Optimal Multiplicity Of Infection Research Articles

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 induction 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, 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, with adsorption rates reaching 80% under all treated conditions. Divalent cations could accelerate phage adsorption, while chloramphenicol expressed little significant effect on phage adsorption. 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.

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.

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

ObjectivesThis study aimed to isolate a phage capable of lysing carbapenem-resistant Klebsiella pneumoniae (CRKP) and to analyze its biological characteristics and whole genome sequence. MethodsThe 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 were determined. The genomic information was analyzed. Evolutionary relationships were established through comparative genomics, proteomics, and phylogenetic analysis. ResultsThe 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 approximately 30 nm and a tail length of about 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 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). ConclusionThe study resulted in the isolation vB_KpnM_KpVB3, a phage demonstrating potential therapeutic efficacy against infections caused by CRKP.

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.

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.

Isolation and Characterization of Bacteriophage VA5 against Vibrio alginolyticus.

Bacteriophages, or phages, can be used as natural biological control agents to eliminate pathogenic bacteria during aquatic product cultivation. Samples were collected from seafood aquaculture water and aquaculture environmental sewage, and phage VA5 was isolated using the double-layer agar plate method, with Vibrio alginolyticus as the host bacteria. The purified phage strain was subjected to genome sequencing analysis and morphological observation. The optimal multiplicity of infection (MOI), the one-step growth curve, temperature stability, and pH stability were analyzed. Phage VA5 was observed to have a long tail. Whole-genome sequencing revealed that the genome was circular dsDNA, with 35,866 bp length and 46% G+C content. The optimal MOI was 1, the incubation period was 20 min, the outbreak period was 30 min, and the cleavage amount was 92.26 PFU/cell. The phage showed good activity at -20 °C, 70 °C, and pH 2-10. Moreover, the phage VA5 exhibited significant inhibitory effects on V. alginolyticus-infected shrimp culture. The isolated phage VA5 has a wide range of host bacteria and is a good candidate for biological control of pathogenic bacteria.

StAP1 phage: an effective tool for treating methicillin-resistant Staphylococcus aureus infections.

Staphylococcus aureus infection has long been a serious concern in the medical field, with methicillin-resistant Staphylococcus aureus (MRSA) posing a considerable challenge to public health. Given the escalating bacterial resistance and the favorable biosafety and environmental properties of phages, the resurgence of phage therapy offers a promising alternative to antibiotics. In this study, we isolated and characterized a MRSA phage named StAP1 from a Chinese hospital. Phenotypic and molecular analyses revealed its broad-spectrum characteristics, genomic background, and potential application in MRSA infection treatment. Morphological examination classified the phage as a member of the Herelleviridae phage family, displaying a typical hexagonal head and a slender fibrous tail. Genomic analysis unveiled a size of ~144,705 bp for the StAP1 genome, encompassing 215 open reading frames (ORFs). The one-step growth curve demonstrated a 20-min incubation period for the phage, with an optimal multiplicity of infection (MOI) of 0.1. Moreover, StAP1 exhibited stability across a wide range of temperatures and pH levels. Further investigation of its broad-spectrum characteristics confirmed its ability to effectively infect all staphylococcal cassette chromosomal mec (SCCmec) types found in MRSA strains, notably displaying a remarkable lysis rate of 76.7% against the prevalent ST239 strain in China. In vivo studies show cased significant efficacy of the StAP1 phage against MRSA infection. Overall, StAP1 phage presents a broad infection spectrum and exhibits strong lytic effects on various MRSA strains, highlighting its tremendous potential as a powerful tool for MRSA infection treatment.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

Isolation, biological and whole genome characteristics of a Proteus mirabilis bacteriophage strain

Proteus mirabilis, a naturally resistant zoonotic bacterium belonging to the Enterobacteriaceae family, has exhibited an alarming increase in drug resistance. Consequently, there is an urgent need to explore alternative antimicrobial agents. Bacteriophages, viruses that selectively target bacteria, are abundant in the natural environment and have demonstrated potential as a promising alternative to antibiotics. In this study, we successfully isolated four strains of Proteus mirabilis phages from sewage obtained from a chicken farm in Sichuan, China. Subsequently, we characterized one of the most potent lytic phages, Q29, by examining its biological and genomic features. Comparative genomic analysis revealed the functional genes and phylogenetic evolution of Q29 phages. Our findings revealed that Proteus mirabilis bacteriophage Q29 possesses an icosahedral symmetrical head with a diameter of 95 nm and a tail length of 240 nm. Moreover, phage Q29 exhibited stability within a temperature range of 37 ℃ to 55 ℃ and under pH conditions ranging from 4 to 9. The optimal multiplicity of infection (MOI) for this phage was determined to be 0.001. Furthermore, the one-step growth curve results indicated an incubation period of approximately 15 min, an outbreak period of approximately 35 min, and an average cleavage quantity of approximately 60 plaque-forming units (PFU) per cell. The genome of phage Q29 was found to have a total length of 58,664 base pairs and encoded 335 open reading frames (ORFs) without carrying any antibiotic resistance genes. Additionally, genetic evolutionary analysis classified phage Q29 within the family Caudalidae and the genus Myotail. This study provides valuable research material for further development of Proteus mirabilis bacteriophage biologics as promising alternatives to antibiotics, particularly in light of the growing challenge of antibiotic resistance posed by this bacterium.

Characteristics and Whole-Genome Analysis of Limosilactobacillus fermentum Phage LFP02.

Limosilactobacillus fermentum is a bacterium widely used in food production, medicine, and industrial fermentation. However, fermentation could fail due to phage contamination. L. fermentum bacteriophage LFP02 can be induced from L. fermentum IMAU 32579 using mitomycin C. To better understand the characteristics of this phage, its physiological and genomic characteristics were evaluated. The results showed that its optimal multiplicity of infection was 0.01, and the burst size was 148.03 ± 2.65 pfu/infective center. Compared to temperature, pH had a more obvious influence on phage viability, although its adsorption capacity was not affected by the divalent cations (Ca2+ and Mg2+) or chloramphenicol. Its genome size was 43,789 bp and the GC content was 46.06%, including 53 functional proteins. Compared to other L. fermentum phages, phage LFP02 had chromosome deletion, insertion, and inversion, which demonstrated that it was a novel phage. This study could expand the knowledge of the biological characteristics of L. fermentum bacteriophages and provide some theoretical basis for bacteriophage prevention during fermentation.

Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands

Phage therapy has potential to combat antibiotic-resistant bacteria causing bovine mastitis. Our objective was to use 3 Klebsiella lytic phages to create a phage cocktail, and to compare bactericidal activity of this phage cocktail versus an individual phage, both in vitro and in vivo. Based on transmission electron microscopy, phage CM_Kpn_HB154724 belonged to Podoviridae and on double agar plates, it formed translucent plaques on the bacterial lawn of Klebsiella pneumoniae KPHB154724. In one-step growth curves, this phage had a latent period of 40 min, an outbreak period of 40 min, a burst size of 1.2 × 107 PFU/mL, and an optimal multiplicity of infection (MOI) of 1. Furthermore, it was inactivated under extreme conditions (pH ≤ 3.0 or ≥ 12.0 and temperatures of 60 or 70 °C). It had a host range of 90% and had 146 predicted genes (Illumine NovaSeq). Based on histopathology and expression of inflammatory factors interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin, phage cocktail therapy had better efficiency than an individual phage in K. pneumoniae-infected murine mammary glands. In conclusion, we used 3 Klebsiella lytic phages to create a phage cocktail and confirmed its effectiveness against K. pneumoniae both in vitro (bacterial lawn) and in vivo (infected murine mammary glands).

Characterization of bacteriophage BUCT631 lytic for K1 Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Severe infections caused by multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) highlight the need for new therapeutics with activity against this pathogen. Phage therapy is an alternative treatment approach for multidrug-resistant K. pneumoniae infections. Here, we report a novel bacteriophage (phage) BUCT631 that can specifically lyse capsule-type K1 K. pneumoniae. Physiological characterization revealed that phage BUCT631 could rapidly adsorb to the surface of K. pneumoniae and form an obvious halo ring, and it had relatively favorable thermal stability (4–50 ​°C) and pH tolerance (pH ​= ​4–12). In addition, the optimal multiplicity of infection (MOI) of phage BUCT631 was 0.01, and the burst size was approximately 303 ​PFU/cell. Genomic analysis showed that phage BUCT631 has double-stranded DNA (total length of 44,812 bp) with a G ​+ ​C content of 54.1%, and the genome contains 57 open reading frames (ORFs) and no virulence or antibiotic resistance related genes. Based on phylogenetic analysis, phage BUCT631 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae. In addition, phage BUCT631 could quickly inhibit the growth of K. pneumoniae within 2 ​h in vitro and significantly elevated the survival rate of K. pneumoniae infected Galleria mellonella larvae from 10% to 90% in vivo. These studies suggest that phage BUCT631 has promising potential for development as a safe alternative for control and treatment of multidrug-resistant K. pneumoniae infection.

Biological and genomic characterization of a polyvalent bacteriophage (S19cd) strongly inhibiting Salmonella enterica serovar Choleraesuis

Bacteriophages are a promising alternative for the control of pathogenic bacteria. In this study, we isolated a virulent bacteriophage, S19cd, from pig gut that could infect both a non-pathogenic bacteria Escherichia coli 44 (EC44) and two pathogenic bacterial strains (ATCC 13312 (SC13312) and CICC 21493 (SC21493)) of Salmonella enterica serovar Choleraesuis (SC). S19cd exhibited strong lytic ability in both SC13312 and SC21493 with an optimal multiplicity of infection (MOI) of 10−6 and 10−5, respectively, and inhibited their growth at an MOI of 10−7 within 24 h. Mice pre-treated with S19cd exhibited protection against the SC13312 challenge. Moreover, S19cd has good heat resistance (80 ℃) and pH tolerance (pH 3–12). Genome analysis revealed that S19cd belongs to the Felixounavirus genus and does not contain any virulence or drug-resistance-related genes. Additionally, S19cd encodes an adenine-specific methyltransferase that has no similarity to methyltransferases from other Felixounavirus phages and shares limited similarity with other methyltransferases in the NCBI protein database. Metagenomic analysis of S19cd genomes from 500 pigs revealed that S19cd-like phages may be widespread in Chinese pig gut. In conclusion, S19cd can be a potential phage therapy targeting SC infections.

Isolation, characterization, and application of a novel, lytic phage vB_SalA_KFSST3 with depolymerase for the control of Salmonella and its biofilm on cantaloupe under cold temperature

This study investigated the efficacy of a novel Salmonella phage with depolymerase activity to control S. Typhimurium (ST) and its biofilm on cantaloupes, for the first time, under simulated cold temperature. vB_SalA_KFSST3 forming a halo zone was isolated and purified from a slaughterhouse with a final concentration of 12.1 ± 0.1 log PFU/mL. Based on the morphological and bioinformatics analyses, vB_SalA_KFSST3 was identified as a novel phage belonging to the family Ackermannviridae. Before employing the phage on cantaloupe, its genetic characteristics, specificity, stability, and bactericidal effect were investigated. Genetic analyses confirmed its safety and identified endolysin and two depolymerase domains possessing antibiofilm potential. In addition, the phage exhibited a broad specificity with great efficiencies toward five Salmonella strains at 4 °C, 22 °C, and 37 °C, as well as stable lytic activity over a wide range of pHs (3 to 11) and temperatures (−20 °C to 60 °C). The optimal multiplicity of infection (MOI) and exposure time of phage were determined to be 100 and 2 h, respectively, based on the highest bacterial reduction of ∼2.7 log CFU/mL. Following the formation of ST biofilm on cantaloupe at 4 °C and 22 °C, the cantaloupe was treated with phage at an MOI of 100 for 2 h. The antibiofilm efficacy of phage was evaluated via the plate count method, confocal laser scanning microscopy, and scanning electron microscopy (SEM). The initial biofilm population at 22 °C was significantly greater and more condensed than that at 4 °C. After phage treatment, biofilm population and the percentage of viable ST in biofilm were reduced by ∼4.6 log CFU/cm2 and ∼90% within 2 h, respectively, which were significantly greater than those at 22 °C (∼2.0 log CFU/cm2 and ∼45%) (P < 0.05). SEM images also confirmed more drastic destruction of the cohesive biofilm architecture at 4 °C than at 22 °C. As a result of its cold temperature-robust lytic activity and the contribution of endolysin and two depolymerases, vB_SalA_KFSST3 demonstrated excellent antibiofilm efficacy at cold temperature, highlighting its potential as a promising practical biocontrol agent for the control of ST and its biofilm.