Phage vB Research Articles

Genomic characterization, in vitro, and preclinical evaluation of two microencapsulated lytic phages VB_ST_E15 and VB_ST_SPNIS2 against clinical multidrug-resistant Salmonella serovars

BackgroundSalmonella infections continue to be one of the essential public health issues threatening millions of people. With the increasing occurrence of resistance against conventionally used antibiotics, the search for alternatives has become crucial. In this study, we aimed to isolate, characterize, and evaluate two lytic bacteriophages against clinically isolated multidrug-resistant (MDR) Salmonella serovars.MethodsScreening for the phage lytic activity was performed using a spot test. Characterization of the isolated phages was done by determining the host range, longevity test, and the effect of temperature, pH, organic solvents, and morphological characterization using a transmission electron microscope. Genomic analysis was performed using Oxford nanopore sequencing. The lytic activities of the free phage lysates and formulated phage as microencapsulated were evaluated both in vitro and in vivo.ResultsTwo phages (VB_ST_E15 and VB_ST_SPNIS2) were successfully isolated and showed lytic strong activities against MDR Salmonella (S.) Typhimurium ATCC 14,028, S. Paratyphi A, and S. Typhi. The two phages survived at the tested temperatures, maintained their infectivity for 90 days, and retained their activity until 60 °C with thermal inactivation at 65 °C. They were lytic at a pH range from 3 to 11 but lost their activities at extremely acidic or alkaline pH. The phages could withstand the organic solvents but were completely inactivated by 100% ethanol. Both phages were classified under the order Caudoviricetes, and Genus: Uetakevirus. Their genomic sequences were assembled, annotated, and submitted to the NCBI GenBank database (OR757455 and OR757456). The preclinical evaluation using the murine animal model revealed that the two-phage cocktail managed MDR Salmonella infection as evidenced by the reduction in the bacterial burden, increased animal weight, and histopathological examination.ConclusionThe two encapsulated phage formulas could be considered promising candidates for the management of MDR Salmonella-associated infections and clinical analysis should be undertaken to evaluate their potential use in humans.

Investigating bacteriophages as a novel multiple-hurdle measure against Campylobacter: field trials in commercial broiler plants

Campylobacter mitigation along the food production chain is considered effective for minimizing the public health burden of human campylobacteriosis. This study is the first combining different measures in a multiple-hurdle approach, using drinking water additives and feed additives in single and combined application schemes in commercial broiler plants. Broiler chickens in the study groups were naturally contaminated with Campylobacter. Application of an organic acid blend via drinking water, consisting of sodium propionate, potassium sorbate, and sodium diacetate, resulted in significant reductions of up to 4.9 log10 CFU/mL in fecal samples and in cecal samples at slaughter. The application of a phage mixture, consisting of Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1, resulted in reductions of up to 1.1 log10 CFU/mL in fecal samples 1 day after dosing. The sole administration of curcumin via feed resulted in small and inconsistent reductions. In the group receiving a combination of all tested measures, reductions of up to 1.1 log10 CFU/mL were observed. Based on the results of our field trials, it was shown that both the sole application and the combined application of mitigation measures in primary production can reduce the Campylobacter load in broiler chickens, while no synergism could be observed.

Characterization of a novel high-efficiency cracking Burkholderia gladiolus phage vB_BglM_WTB and its application in black fungus

Burkholderia gladiolus (B. gladiolus) is foodborne pathogenic bacteria producing bongkrekic acid (BA), which causes food poisoning and has a mortality rate of up to 40 % or more. However, no drugs have been reported in the literature for the prevention and treatment of this infection. In this study, a phage was identified to control B. gladiolus. The novel phage vB_BglM_WTB (WTB), which lyse B. gladiolus with high efficiency, was isolated from sewage of Huaihe Road Throttle Well Sewage Treatment Plant in Hefei. Transmission electron microscopy showed that WTB had an icosahedral head (69 ± 2 nm) and a long retractable tail (108 ± 2 nm). Its optimal temperature and pH ranges to control B. gladiolus were 25 °C -65 °C and 3–11 respectively. The phage WTB was identified as a linear double-stranded DNA phage of 68, 541 bp with 60.04 % G + C content, with a long latent period of 60 min. Phylogenetic analysis and comparative genetic analysis indicated that phage WTB has low identity (<50 %) with other phages, with the highest similarity to Burkholderia phage Maja (25.7 %), which showed that it does not belong to any previous genera recognized by the International Committee on Taxonomy of Viruses (ICTV) and was a candidate for a new genus within the Caudoviricetes. We have submitted a new proposal to ICTV to create a new genus, Bglawtbvirus. No transfer RNA (tRNA), virulence associated and antibiotic resistance genes were detected in phage WTB. Experimental results indicated that WTB at 4 °C and 25 °C had excellent inhibition activity against B. gladiolus in the black fungus, with an inhibition efficiency of over 99 %. The amount of B. gladiolus in the black fungus was reduced to a minimum of 89 CFU/mL when treated by WTB at 25 °C for 2 h. The inhibition rate remained at 99.97 % even after 12 h. The findings showed that the phage WTB could be applied as a food-cleaning agent for enhancing food safety and contributed to our understanding of phage biology and diversity.

Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d’Hérelle’s first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.

Genome sequence of Shiga toxin-producing Escherichia coli jumbo bacteriophage vB_EcoM_JNE01.

Bacteriophage vB_EcoM_JNE01 was isolated from chicken farm sewage using Escherichia coli O157:H7 as the host bacteria. The total length of the vB_EcoM_JNE01 genome is 355,583 bp, with 584 open reading frames and 36% G+C content. It shares an 80% nucleotide identity with 59% query coverage with the bacteriophage PBECO4 (NC_027364).

A new Rogue-like Escherichia phage UDF157lw to control Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is one of the notorious foodborne pathogens causing high mortality through the consumption of contaminated food items. The food safety risk from STEC pathogens could escalate when a group of bacterial cells aggregates to form a biofilm. Bacterial biofilm can diminish the effects of various antimicrobial interventions and enhance the pathogenicity of the pathogens. Therefore, there is an urgent need to have effective control measurements. Bacteriophages can kill the target bacterial cells through lytic infection, and some enzymes produced during the infection have the capability to penetrate the biofilm for mitigation compared to traditional interventions. This study aimed to characterize a new Escherichia phage vB_EcoS-UDF157lw (or UDF157lw) and determine its antimicrobial efficacy against E. coli O157:H7. Phage characterization included biological approaches, including phage morphology, one-step growth curve, stability tests (pH and temperature), and genomic approaches (whole-genome sequencing). Later, antimicrobial activity tests, including productive infection against susceptible bacterial strains, in vitro antimicrobial activity, and anti-biofilm, were conducted. UDF157lw is a new member of the phages belonging to the Rogunavirus genus, comprising a long and non-contractile tail, isolated from bovine feces and shares close genomic evolutionary similarities with Escherichia phages vB_EcoS-BECP10 and bV_EcoS_AKS96. When used against E. coli O157:H7 (ATCC35150), phage UDF157lw exhibited a latent period of 14 min and a burst size of 110 PFU per infected cell. The phage remained viable in a wide range of pH values (pH 4-11) and temperatures (4-60°C). No virulence genes, such as stx, lysogenic genes, and antibiotic resistance genes, were found. Phage UDF157lw demonstrated high infection efficiencies against different E. coli O157:H7 and generic E. coli strains. In addition, UDF157lw encoded a unique major tail protein (ORF_26) with prominent depolymerase enzyme activity against various E. coli O157:H7 strains, causing large plaque sizes. In contrast to the phage without encoding depolymerase gene, UDF157lw was able to reduce the 24-h and 48-h E. coli O157:H7 biofilm after 1-h phage treatment. The findings of this study provide insights into a new member of the Rogunavirus phages and demonstrate its antimicrobial potential against E. coli O157:H7 in vitro.

Two novel phages, Klebsiella phage GADU21 and Escherichia phage GADU22, from the urine samples of patients with urinary tract infection.

Phages are found in a wide variety of places where bacteria exist including body fluids. The aim of the present study was to isolate phages from the urine samples of patients with urinary tract infection. The 10 urine samples were cultured to isolate bacteria and also used as phage sources against the isolated bacteria. From 10 urine samples with positive cultures, 3 phages were isolated (33%) and two of them were further studied. The Klebsiella phage GADU21 and Escherichia phage GADU22 phages infected Klebsiella pneumonia and Escherichia coli, respectively. Among the tested 14 species for host range analysis, the Klebsiella phage GADU21 was able to infect two species which are Klebsiella pneumonia and Proteus mirabilis, and Escherichia phage GADU22 was able to infect four species which are Shigella flexneri, Shigella sonnei and Escherichia coli. Among different isolates of the indicator bacteria for each phage, GADU21 infected half of the tested 20 Klebsiella pneumonia isolates while GADU22 infected 85% of the tested 20 E. coli isolates. The genome sizes and GC ratios were 75,968bp and 44.4%, and 168,023bp and 35.3% for GADU21 and GADU22, respectively. GADU21 and GADU22 were both lytic and had no antibiotic resistance and virulence genes. GADU21 was homologue with Klebsiella phage vB_KpP_FBKp27 but only 88% of the genome was covered by this phage. The non-covered parts of the GADU21 genome included genes for tail-fiber-proteins and HNH-endonuclease. GADU22 had 94.8% homology with Escherichia phage vB_Eco_OMNI12 and had genes for immunity proteins. Phylogenetic analysis showed GADU21 and GADU22 were members of Schitoviridae family and Efbeekayvirus genus and Straboviridae family and Tevenvirinae genus, respectively. VIRIDIC analysis classified these phages in new species clusters. Our study demonstrated the possibility to use infected body fluids as phage sources to isolate novel phages. GADU21 is the first reported Klebsiella phage isolated from human body fluid. The absence of virulence and antibiotic resistance genes in their genomes makes the phages a potential therapeutic tool against infections.

Shewanella phage encoding a putative anti-CRISPR-like gene represents a novel potential viral family.

Shewanella is a prevalent bacterial genus in deep-sea environments including marine sediments, exhibiting diverse metabolic capabilities that indicate its significant contributions to the marine biogeochemical cycles. However, only a few Shewanella phages were isolated and deposited in the NCBI database. In this study, we report the isolation and characterization of a novel Shewanella phage, vB_SbaS_Y11, that infects Shewanella KR11 and was isolated from the sewage in Qingdao, China. Transmission electron microscopy revealed that vB_SbaS_Y11 has an icosahedral head and a long tail. The genome of vB_SbaS_Y11 is a linear, double-stranded DNA with a length of 62,799 bp and a G+C content of 46.9%, encoding 71 putative open reading frames. No tRNA genes or integrase-related feature genes were identified. An uncharacterized anti-CRISPR AcrVA2 gene was detected in its genome. Phylogenetic analysis based on the amino acid sequences of whole genomes and comparative genomic analyses indicate that vB_SbaS_Y11 has a novel genomic architecture and shares low similarity to Pseudomonas virus H66 and Pseudomonas phage F116. vB_SbaS_Y11 represents a potential new family-level virus cluster with eight metagenomic assembled viral genomes named Ranviridae.IMPORTANCEThe Gram-negative Shewanella bacterial genus currently includes about 80 species of mostly aquatic Gammaproteobacteria, which were isolated around the globe in a multitude of environments, such as freshwater, seawater, coastal sediments, and the deepest trenches. Here, we present a Shewanella phage vB_SbaS_Y11 that contains an uncharacterized anti-CRISPR AcrVA2 gene and belongs to a potential virus family, Ranviridae. This study will enhance the knowledge about the genome, diversity, taxonomic classification, and global distribution of Shewanella phage populations.

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 Salmonella phages isolated from poultry coops and its effect with nisin on food bio-control.

Salmonella is a bacterium associated with food contaminated by various animals, primarily poultry. Interest and research on bacteriophages are increasing because they can be used as an alternative against increasing antibiotic resistance. In our study, eight Salmonella-specific lytic bacteriophages were isolated from chicken feces. Two of the isolated phages (AUFM_Sc1 and AUFM_Sc3) were chosen for their characterization due to their broader host range. Based on morphological and genomic analysis, AUFM_Sc1 was identified to be close to similar Enterobacteria spp. CC31 (Myoviridae) and AUFM_Sc3 was identified to be close to Salmonella phage vB_Sen_I1 (Demerecviridae (formerly Siphoviridae)). Although these phages have shown promise for use in phage therapy applications for chickens, further studies are needed on their suitability. When a cocktail of these phages (AUFM_Sc1 + AUFM_Sc3) and nisin combination was applied on chicken breast meat, it was determined that it was effective against Salmonella contamination and while a good inhibitory effect was observed on the food, especially during the first 48 h, the effect decreased later, but the bacterial concentration was still low compared to the control group. Therefore, it is considered that the combination of AUFM_Sc1 + AUFM_Sc3 + nisin can be used as a food preservative against Salmonella.

Genome analysis of vB_SupP_AX, a novel N4-like phage infecting Sulfitobacter.

Sulfitobacter is a bacterium recognized for its production of AMP-independent sulfite oxidase, which is instrumental in the creation of sulfite biosensors. This capability underscores its ecological and economic relevance. In this study, we present a newly discovered phage, Sulfitobacter phage vB_SupP_AX, which was isolated from Maidao of Qingdao, China. The vB_SupP_AX genome is linear and double-stranded and measures 75,445 bp with a GC content of 49%. It encompasses four transfer RNA (tRNA) sequences and 79 open reading frames (ORFs), one of which is an auxiliary metabolic gene encoding thioredoxin. Consistent with other N4-like phages, vB_SupP_AX possesses three distinct RNA polymerases and is characterized by the presence of four tRNA molecules. Comparative genomic and phylogenetic analyses position vB_SupP_AX and three other viral genomes from the Integrated Microbial Genomes/Virus v4 database within the Rhodovirinae virus subfamily. The identification of vB_SupP_AX enhances our understanding of virus-host interactions within marine ecosystems.

Biological and genomic characterization of three psychrophilic Y. enterocolitica phages.

Yersinia (Y.) enterocolitica is an important foodborne pathogenic species that is mainly transmitted by the consumption of contaminated meat, particularly pork. To combat the bacteria along the food chain, the application of strictly lytic phages may be a promising tool. As the temperatures in the gut of animals and during food processing can differ significantly, a phage cocktail intended to be used for applications should comprise phages that are active at various temperatures. In this study, we isolated and characterized three phages with a myoviridal morphology (vB_YenM_P8, vB_YenM_P744 and vB_YenM_P778), which lysed the most important Y. enterocolitica serotypes O:3, O:9 and O:5,27 at a low multiplicity of infection (MOI) and at low temperatures down to 6°C. While vB_YenM_P8 is a member of the T4 family, vB_YenM_P744 and vB_YenM_P778 are novel phages that do not show relationship to known phages. The three phages were mixed in a cocktail with the already described phages vB_YenM_P281 and vB_YenP_Rambo. The cocktail revealed a strong lytic activity and lysed a mixture of Y. enterocolitica serotypes at room temperature (RT) within few hours with a reduction of up to 4.8 log10 units. Moreover, at even lower temperatures the mixture was significantly reduced after incubation overnight. The strongest reductions were determined at 6°C (4.0 log10 units) suggesting that the cocktail can lyse the psychrophilic Y. enterocolitica also during food processing. To determine possible phage resistance, 100 colonies that survived the infection by the phages were isolated and analysed regarding their serotype and phage susceptibility. Most isolates belonged to serotype O:9, but all of them were still sensitive to at least one phage of the cocktail.

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.

Isolation and characterization of two novel bacteriophages against carbapenem-resistant Klebsiella pneumoniae.

The increase of antibiotic-resistant bacteria has become a global health emergency and the need to explore alternative therapeutic options arises. Phage therapy uses bacteriophages to target specific bacterial strains. Phages are highly specific and can target resistant bacteria. Currently, research in this regard is focused on ensuring reliability and safety to bring this tool into clinical practice. The first step is to conduct comprehensive preclinical research. In this work, we present two novel bacteriophages vB_Kpn_F13 and vB_Kpn_F14 isolated against clinical carbapenem-resistant Klebsiella pneumoniae strains obtained from hospital sewage. Multiple studies in vitro were conducted, such as sequencing, electron microscopy, stability, host range infectivity, planktonic effect and biofilm inhibition in order to discover their ability to be used against carbapenem-resistant K. pneumoniae pathogens causing difficult-to-treat infections.

Isolation of bacteriophages specific to bovine mastitis-causing bacteria and characterization of their lytic activity in pasteurized milk.

Bovine mastitis is one of the most serious issues in dairy production. It is caused by contagious and coliform pathogens such as Staphylococcus spp., Escherichia coli, and Klebsiella pneumoniae. In addition, the emergence of drug-resistant bacteria raises urgent concerns in the field of drug treatment, thus requiring the exploration of alternative treatments. Bacteriophage therapy has been shown to be a promising alternative approach for the control of antibiotic-resistant pathogens. In this study, we aimed to isolate phages specific to contagious mastitis and coliform mastitis, characterize the isolated phages, and examine their ability to lyse bacteria in pasteurized milk samples. The Staphylococcus phage vB_Sau-RP15 isolated from raw milk in our previous study was used in this study. Other three phages, vB_Eco-RN12i1, vB_Kpn-RN14i1, and vB_Ssc-RN20i3, were isolated from wastewater using E. coli 5823, K. pneumoniae 194, and Staphylococcus sciuri MM01 as hosts, respectively. The host range and efficiency of plating (EOP) were determined following phage isolation. Moreover, the lysis activities of these phages against their hosts were investigated in pasteurized milk using a multiplicity of infections (MOIs) of 10 and 100 at 37°C. Phages were applied using individual and combination phages. According to the EOP results, all phages showed high specificity to their respective hosts. They are tailed phages with distinct morphologies. Individual phage treatments in spiked pasteurized milk with their respective bacterial hosts significantly reduced the bacterial counts in both MOI conditions during the first 2 h of the treatment (approximately 1-8 log reduction compared to the control). Although these phages specifically infected only their hosts, the phage cocktail resulted in a better result compared to the use of individual phage. However, bacterial regrowth was observed in all experiments, which may be related to the development of phage-insensitive mutants. Our findings suggest that the application of phages could be used to treat bovine mastitis. Phage cocktail is suitable to promote the efficacy of phage treatment in pasteurized milk. However, when considering the use of phages in dairy cows, certain phage properties in raw milk and in vivo and ex vivo should be highlighted to ensure their effectiveness as biocontrol agents for bovine mastitis treatment.

Characterization of the broad-spectrum phage vB_CsaM_CBT2 and its inhibition of multidrug-resistant Cronobacter sakazakii in powered infant formula

Cronobacter is a group of foodborne pathogens linked to serious and deadly infections in newborns and babies through the consumption of contaminated powdered infant formula (PIF). Meanwhile, antimicrobial misuse has led to emergence of multi-drug resistant (MDR) Cronobacter strains, affecting food safety. Here, a lytic phage vB_CsaM_CBT2 (CBT2) was isolated from a wastewater treatment plant in Hefei as an effective antimicrobial alternative to antibiotics. Phage CBT2 has a broad host spectrum. It can target and lyse 17 strains including MDR clinical strain C. sakazakii GZcsf-1 from four species of C. sakazakii, C. turicensis, C. muytjensii and C. condimenti in the genus Cronobacter. Some Cronobacter phages have a narrow host range and have only been confirmed to infect a single strain of Cronobacter. In addition, excellent tolerance to temperature (25 °C–70 °C) and pH values (3-11) of phage CBT2 were determined. Based on genomic and phylogenetic analyses, the phage CBT2 was successfully identified belonging to the Straboviridae, and genes related with drug resistance and virulence were not observed. After treatment of the phage CBT2 in four brands of rehydrated PIF, the viable counts of host bacteria (C. sakazakii GZcsf-1) were reduced 3.0 log CFU/mL (＞99.9%) after 6 h at 37 °C. The findings in this study provided an alternative tool for prevention and control of Cronobacter contamination in PIF.

A newly isolated Pseudomonas otitidis phage, vB_PotS-PotUPM1 from tilapia in Malaysia

A new Pseudomonas species, Pseudomonas otitidis was first known as an otic infectious agent in human. The presence of an inherent metallo-β-lactamase gene and its recent emergence in aquaculture underscore its potential zoonotic pathogenicity. The present study is aimed to characterize P. otitidis NK1 and its bacteriophage vB_PotS-PotUPM1 (PotUPM1), which were isolated from an infected tilapia fish farm in Malaysia. P. otitidis NK1 showed beta-hemolysis on blood agar and is resistant to several antibiotics. Its genome size was 6067,534 bp, with an average nucleotide identity of less than 99 % to all P. otitidis genome assemblies in GenBank, making it a novel strain of P. otitidis. The phage PotUPM1 has a latent period of 45 min and a burst size of 405 phage particles per bacterium. It effectively inhibited the growth of P. otitidis NK1 even at a low multiplicity of infection of 0.001 up to 9-hour post-inoculation. Transmission electron microscopy showed that it has a prolate head and a long, non-contractile tail, resembling those in the Siphoviridae family. Based on bioinformatic analysis, PotUPM1 has no resistance or virulence genes and is a lysogenic phage based on the presence of an integrase gene in the phage genome. Similarity analysis showed that PotUPM1 may represent a novel viral species with less than 95 % nucleotide sequence similarity to other characterized phages. The findings of this study could provide an insight on the occurrence of P. otitidis in the aquaculture industry and lay a basis for the application of phage or phage-derived proteins against pathogenic P. otitidis to alleviate antimicrobial resistance.

Genomic and biological characteristics of a novel lytic phage, vB_MscM-PMS3, infecting the opportunistic zoonotic pathogen Mammaliicoccus sciuri.

Mammaliicoccus sciuri is an opportunistic zoonotic pathogen in humans and animals. We isolated the Mammaliicoccus phage vB_MscM-PMS3, which was also able to infect and lyse M. sciuri and M. lentus. The phage genome is a linear dsDNA that is 147,811 bp in length and contains 206 ORFs and three tRNA genes. It showed low genome coverage (< 17%) and sequence identity (< 91.3%) to other phage genomes. Phylogenetic analysis based on the whole genome and major capsid protein revealed that this phage clustered with members of the subfamily Twortvirinae of the family Herelleviridae, but it was distinctly separated from the other members, indicating its uniqueness.

LysZX4-NCA, a new endolysin with broad-spectrum antibacterial activity for topical treatment

The prevalence of multidrug-resistant highly virulent Klebsiella pneumoniae (MDR-hvKP) requires the development of new therapeutic agents. Herein, a novel lytic phage vB_KpnS_ZX4 against MDR-hvKP was discovered in hospital sewage. Phage vB_KpnS_ZX4 had a short latent period (5 min) and a large burst size (230 PFU/cell). It can rapidly reduce the number of bacteria in vitro and improve survival rates of bacteremic mice in vivo from 0 to 80 % with a single injection of 108 PFU. LysZX4, an endolysin derived from vB_KpnS_ZX4, exhibits potent antimicrobial activity in vitro in combination with ethylenediaminetetraacetic acid (EDTA). The antimicrobial activity of LysZX4 was further enhanced by the fusion of KWKLFKI residues from cecropin A (LysZX4-NCA). In vitro antibacterial experiments showed that LysZX4-NCA exerts broad-spectrum antibacterial activity against clinical Gram-negative bacteria, including MDR-hvKP. Moreover, in the mouse model of MDR-hvKP skin infection, treatment with LysZX4-NCA resulted in a three-log reduction in bacterial burden on the skin compared to the control group. Therefore, the novel phages vB_KpnS_ZX4 and LysZX4-NCA are effective reagents for the treatment of systemic and local MDR-hvKP infections.

How Do Phages Disrupt the Structure of Enterococcus faecalis Biofilm?

Biofilms are composed of multicellular communities of microbial cells and their self-secreted extracellular polymeric substances (EPS). The viruses named bacteriophages can infect and lyze bacterial cells, leading to efficient biofilm eradication. The aim of this study was to analyze how bacteriophages disrupt the biofilm structure by killing bacterial cells and/or by damaging extracellular polysaccharides, proteins, and DNA. The use of colorimetric and spectrofluorimetric methods and confocal laser scanning microscopy (CLSM) enabled a comprehensive assessment of phage activity against E. faecalis biofilms. The impact of the phages vB_Efa29212_2e and vB_Efa29212_3e was investigated. They were applied separately or in combination on 1-day and 7-day-old biofilms. Phages 2e effectively inhibited the growth of planktonic cells with a limited effect on the biofilm. They did not notably affect extracellular polysaccharides and proteins; however, they increased DNA levels. Phages 3e demonstrated a potent and dispersing impact on E. faecalis biofilms, despite being slightly less effective than bacteriophages 2e against planktonic cells. Phages 3e reduced the amount of extracellular polysaccharides and increased eDNA levels in both 1-day-old and 7-day-old biofilm cultures. Phage cocktails had a strong antimicrobial effect on both planktonic and biofilm-associated bacteria. A significant reduction in the levels of polysaccharides, proteins, and eDNA in 1-day-old biofilm samples was noted, which confirms that phages interfere with the structure of E. faecalis biofilm by killing bacterial cells and affecting extracellular polymer levels.