Optimal Multiplicity Of Infection Research Articles

Characterization and Potential Application of Phage vB_PmuM_CFP3 for Phage Therapy Against Avian Pasteurella multocida

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera.

Characterization of phage HZY2308 against Acinetobacter baumannii and identification of phage-resistant bacteria

Background Acinetobacter baumannii (AB) is a notable cause of hospital-acquired infections, with carbapenem-resistant Acinetobacter baumannii (CRAB) classified as a high-priority critical pathogen. Bacteriophage therapy is emerging as a promising alternative to combat drug-resistant bacterial infections. In this study, a lytic phage, HZY2308, was isolated from hospital sewage, and the biological properties, biosafety and anti-biofilm properties of phage HZY2308 were characterized and identified. Moreover, the antibacterial effect of phage HZY2308 in combination with antibiotics was investigated, and the apparent characteristics of phage-resistant strain AB48-R were demonstrated, which provided data support for further studies to elucidate the mechanism of generating phage resistance.MethodsPhage HZY2308 was isolated by double agar plate method using clinical strain AB48 as the host bacterium. The morphology of phage HZY2308 was identified by transmission electron microscopy (TEM), and biological characteristics of phage HZY2308 were identified by host range, the efficiency of plating (EOP), sensitivity to temperature, pH, and chloroform, one-step growth curve, the optimal multiplicity of infection (MOI), and detection of endotoxin and cytotoxicity. Besides, the complete genome map of HZY2308 was constructed using CGview, and the phylogenetic tree of HZY2308 was constructed with MEGA. Additionally, the full genomic sequence of phage HZY2308 and the selected phage were compared using Easyfig. Checkerboard test of phage HZY2308 in combination with tigecycline (TGC) was performed to investigate their synergistic effect and bactericidal kinetics. The effect of HZY2308 on biofilm was investigated by semi-quantitative staining of biofilm with crystal violet, determination of bacterial activity in biofilm by 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl) -2 H-tetrazolium-5-carboxanilide (XTT) assay and observation of biofilm structure by fluorescence microscopy. Finally, Phage-resistant bacteria AB48-R were characterized by colony-forming capacity, morphology, growth curves, adsorption efficiency, and antibiotic susceptibility assays.ResultsA lytic phage, HZY2308, was isolated from hospital sewage, which exhibited advantageous traits such as a brief incubation period, large burst size, and robust stability. Safety assessments conducted at both genetic and cellular levels also have yielded positive outcomes. Besides, phage HZY2308 effectively inhibited AB biofilm formation and disrupted established biofilm structures. Furthermore, a synergistic antibacterial effect was noted when phage HZY2308 was combined with tigecycline. Interestingly, the phage-resistant strain, AB48-R was screened through natural selection. Compared to the wild strain AB48, the adsorption efficiency of the phage to AB48-R diminished. However, AB48-R’s sensitivity to antibiotics such as cefepime, gentamicin, amikacin, and tobramycin increased, indicating an evolutionary trade-off.ConclusionsPhage HZY2308 shows strong antimicrobial potential, especially in combination with tigecycline, and the phage-resistant strain exhibits increased antibiotic sensitivity.

Characterization of novel phage pK3–24 targeting multidrug-resistant Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Klebsiella pneumoniae is a common, conditionally pathogenic bacterium that often has a multidrug-resistant phenotype, leading to failure of antibiotic therapies. It can therefore induce serious diseases, including community-acquired pneumonia and bloodstream infections. As an emerging alternative to antibiotics, phages are considered key to solving the problem of drug-resistant bacterial infections. Here, we report a novel phage, pK3–24, that mainly targets ST447 K. pneumoniae. Phage pK3–24 is a T7-like short-tailed phage with a fast adsorption capacity that forms translucent plaques with halos on bacterial lawns. The optimal multiplicity of infection (MOI) is 0.01, and the average burst size is 50 PFU/mL. Phage pK3–24 shows environmental stability, surviving at below 50 °C and at pH values of 6–10. It has a double-stranded DNA genome of 40,327 bp and carries no antibiotic-resistance, virulence, or lysogeny genes. Phylogenetic analysis assigned phage pK3–24 to the genus Przondovirus as a new species. Phage pK3–24 inhibited the production of biofilm. Moreover, treatment with pK3–24 at doses with an MOI > 1 effectively reduced the mortality of Galleria mellonella larvae infected with ST447 K. pneumoniae.

Characterisation of a new virulent phage isolated from Hainan Island with potential against multidrug-resistant Pseudomonas aeruginosa infections

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a serious life-threatening pathogen. The rise in P. aeruginosa resistance rates has renewed interest in phages as an alternative therapeutic approach for treating bacterial infections. In this study, we investigated the characteristics of the first Pseudomonas phage, vB_PaP_HN01, isolated from Hainan, the only tropical island in China. The lytic rate of this phage against P. aeruginosa reached 64.3 % (27/42). Under the optimal multiplicity of infection (MOI) of 0.1, more than 90 % of phage particles absorb onto the host cell within 10 min, with an eclipse period of around 15 min, and a high titer phage production (1011 PFU/ml) within 90 min was demonstrated. vB_PaP_HN01 maintains a robust titer after 1 h exposure to pH values and temperatures (up to 50 °C). Genome annotation revealed that vB_PaP_HN01 did not contain drug-resistance or lysogeny-associated genes. It can effectively inhibit the formation of biofilms of MDR P. aeruginosa and eliminated aggressive biofilms (removal rate about 70 %). In the in vivo infection models, it was demonstrated that the survival rate and lifespan of Galleria mellonella larvae were increased alongside the injection of vB_PaP_HN01. These data revealed the potential of vB_PaP_HN01 against P. aeruginosa in clinic.

Characterization of the novel bequatrovirus vB-BcgM and its antibacterial effects in a food matrix.

Bacteria belonging to the Bacillus cereus group are ubiquitous in nature, causing food spoilage and food poisoning cases. A bequatrovirus, vB-BcgM, belonging to the C3 cluster infecting B. cereus group members, was isolated and characterized. Its 160-kb linear dsDNA genome contains a number of replication-related coding sequences (CDSs) and displays a collinear relationship with that of the virulent phage B4, with variations in its structural and replication regions. vB-BcgM has a relatively broad host range, with the ability to infect 33.3% of the B. cereus group isolates tested, including B. cereus, B. thuringiensis, B. anthracis, B. paranthracis, B. mycoides, and B. cytotoxicus. Moreover, vB-BcgM displays efficient infection and high replication capacity. It was found that 96.5% of the virions complete the adsorption process within 5 min. The optimal multiplicity of infection (MOI) is 10-7, and the burst size is 63 plaque-forming units (PFU)/cell. This phage showed stability over a broad pH range (4-12) and at temperatures up to 70 °C. Furthermore, vB-BcgM displays significant antibacterial effects in processed food matrices (ultra-high temperature [UHT] sterilized milk [GB 25190], UHT refrigerated milk [GB 25190], pasteurized milk [GB 19645], mashed meat, and cereals) and fresh foods (lettuce, apple, and potato). The antibacterial effects were found to be dependent on the dose of viral inoculum, incubation conditions (food matrix and temperature), and time. The data indicate that vB-BcgM has good potential as an antibacterial agent.

Biological and genomic characterization of a polyvalent phage PSH-1 against multidrug-resistant Salmonella Enteritidis

BackgroundBacteriophage has been renewed attention as a new antibacterial agent due to the limitations of antibiotic treatment. Bacteriophages are generally thought to be highly host specific and even strain specific, but a small number of polyvalent bacteriophages have been found to infect bacteria of different genera.ResultsIn this study, a virulent lytic bacteriophage (named Salmonella phage PSH-1) of Salmonella Enteritidis was isolated from the sewage samples of a large-scale pig farm, PSH-1 demonstrated lytic activity against four multidrug-resistant Salmonella Enteritidis isolates and Escherichia coli, and then its biological characteristics, genome and bacteriostatic ability were investigated. The results showed that the initial titer of PSH-1 was 1.15 × 1010 PFU/mL and the optimal multiplicity of infection (MOI) was 0.01, PSH-1 has stable activity in the range of pH 3.0–11.0. One-step growth curve showed that its latent period was 20 min, burst time was 80 min, and the burst was 495 particles. The whole-genome sequencing results revealed phage PSH-1 had a linear dsDNA with 48,466 bp length. The G/C content was 45.33%. Non-coding RNA genes and virulence factors were not found. Eighty- five open reading frames (ORFs) were identified after online annotation. By tests, the use of phage could succeed in controlling the artificial Salmonella contamination in milk at a range of temperatures.ConclusionsThis study reports a novel Salmonella Enteritidis phage PSH-1, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of PSH-1 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling of salmonellosis.

Characterization of the novel broad-spectrum lytic phage Phage_Pae01 and its antibiofilm efficacy against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is present throughout nature and is a common opportunistic pathogen in the human body. Carbapenem antibiotics are typically utilized as a last resort in the clinical treatment of multidrug-resistant infections caused by P. aeruginosa. The increase in carbapenem-resistant P. aeruginosa poses an immense challenge for the treatment of these infections. Bacteriophages have the potential to be used as antimicrobial agents for treating antibiotic-resistant bacteria. In this study, a new virulent P. aeruginosa phage, Phage_Pae01, was isolated from hospital sewage and shown to have broad-spectrum antibacterial activity against clinical P. aeruginosa isolates (83.6%). These clinical strains included multidrug-resistant P. aeruginosa and carbapenem-resistant P. aeruginosa. Transmission electron microscopy revealed that the phage possessed an icosahedral head of approximately 80 nm and a long tail about 110 m, indicating that it belongs to the Myoviridae family of the order Caudovirales. Biological characteristic analysis revealed that Phage_Pae01 could maintain stable activity in the temperature range of 4~ 60°C and pH range of 4 ~ 10. According to the in vitro lysis kinetics of the phage, Phage_Pae01 demonstrated strong antibacterial activity. The optimal multiplicity of infection was 0.01. The genome of Phage_Pae01 has a total length of 93,182 bp and contains 176 open reading frames (ORFs). The phage genome does not contain genes related to virulence or antibiotic resistance. In addition, Phage_Pae01 effectively prevented the formation of biofilms and eliminated established biofilms. When Phage_Pae01 was combined with gentamicin, it significantly disrupted established P. aeruginosa biofilms. We identified a novel P. aeruginosa phage and demonstrated its effective antimicrobial properties against P. aeruginosa in both the floating and biofilm states. These findings offer a promising approach for the treatment of drug-resistant bacterial infections in clinical settings.

Isolation, Identification, and Biological Characterization of Phage vB_KpnM_KpVB3 Targeting Carbapenem-Resistant Klebsiella pneumoniae ST11

This study aimed to isolate a phage capable of lysing carbapenem-resistant Klebsiella pneumoniae (CRKP) and to analyse its biological characteristics and whole-genome sequence. The phage was isolated and purified from the sewage. Transmission electron microscopy (TEM) was employed to observe the bacteriophage's morphology. Phenotypic characterization of the bacteriophages was determined. The genomic information was analysed. Evolutionary relationships were established through comparative genomics, proteomics, and phylogenetic analysis. The isolation of a virulent phage, named Klebsiella phage vB_KpnM_KpVB3, was notable for forming 6-7 mm transparent circular zones, each surrounded by a distinct halo. The phage had a head diameter of ca. 30 nm and a tail length of ca. 20 nm, being identified as a member of the Myoviridae family and the Caudovirales order. The optimal multiplicity of infection (MOI) was 0.00001, with an incubation period of 20 minutes and a lysis period of 60 minutes, and the number of released phages after lysis was 133±35 PFU/cell. The phage was relatively stable at temperatures ranging from 10°C to 40°C and at pH values ranging from 3 to 11. Its lytic efficiency against CRKP was 30.30%. It has been shown to be able to destroy the biofilm of host bacteria. The bacteriophage genome consists of double-stranded DNA (dsDNA) with a total length of 48,394 base pairs, a GC content of 48.99%, and 78 open reading frames (ORFs). The study resulted in the isolation vB_KpnM_KpVB3, a phage demonstrating potential therapeutic efficacy against infections caused by CRKP.

Biological characteristics of the bacteriophage LDT325 and its potential application against the plant pathogen Pseudomonas syringae.

Bud blight disease caused by Pseudomonas syringae is a major bacterial disease of tea plants in China. Concerns regarding the emergence of bacterial resistance to conventional copper controls have indicated the need to devise new methods of disease biocontrol. Phage-based biocontrol may be a sustainable approach to combat bacterial pathogens. In this study, a P. syringae phage was isolated from soil samples. Based on morphological characteristics, bacteriophage vB_PsS_LDT325 belongs to the Siphoviridae family; it has an icosahedral head with a diameter of 53 ± 1 nm and nonretractable tails measuring 110 ± 1 nm. The latent period and burst size of the phage were 10 min and 17 plaque-forming units (PFU)/cell, respectively. Furthermore, an analysis of the biological traits showed that the optimal multiplicity of infection (MOI) of the phage was 0.01. When the temperature exceeded 60°C, the phage titer began to decrease. The phage exhibited tolerance to a wide range of pH (3-11) and maintained relatively stable pH tolerance. It showed a high tolerance to chloroform, but was sensitive to ultraviolet (UV) light. The effects of phage LDT325 in treating P. syringae infections in vivo were evaluated using a tea plant. Plants were inoculated with 2 × 107 colony-forming units (CFU)/mL P. syringae using the needle-prick method and air-dried. Subsequently, plants were inoculated with 2 × 107 PFU/mL LDT325 phage. Compared with control plants, the bacterial count was reduced by 1 log10/0.5 g after 4 days in potted tea plants inoculated with the phage. These results underscore the phage as a potential antibacterial agent for controlling P. syringae.

Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli.

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

Biological and genomic characteristics of three novel bacteriophages and a phage-plasmid of Klebsiella pneumoniae.

Bacteriophages have emerged as promising candidates for the treatment of difficult-to-treat bacterial infections. The aim of this study is to isolate and characterize phages infecting carbapenem-resistant and extended-spectrum beta-lactamase producer Klebsiella pneumoniae isolates. Water samples were taken for the isolation of bacteriophages. One-step growth curve, the optimal multiplicity of infection (MOI), thermal and pH stabilities, transmission electron microscopy and whole-genome sequencing of phages were studied. Four phages were isolated and named Klebsiella phage Kpn02, Kpn17, Kpn74, and Kpn13. The optimal MOI and latent periods of phage Kpn02, Kpn17, Kpn74, and Kpn13 were 10, 1, 0.001, and 100 PFU/CFU and 20, 10, 20, and 30min, respectively. Burst sizes ranged from 811 to 2363. No known antibiotic resistance and virulence genes were identified. No tRNAs were detected except Klebsiella phage Kpn02 which encodes 24 tRNAs. Interestingly, Klebsiella phage Kpn74 was predicted to be a lysogenic phage whose prophage is a linear plasmid molecule with covalently closed ends. Of the Klebsiella-infecting phages presented in current study, virulent phages suggest that they may represent candidate therapeutic agents against MDR K. pneumoniae, based on short latent period, high burst sizes and no known antibiotic resistance and virulence genes in their genomes.

Bacterial isolation and genome analysis of a novel Klebsiella quasipneumoniae phage in southwest China’s karst area

BackgroundSouthwest China is one of the largest karst regions in the world. Karst environment is relatively fragile and vulnerable to human activities. Due to the discharge of sewage and domestic garbage, the karst system may be polluted by pathogenic bacteria. The detection of bacterial distribution and identification of phage capable of infecting them is an important approach for environmental assessment and resource acquisition.MethodsBacteria and phages were isolated from karst water in southwest China using the plate scribing and double plate method, respectively. Isolated phage was defined by transmission electron microscopy, one-step growth curve and optimal multiplicity of infection (MOI). Genomic sequencing, phylogenetic analysis, comparative genomic and proteomic analysis were performed.ResultsA Klebsiella quasipneumoniae phage was isolated from 32 isolates and named KL01. KL01 is morphologically identified as Caudoviricetes with an optimal MOI of 0.1, an incubation period of 10 min, and a lysis period of 60 min. The genome length of KL01 is about 45 kb, the GC content is 42.5%, and it contains 59 open reading frames. The highest average nucleotide similarity between KL01 and a known Klebsiella phage 6939 was 83.04%.ConclusionsKL01 is a novel phage, belonging to the Autophagoviridae, which has strong lytic ability. This study indicates that there were not only some potential potentially pathogenic bacteria in the karst environment, but also phage resources for exploration and application.

Transcriptional Response of Salmonella enterica to Bacteriophage Treatments with Differential Multiplicities of Infection

Salmonella enterica (S. enterica) is a causative agent of numerous foodborne outbreaks, as current industrial measures may be <90% effective. Therefore, bacteriophages have been suggested as an antimicrobial treatment against S. enterica, but it is currently unclear if there is an optimal bacteriophage multiplicity of infection (MOI) against S. enterica. Two bacteriophage cocktails at MOIs 1, 10, 100, 1000 and 10,000 were co-inoculated against four S. enterica strains (S. Enteritidis, S. Newport, S. Muenchen and S. Typhimurium), and populations were estimated on days 0–3. The transcriptional profiles of 20 genes previously indicated to be differentially expressed after bacteriophage treatment were studied by extracting RNA from all four S. enterica strains after bacteriophage SE14, SF5 and SF6 treatment on days 0, 1 and 3, and RT-qPCR was conducted to determine the expression of the 20 selected genes. The results showed that an MOI of 1000 was the most optimal in reducing S. Enteritidis populations to undetectable levels from day 0 to 3. The cas1 (SOS response) and mod (DNA modification and recombination) genes were highly upregulated between 2.5- and 5-fold on day 0 for S. Enteritidis S5-483 and S. Typhimurium S5-536 at MOIs of 1000 and 10,000. On day 3, hsdS (DNA modification and recombination) was upregulated by ~1-fold for S. enteritidis S5-483 after an MOI of 1000. Understanding an optimal bacteriophage MOI can be beneficial to implementing effective and optimal bacteriophage treatments in the industry. Knowledge of S. enterica’s transcriptional response after bacteriophage treatment provides further insight into how S. enterica can survive bacteriophage infection.

A bacteriophage against Citrobacter braakii and its synergistic effect with antibiotics.

A bacteriophage BD49 specific for Citrobacter braakii was screened out and purified by double-layer plate method. It consists of a polyhedral head of 93.1 ± 1.2nm long and 72.9 ± 4.2nm wide, tail fibers, collar, sheath and baseplate. The bacteriophage was identified by morphology observed with transmission electron microscope (TEM), whole genome sequencing carried out by Illumina next generation sequencing (NGS) technique, and gene annotation based on Clusters of Orthologous Groups of proteins (COG) database. It was identified primarily as a member of Caudovirales by morphology and further determined as Caudovirales, Myoviridae, and Citrobacter bacteriophage by alignment of its whole genome sequence with the NCBI database and establishment of phylogenetic tree. The bacteriophage showed good environmental suitability with optimal multiplicity of infection (MOI) of 0.01, proliferation time of 80min, optimum living temperature of 30-40°C, and living pH of 5-10. In addition, it exhibited synergistic effect with ciprofloxacin against C. braakii in antibacterial tests.

Genomic and biological characteristics of a novel phage induced from Limosilactobacillus fermentum

Bacteriophage infection of starter cultures remains a major threat to the food fermentation industry. To date, adequate information regarding the genome and characteristics of the Limosilactobacillus fermentum (L. fermentum) phage is lacking. The phage LFP01 was obtained by inducing L. fermentum IMAU32646 with mitomycin-C. The results showed that this phage contained a linear double-stranded DNA genome spanning 40,562 bp with a GC content of 46.25%. It comprises 54 coding sequences, which are grouped into three potential functional modules: DNA replication, cell lysogeny/lysis, and virion morphogenesis. Taxonomic analysis further confirmed that this phage belonged to a novel virus genus within the Caudoviricetes class. The optimal multiplicity of infection was 0.1, the latent period was 45 min, and the burst size was 158 ± 2.65 PFU/cell. This phage exhibited good environmental tolerance at 0–42 °C and a pH of 4–11. High temperature (50 °C) and strongly acidic (pH 2–3) conditions significantly influenced the adsorption capacity of the phage, but divalent cations and chloramphenicol had no similar effect. The findings of this study could enrich the database of phages and broaden the understanding of phage diversity.

Experimental Feasibility Study of TRAIL Gene Transfected into Neural Stem Cells.

To investigate the feasibility of transfecting the TNF-related apoptosis-inducing ligand (TRAIL) gene into neural stem cells (NSCs) in vitro, and explore whether NSCs retain their proliferative and differentiated activities after transfection. NSCs were obtained from fetal mouse brains, cultured in serum-free medium and identified by immunofluorescence staining. Lentivirus vector solution containing green fluorescent protein (GFP) gene was added to the NSCs based on the multiplicity of infection (MOI). The transfection efficiency of GFP was observed using a fluorescence microscope and detected by flow cytometry. NSCs were transfected with GFP-TRAIL fusion genes mediated by the optimized MOI lentivirus solution. The expression of TRAIL proteins in NSCs was detected by immunofluorescence and Western blot analysis. The differentiation of NSCs were induced and identified by immunofluorescence staining. The optimal MOI value of virus transfection was 10, resulting in a transfection rate was higher than 90%. GFP fluorescence could be observed at 24 hours after transfecting GFP-TRAIL genes into NSCs with an MOI of 10, and reached the maximum value at 72 hours. Immunofluorescence and Western-blot assays confirmed that GFP-TRAIL fusion proteins could be continuously expressed stably. Transfected NSCs could differentiate into neurons and glial cells without any statistically significant difference compared to the non-transfected group. Neural stem cells retained their proliferative and differentiated potential after being transfected with the TRAIL gene while sustainably expressing TRAIL protein.

Phenotypic characterization and genomic analysis of Limosilactobacillus fermentum phage

Limosilactobacillus (L.) fermentum is widely utilized for its beneficial properties, but lysogenic phages can integrate into its genome and can be induced to enter the lysis cycle under certain conditions, thus accomplishing lysis of host cells, resulting in severe economic losses. In this study, a lysogenic phage, LFP03, was induced from L. fermentum IMAU 32510 by UV irradiation for 70 s. The electron microscopy showed that this phage belonged to Caudoviricetes class. Its genome size was 39,556 bp with a GC content of 46.08%, which includes 20 functional proteins. Compared with other L. fermentum phages, the genome of phage LFP03 exhibited deletions, inversions and translocations. Biological analysis showed that its optimal multiplicity of infection was 0.1, with a burst size of 133.5 ± 4.9 PFU/infective cell. Phage LFP03 was sensitive to temperature and pH value, with a survival rate of 48.98% at 50 °C. It could be completely inactivated under pH 2. The adsorption ability of this phage was minimally affected by temperature and pH value, with adsorption rates reaching 80% under all treated conditions. Divalent cations could accelerate phage adsorption, while chloramphenicol expressed little influence. This study might expand the related knowledge of L. fermentum phages, and provide some theoretical basis for improving the stability of related products and establishing phage control measures.

Biologic and genomic characterization of a novel virulent Aeromonas hydrophila phage phiA051, with high homology to prophages.

Aeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature. Water samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage. A novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30-50°C or pH 6.0-10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs. The phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages.

Characterization and Preliminary Application of a Novel Lytic Vibrio parahaemolyticus Bacteriophage vB_VpaP_SJSY21.

Litopenaeus vannamei is one of the most economically significant aquatic species globally. However, the emergence of acute hepatopancreatic necrosis disease (AHPND) in recent years has resulted in substantial losses within the L. vannamei farming industry. Phage therapy holds promise as an effective strategy for preventing and controlling bacterial infections like AHPND, thereby promoting the healthy and sustainable growth of the shrimp aquaculture sector. In this study, a novel and unique Vibrio parahaemolyticus bacteriophage, named vB_VpaP_SJSY21, was successfully isolated from sewage samples. Using transmission electron microscopy, it was observed that phage SJSY21 has an elongated shell. Notably, phage SJSY21 exhibited high infection efficiency, with an optimal multiplicity of infection (MOI) of only 0.01 and a remarkably short latent period of 10 min, resulting in a lysis quantity of 508. Furthermore, phage SJSY21 demonstrated notable heat resistance and the capacity to withstand high temperatures during preservation, thus holding potential for application in phage therapy. Whole-genome sequencing and analysis confirmed that phage SJSY21 has a genome size of 110,776 bp, classifying it as a new member of the short-tailed bacteriophage family. Additionally, cultivation experiments indicated that phage SJSY21 has the potential to enhance the survival of L. vannamei in culture systems, thereby offering innovative prospects for the application of phage therapy in aquaculture.

Isolation and characterization of a novel temperate bacteriophage infecting Aeromonas hydrophila isolated from a Macrobrachium rosenbergii larvae pond

Aeromonas hydrophila is an opportunistic pathogen that frequently leads to significant mortality in various commercially cultured aquatic species. Bacteriophages offer an alternative strategy for pathogens elimination. In this study, we isolated, identified, and characterized a novel temperate A. hydrophila phage, designated as P05B. The bacteriophage P05B is a myovirus based on its morphological features, and possesses the capability to lyse A. hydrophila strains isolated from shrimp. The optimal multiplicity of infection (MOI), adsorption rate, latent period, and burst size for phage P05B were determined to be 0.001, 91.7 %, 20 min, and 483 PFU/cell, respectively. Phage P05B displayed stability across a range of temperatures (28–50 °C) and pH values (4.0–10.0). Sequence analysis unveiled that the genome of phage P05B comprises 32,302 base pairs with an average G + C content of 59.4 %. A total of 40 open reading frames (ORF) were encoded within the phage P05B genome. The comparative genomic analyses clearly implied that P05B might represent a novel species of the genus Bielevirus under Peduoviridae family. A phylogenetic tree was reconstructed, demonstrating that P05B shares a close evolutionary relationship with other Aeromonas and Aeromonas phages. In conclusion, this study increased our knowledge about a new temperate phage of A. hydrophila with strong lytic ability.