 phages were isolated, and two lytic bacteriophages, vB_AbaP_HB01 (henceforth called C2 phage) and vB_AbaM_HB02 (henceforth called K3 phage), were selected for characterization. Electron microscopy scans revealed that the C2 and K3 phages were members of the Podoviridae and Myoviridae families, respectively. Whole-genome sequencing revealed that the sequence of the C2 phage is available in the NCBI database (accession number: OP917929.1), and it was found sequence identity with Acinetobacter phage AB1 18%, the K3 phage DNA sequence is closely related to Acinetobacter phage vB_AbaM_phiAbaA1 (94% similarity). The cocktail of C2 and K3 phages demonstrated a promising decrease in the bacterial cell counts of the biofilm after 4 h. Under a scanning electron microscope, the cocktail treatment destructed the biofilm on the catheter. We propose that the phage cocktail could be a strong alternative to antibiotics to control the A. baumannii biofilm in catheter infections.

Isolation, characterization and antibiofilm efficacy of a novel Klebsiella pneumoniae phage

The multidrug-resistant (MDR) Klebsiella pneumoniae is an important pathogen that threatens public health directly with life-threatening infections. The need for the development of new effective and well tolerated alternative treatments for these infections is crucial. Therefore, the interest in phage therapy as a promising alternative is increasing. Here, a novel phage named ΦAYH was isolated from the Tigris River water, Baghdad, IRAQ near sewage of Baghdad Medical City with its specific host from the same site. Phage ΦAYH belongs to Podoviridae family in the order Caudovirales. The ΦAYH maintained stability at different temperatures (−10 to 60°C) and pH values (5–11). The phage was able to lyse 8 from 32 clinical K. pneumoniae isolates in vitro with no lytic activity against other tested species. For one-step growth, latent period was 10 min with burst size ∼64 virions/cell at multiplicity of infection (MOI) 10. The SDS-PAGE test revealed one major structural protein and different structural proteins ranging from 28 to 89 kDa in size. The phage host and 32 clinical K. pneumoniae isolates were tested for phenotypic identification and antibiotics profile by VITEK-2 system and genotypically using rpob gene. All clinical K. pneumoniae isolates showed resistance to the most antibiotics tested while phage host was resistant only to amoxicillin. Biofilm production by all clinical isolates including the host isolate was tested. These isolates showed different ability as following: 72.72% as weak, 6.06% as moderate, and 21.21% as strong biofilm producer. Together these results demonstrate that ΦAYH is a promising alternative against MDR K. pneumoniae.

Characterization of selected phages for biocontrol of food-spoilage pseudomonads.

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30°C and 4°C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30°C, 8°C, and 4°C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

Characterization and genomic analysis of a novel E. coli lytic phage with extended lytic activity against S. Enteridis and S. Typhimurium

In order to effectively use of phages as antimicrobial agents for controlling multidrug-resistant E. coli, it is important to understand phage biology. The isolation and research of novel bacteriophages are urgently needed for food safety and animal health. Phage is considered as a novel alternative antibacterial agents and a new way of prevent, control and treat pathogens. In this paper, we isolated and characterized a novel lytic phage from chicken samples, named vB_EscP_vE20 (in brief vE20). We identified vE20 as belonging to the Podoviridae family through morphological and phylogenetic analysis. It had lytic activities against 24 out of 51 different serotypes of clinical E. coli strains, interestingly it also can lyse Salmonella species, such as Salmonella Enteritidis (S. Enteritidis) and Samonella Typhimurium (S. Typhimurium). One step growth curve showed that the latent period and lysis period was 10 min and 60 min, respectively. The burst size of vE20 was about 60 PFU/cell. The phage vE20 survived in a wide range of temperatures (30–60 °C) and pH (3–11). The whole genome size of phage vE20 is 77,938 bps, which is double stranded DNA. There are 121 Open reading frame (ORFs) in total, and the GC-content is 42.17%, without virulence-associated, antibiotic and lysogeny related genes. Phage vE20 has high bactericidal activity in killing E. coli EXG20-1 in cultures containing 4 × 109 cfu/mL in several MOIs culture. The results revealed the promising potential of phage vE20 as attractive candidates for the control of E. coli infections.Graphical

Effect of wastewater collection and concentration methods on assessment of viral diversity

Monitoring of potentially pathogenic human viruses in wastewater is of crucial importance to understand disease trends in communities, predict potential outbreaks, and boost preparedness and response by public health departments. High throughput metagenomic sequencing opens an opportunity to expand the capabilities of wastewater surveillance. However, there are major bottlenecks in the metagenomic enabled wastewater surveillance, including the complexities in selecting appropriate sampling and concentration/virus enrichment methods as well as in bioinformatic analysis of complex samples with low human virus concentrations. To evaluate the abilities of two commonly used sampling and concentration methods in virus identification, virus communities concentrated with Virus Adsorption-Elution (VIRADEL) and PolyEthylene Glycol (PEG) precipitation were compared for three interceptor sites. Results indicated that more viral reads were obtained by the VIRADEL concentration method, with 2.84 ± 0.57 % viral reads in the sample. For samples concentrated with PEG, the average proportion of viral reads in the sample was 0.63 ± 0.19 %. In all wastewater samples, bacteriophage affiliated with the families Siphoviridae, Myoviridae and Podoviridae were found to be the abundant populations. Comparison against a custom Swiss-Prot human virus database indicated that the relatively abundant human viruses (average proportions in human virus community greater than 1.00 %) in samples concentrated with the VIRADEL method were Orthopoxvirus, Rhadinovirus, Parapoxvirus, Varicellovirus, Hepatovirus, Simplexvirus, Molluscipoxvirus, Parechovirus, Lymphocryptovirus, and Spumavirus. In samples concentrated with the PEG method, fewer human viruses were found to be relatively abundant. These were Orthopoxvirus, Rhadinovirus, Varicellovirus, Simplexvirus, Molluscipoxvirus, Lymphocryptovirus, and Betacoronavirus. Contigs of Betacoronavirus, which contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were identified in VIRADEL and PEG samples. Our study demonstrates the feasibility of using metagenomics in wastewater surveillance as a first screening tool and the need for selecting the appropriate virus concentration methods and optimizing bioinformatic approaches in analyzing metagenomic data of wastewater samples.

A novel phage carrying capsule depolymerase effectively relieves pneumonia caused by multidrug-resistant Klebsiella aerogenes

BackgroundKlebsiella aerogenes can cause ventilator-associated pneumonia by forming biofilms, and it is frequently associated with multidrug resistance. Phages are good antibiotic alternatives with unique advantages. There has been a lack of phage therapeutic explorations, kinetic studies, and interaction mechanism research targeting K. aerogenes.MethodsPlaque assay, transmission electron microscopy and whole-genome sequencing were used to determine the biology, morphology, and genomic characteristics of the phage. A mouse pneumonia model was constructed by intratracheal/endobronchial delivery of K. aerogenes to assess the therapeutic effect of phage in vivo. Bioinformatics analysis and a prokaryotic protein expression system were used to predict and identify a novel capsule depolymerase. Confocal laser scanning microscopy, Galleria mellonella larvae infection models and other experiments were performed to clarify the function of the capsule depolymerase.ResultsA novel lytic phage (pK4-26) was isolated from hospital sewage. It was typical of the Podoviridae family and exhibited serotype specificity, high lytic activity, and high environmental adaptability. The whole genome is 40,234 bp in length and contains 49 coding domain sequences. Genomic data show that the phage does not carry antibiotic resistance, virulence, or lysogenic genes. The phage effectively lysed K. aerogenes in vivo, reducing mortality and alleviating pneumonia without promoting obvious side effects. A novel phage-derived depolymerase was predicted and proven to be able to digest the capsule, remove biofilms, reduce bacterial virulence, and sensitize the bacteria to serum killing.ConclusionsThe phage pK4-26 is a good antibiotic alternative and can effectively relieve pneumonia caused by multidrug-resistant K. aerogenes. It carries a depolymerase that removes biofilms, reduces virulence, and improves intrinsic immune sensitivity.

Isolation and characterization of lytic bacteriophage from waste water to control clinical multidrug resistant Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic bacterium which can cause nosocomial, community acquired, and respiratory infections. It also has the biofilm forming ability which is difficult to treat through antimicrobial drugs. Therefore, an alternative therapy to antibiotics, such as bacteriophages, are required to control this biofilm producing, multi-drug resistant bacterium. The aim of this study was to isolate and characterize a lytic bacteriophage to control the multi drug resistant K. pneumoniae-3206 planktonic cells and biofilm. Phage RAM-1 was isolated from wastewater. It was characterized through transmission electron microscopy and was classified to be a member of the Podoviridae family. SDS-PAGE analysis showed that the phage consisted of approximately eighteen different proteins (13–200 ​kDa). Its genome was found to be ds DNA of about of ≈36 ​kb. Phage RAM-1 displayed good heat (37–65 ​°C) and pH (5–11) stability. Phage RAM-1 showed promising activity against the 24-, 48-, and 72-h biofilms made (underneath static conditions along with media renewal) by K. pneumoniae-3206. After a 6-h phage treatment, about 2.5, 3.6 and 4-log reductions were observed respectively. Thus, we concluded from this study that RAM-1 has potential in eradicating both bacterial planktonic cells and biofilm. However, to achieve complete bacterial eradication, a phage cocktail (a combination of two or more phages) will be more promising.

Viruses participate in the organomineralization of travertines

Travertines, which precipitate from high temperature water saturated with calcium carbonate, are generally considered to be dominated by physico-chemical and microbial precipitates. Here, as an additional influence on organomineral formation, metagenomic data and microscopic analyses clearly demonstrate that highly diverse viral, bacterial and archaeal communities occur in the biofilms associated with several modern classic travertine sites in Europe and Asia, along with virus-like particles. Metagenomic analysis reveals that bacteriophages (bacterial viruses) containing icosahedral capsids and belonging to the Siphoviridae, Myoviridae and Podoviridae families are the most abundant of all viral strains, although the bacteriophage distribution does vary across the sampling sites. Icosahedral shapes of capsids are also the most frequently observed under the microscope, occurring as non-mineralized through to mineralized viruses and virus-like particles. Viruses are initially mineralized by Ca-Si amorphous precipitates with subordinate Mg and Al contents; these then alter to nanospheroids composed of Ca carbonate with minor silicate 80–300 nm in diameter. Understanding the roles of bacteriophages in modern carbonate-saturated settings and related organomineralization processes is critical for their broader inclusion in the geological record and ecosystem models.

Isolation and characterization of bacteriophages specific to selected equine genital pathogens

Chronic bacterial endometritis is among the major problems in equine reproduction. Streptococcus equi ssp. zooepidemicus (SEZ), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA) are among the most important pathogens for the condition. Bacteriophages (phages) are the viruses of bacteria, having a species- and strain-specific lytic activity. Due to their bactericidal capacities, phages can be used as an alternative treatment approach for bacterial infections. The study aimed to isolate and characterize phages with lytic activity against Streptococcus equi ssp. zooepidemicus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phage samples were obtained from manure, uterine lavage fluid and drain water of horse husbandries (n=14). For isolation of phages, bacterial isolates of the equine genital tract (SEZ (n=1), KP (n=1), and PA (n=1)) were inoculated with the phage samples. For purification, bacteriophage plaques were repeatedly cultivated, harvested and filtered. For determining the host-range, the obtained phages were inoculated with bacterial isolates (SEZ (n=37), KP (n=26), and PA (n=33)). In total, 28 bacteriophages were isolated (against SEZ (n=13), KP (n=6), and PA (n=9)). Bacteriolysis was achieved in 32/37 Streptococcus-, 16/26 Klebsiella-, and 14/33 Pseudomonas-isolates. The bacteriophage showing the broadest host range covered 81% of all tested Streptococcus-isolates (30/37). Host ranges of Klebsiella pneumoniae- (9/26) and Pseudomonas aeruginosa-specific bacteriophages (14/33) covered a lower proportion of tested isolates. Seven bacteriophages (SEZ (n=3), KP (n=3), PA (n=1)) were selected according to theirhost ranges and submitted to electron microscopy for further characterization. Electron micrographs revealed that the selected bacteriophages belonged either to the family Demerecviridae (n=1), Myoviridae (n=1), Podoviridae (n=3) or Siphoviridae (n=2), which was confirmed by sequencing. Next, planktonic killing assay was conducted, incubating liquid cultures of Streptococcus equi ssp. zooepidemicus-, Klebsiella pneumoniae- and Pseudomonas aeruginosa-isolates in presence and absence of a specific bacteriophage and monitoring growth by optical density measurements for 24h. Significantly decreased bacterial growth, as determined by comparing the area under the curve (AUC), was observed in presence of 0/3 Streptococcus equi ssp. zooepidemicus-, 1/1 Pseudomonas aeruginosa- and 3/3 Klebsiella pneumoniae-specific phages, using a multiplicity of infection of 1 (MOI1; one phage per host cell). At MOI10 the most effective Streptococcus-phage reduced the AUC by 61%; the most effective Klebsiella-phage resulted in 95% reduction using MOI1 and for the Pseudomonas-phage AUC was reduced by 14% at MOI100. In conclusion, isolation of phages specific to equine uterine pathogens resulted in 28 purified phages. Seven phages were selected and further characterized by efficacy testing, sequencing and electron microscopy. Results of the in vitro host range determination and planktonic killing assay are promising, suggesting further investigations and the development of an alternative treatment approach for uterine infections in mares.

Metavirome Profiling and Dynamics of the DNA Viral Community in Seawater in Chuuk State, Federated States of Micronesia.

Despite their abundance and ecological importance, little is known about the diversity of marine viruses, in part because most cannot be cultured in the laboratory. Here, we used high-throughput viral metagenomics of uncultivated viruses to investigate the dynamics of DNA viruses in tropical seawater sampled from Chuuk State, Federated States of Micronesia, in March, June, and December 2014. Among the identified viruses, 71-79% were bacteriophages belonging to the families Myoviridae, Siphoviridae, and Podoviridae (Caudoviriales), listed in order of abundance at all sampling times. Although the measured environmental factors (temperature, salinity, and pH) remained unchanged in the seawater over time, viral dynamics changed. The proportion of cyanophages (34.7%) was highest in June, whereas the proportion of mimiviruses, phycodnaviruses, and other nucleo-cytoplasmic large DNA viruses (NCLDVs) was higher in March and December. Although host species were not analysed, the dramatic viral community change observed in June was likely due to changes in the abundance of cyanophage-infected cyanobacteria, whereas that in NCLDVs was likely due to the abundance of potential eukaryote-infected hosts. These results serve as a basis for comparative analyses of other marine viral communities, and guide policy-making when considering marine life care in Chuuk State.

Characterization of thermostable bacteriophage CPD2 and its endolysin LysCPD2 as biocontrol agents against Clostridium perfringens.

Clostridium perfringens is one of the major foodborne pathogens in humans and animals. With the prevalence of antibiotic-resistant C. perfringens strains, bacteriophages and their endolysins have received considerable attention as promising alternatives to antibiotics. In this study, C. perfringens phage CPD2 was isolated from retail chicken samples. CPD2 belongs to the Podoviridae family and exhibits remarkable thermostability. While CPD2 has narrow host specificity, its endolysin LysCPD2 showed a broader lytic range, killing not only C. perfringens strains but other Gram-positive bacteria, such as B. cereus and B. subtilis. In addition, due to its exceptional thermal stability, LysCPD2 showed significant antibacterial ability against germinating C. perfringens spores during the heat activation process (75°C for 20min). Taken together, these results indicate that both thermostable phage CPD2 and its endolysin LysCPD2 can be used as efficient antimicrobial agents to control C. perfringens during thermal processing of foods.

Isolation of bacteriophages specific to Pseudomonas mosselii for controlling milk spoilage

Bacteriophages controlling heat-stable protease producing Pseudomonas mosselii were characterised and applied. Two Podoviridae family phages, designated ΦC106 and Φ21A, were isolated and shown to exhibit a broad host range. The optimal multiplicity of infection of both phages generating maximum titres was 10, latent periods were at 20 min and burst sizes were 1862 and 331 plaque-forming units per infected cell for ΦC106 and Φ21A, respectively. ΦC106 and Φ21A genomes did not encode known toxins, antibiotic-resistant genes, virulent factors of bacterial origin, or lysogenic markers such as integrase, recombinase, repressor/anti-repressor protein, and excisionase, guaranteeing their safety for addition into milk and dairy products. In vitro lytic activity and proteolytic activity of ΦC106 and Φ21A revealed a remarkable decrease in bacterial count and enzyme activity. These data suggest that ΦC106 and Φ21A are efficient in controlling the prevalence of spoilage-causing P. mosselii strains in dairy- and food-related environments.

Escherichia coli phages isolated from broiler chickens showed ideal characteristics in gut modulation

Phage has gained interest as an alternative antibiotic growth promoter (AGPs) in poultry production. Most phage studies only focus on phages that target pathogens. In this study, we isolated and characterised phages that target non-pathogens from chicken intestines. This study aimed to isolate and characterise phages that target non-pathogenic Escherichia coli for gut modulation study in broiler chickens. Based on a morphological study, the C1 phage belonged to the Podoviridae family, whereas C2, C3, and C4 phages belonged to the Siphoviridae family. The C1, C2, C3, and C4 phages appeared to be unique based on restriction fragment length polymorphisms (RFLPs), amplification of phage signature genes, and protein profiling (SDS-PAGE). The C1 phage had an ideal multiplicity of infection (MOI) of 0.001, followed by 0.1 for the C2, C3, and C4 phages. C1 had the highest adsorption rate of 99.7% in 1 min, followed by C2 (98%), C3 (98.7%), and C4 (98.2%), all of which were within 2 min. C1 also exhibited the largest burst size (72 PFU/infected cell) and the shortest latent period (5 min). The latent period of the C2, C3, and C4 phages was longer, lasting 10 minutes, and their burst sizes were 70, 77, and 46 PFU/infected cells, respectively. All phages had optimum lytic activity at pH 7 and 37°C. Each phage was unique and possessed favourable lytic characteristics, making all of them suitable for gut modulation study in chickens.