Large Burst Size Research Articles

Isolation, Characterization, and Anti-Biofilm Activity of a Novel Kaypoctavirus Against K24 Capsular Type, Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates

Background/Objectives: The significant outbreak of multidrug-resistant Klebsiella pneumoniae has emerged as a primary global concern associated with high morbidity and mortality rates. Certain strains of K. pneumoniae are highly resistant to most antibiotics available in clinical practice, exacerbating the challenge of bacterial infections. Methods: Phage vB_KpnP_PW7 (vKPPW7) was isolated and characterized. Its morphology, stability, adsorption rate, one-step growth curve, lytic activity, whole-genome sequence analysis, and antibacterial and antibiofilm activities were evaluated. Results: The virulent phage has a 73,658 bp linear dsDNA genome and was classified as a new species of the genus Kaypoctavirus, subfamily Enquatrovirinae, and family Schitoviridae. Phage vKPPW7 has a high adsorption rate, a short latent period, and a large burst size. The phage showed activity against 18 K. pneumoniae isolates with the K24 capsular type but was unable to lyse K. pneumoniae isolates whose capsular type was not classified as K24. Additionally, phage vKPPW7 demonstrated strong stability across various temperatures and pH values. The phage exhibited antibacterial activity, and scanning electron microscopy (SEM) confirmed its ability to lyse MDR K. pneumoniae with the K24 capsular type. Furthermore, phage vKPPW7 effectively removed preformed biofilm and prevented biofilm formation, resulting in reduced biofilm biomass and biofilm viability compared to controls. The architecture of phage-treated biofilms was confirmed under SEM. Conclusions: These findings suggest that phage vKPPW7 holds promise for development as a therapeutic or biocontrol agent.

Characterization of Broad Spectrum Bacteriophage vB ESM-pEJ01 and Its Antimicrobial Efficacy Against Shiga Toxin-Producing Escherichia coli in Green Juice.

Shiga toxin-producing Escherichia coli (STEC) infections have increased in humans, animals, and the food industry, with ready-to-eat (RTE) food products being particularly susceptible to contamination. The prevalence of multidrug-resistant strains has rendered the current control strategies insufficient to effectively control STEC infections. Herein, we characterized the newly isolated STEC phage vB_ESM-pEJ01, a polyvalent phage capable of infecting Escherichia and Salmonella species, and assessed its efficacy in reducing STEC in vitro and food matrices. The phage, belonging to the Tevenvirinae, exhibits effective bacteriolytic activity, a short latent period, large burst size, and stability under a broad pH range and moderate temperatures. Moreover, the phage demonstrated strong anti-biofilm efficacy even at low concentrations. Genomic analysis revealed that the phage was similar to the well-characterized RB49 phage (T4-like phage) but possesses distinct host-specificity-related genes that potentially contribute to its extensive host range. The efficacy of phage vB_ESM-pEJ01 was evaluated in artificially STEC-inoculated green juice samples, where it significantly reduced STEC and the abundance of Shiga toxin-producing genes at 4 and 25 °C. Therefore, these results suggest that the polyvalent phage vB_ESM-pEJ01 is a promising biocontrol agent for foodborne pathogens in RTE foods such as fresh juices.

From Isolation to Application: Utilising Phage-Antibiotic Synergy in Murine Bacteremia Model to Combat Multidrug-Resistant Enterococcus faecalis.

Enterococcus species, natural inhabitants of the human gut, have become major causes of life-threatening bloodstream infections (BSIs) and the third most frequent cause of hospital-acquired bacteremia. The rise of high-level gentamicin resistance (HLGR) in enterococcal isolates complicates treatment and revives bacteriophage therapy. This study isolated and identified forty E. faecalis clinical isolates, with 30% exhibiting HLGR. The HLGR5 isolate, resistant to fosfomycin, vancomycin, and linezolid, was used to isolate the vB_EfaS_SZ1 phage from effluent water. This phage specifically lysed 42% of HLGR isolates. vB_EfaS_SZ1 demonstrated beneficial traits, including thermal stability, acid-base tolerance, a short latent period, and a large burst size. The phage genome comprises a 40,942 bp linear double-stranded DNA with 65 open reading frames (ORFs). The genome closely resembled Enterococcus phages, classifying it within the Efquatrovirus genus. Phage-antibiotic synergy was assessed using checkerboard assays and time-killing analyses, revealing enhanced bacteriolytic activity of ampicillin and fosfomycin, with significant reductions in minimum inhibitory concentration values. In a mouse bacteremia model, phage-antibiotic combinations significantly reduced E. faecalis liver burden compared to monotherapies. Histopathological analysis confirmed therapeutic synergy, showing reduced inflammation and improved hepatocyte regeneration. These findings underscore the potential of phage vB_EfaS_SZ1 as an adjunct to antibiotic therapy for resistant enterococcal bacteremia.

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

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

Isolation and Characterization of a Lytic Phage PaTJ Against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major global threat to human health, and phage therapy has emerged as a promising strategy for treating infections caused by multidrug-resistant pathogens. In this study, we isolated and characterized a Pseudomonas lytic phage, PaTJ, from wastewater. PaTJ belongs to the phage family Mesyanzhinovviridae, and is featured by short latency (30 min) and large burst size (103 PFU per infected cell). Our investigation revealed that PaTJ utilizes the type IV Pili (T4P) as a receptor. Transcriptome analysis of PaTJ infected host at latent stage showed distinct expression patterns of PaTJ encoding genes involved in replication and structure assembly, without expression of the majority of toxic accessory genes responsible for phage release. In addition, host bacteria exhibited specific induction of host metabolism-related genes in response to the PaTJ's infection. Furthermore, our findings demonstrated the PaTJ's potential in degrading biofilms. This work sheds light on the multifaceted impact of this lytic phage PaTJ on P. aeruginosa, presenting potential applications in both gene expression modulation and biofilm management.

A novel Alteromonas phage with tail fiber containing six potential iron-binding domains.

Viruses play a vital role in regulating microbial communities, contributing to biogeochemical cycles of carbon, nitrogen, and essential metals. Alteromonas is widespread and plays an essential role in marine microbial ecology. However, there is limited knowledge about the interactions of Alteromonas and its viruses (alterophages). This study isolated a novel podovirus, vB_AmeP-R22Y (R22Y), which infects Alteromonas marina SW-47 (T). Phylogenetic analysis suggested that R22Y represented a novel viral genus within the Schitoviridae family. R22Y exhibited a broad host range and a relatively large burst size, exerting an important impact on the adaptability and dynamics of host populations. Two auxiliary metabolic genes, encoding Acyl carrier protein and AAA domain-containing protein, were predicted in R22Y, which may potentially assist in host fatty acid metabolism and VB12 biosynthesis, respectively. Remarkably, the prediction of the R22Y tail fiber structure revealed six conserved histidine residues (HxH motifs) that could potentially bind iron ions, suggesting that alterophages may function as organic iron-binding ligands in the marine environment. Our isolation and characterization of R22Y complements the Trojan Horse hypothesis, proposes the possible role of alterophages for marine iron biogeochemical cycling, and provides new insights into phage-host interactions in the iron-limited ocean.IMPORTANCEIron (Fe), as an essential micronutrient, is often a limiting factor for microbial growth in marine ecosystems. The Trojan Horse hypothesis suggests that iron in the phage tail fibers is recognized by the host's siderophore-bound iron receptor, enabling the phage to attach and initiate infection. The potential role of phages as iron-binding ligands has significant implications for oceanic trace metal biogeochemistry. In this study, we isolated a new phage R22Y with the potential to bind iron ions, using Alteromonas, a major siderophore producer, as the host. The tail fiber structure of R22Y exhibits six conserved HxH motifs, suggesting that each phage could potentially bind up to 36 iron ions. R22Y may contribute to colloidal organically complexed dissolved iron in the marine environment. This finding provides further insights into the Trojan Horse hypothesis, suggesting that alterophages may act as natural iron-binding ligands in the marine environment.

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.

Novel Bacteriophage KG853 Exhibits Potent Lytic Activity and Biofilm Inhibition Against Pseudomonas aeruginosa

This study reports the isolation and characterization of a novel bacteriophage, KG853, specifically targeting Pseudomonas aeruginosa ATCC 27853. Morphological analysis using transmission electron microscopy revealed that bacteriophage KG853 belongs to the Bruynoghevirus genus. The phage demonstrated favorable characteristics for potential therapeutic applications, including a short latent period of 30 minutes and a large burst size of 136 plaque-forming units (PFU) per cell. KG853 exhibited stability across various temperatures and pH values, indicating its robustness under various environmental conditions. Genomic analysis showed that KG853 possesses a circular DNA genome of 45,390 base pairs with a GC content of 52.2%. No lysogenic or virulence genes were detected among the 84 open reading frames annotated in the genome, suggesting its safety for potential therapeutic use. Phylogenetic analysis revealed that phage KG853 is closely related to phage PaP3. Notably, KG853 demonstrated the ability to inhibit the formation of 4-hour biofilms by P. aeruginosa, a critical virulence factor in many infections. Host range analysis showed that KG853 is specific to P. aeruginosa, an important characteristic for targeted therapy. These findings suggest that bacteriophage KG853 represents a promising candidate for combating drug-resistant P. aeruginosa infections. Its specific host range, robust physical characteristics, lack of harmful genes, and anti-biofilm activity make it a potential alternative to conventional antibiotics. Further research is warranted to explore its efficacy in in vivo models and potential clinical applications.

Isolation and evaluation of bacteriophage cocktail for the control of colistin-resistant Escherichia coli

The frequent emergence of colistin-resistant E. coli worldwide drives the exploration of alternative therapies, and bacteriophages (phages) have emerged as promising candidates to tackle this challenge. In this study, three E. coli phages were isolated, screened, and evaluated against 96 colistin-resistant strains obtained from diverse sources. The combined recognition rate for these strains was 43.6%, while individually it ranged from 17.0% to 24.5%. Notably, among the tested phages (FJ3-79, SD1-92L, and FJ4-63), FJ4-63 demonstrated exceptional characteristics in regulating host population dynamics upon infection by exhibiting a shorter latent period (20min) and a larger burst size (95.99±3.61PFU/cell). Furthermore, it exhibited relative stability at pH 3-11 and below 60°C. Transmission electron microscopy and genomic analysis classified phage FJ4-63 belongs to the Dhakavirus genus within the Straboviridae family. Its genome comprised a linear double-stranded DNA measuring 169,669 bp (containing 272 coding sequences) with a GC content of 39.76%, of which 93 (34.2%) had known functions, and the remaining 177 were annotated as hypothetical proteins. Additionally, two tRNAs were recognized, possess the "holin-endolysin" lytic system, and no resistance or virulence genes were detected. The phylogenetic tree and average nucleotide identity (ANI) analysis revealed that phage FJ4-63 exhibited the highest similarity to Escherichia phage C6 (679410.1), indicating a consistent close relationship within the same branch. The cocktail comprising three phages exhibits enhanced in vitro bactericidal efficacy compared to a single phage. At high doses with MOI=100, it rapidly and completely eradicates bacteria within 1h while significantly reducing bacterial biofilms. All this evidence suggests that lytic phages offer an effective solution for clinical treatment, with a phage cocktail demonstrating greater potential in the alternative management of colistin-resistant E. coli infections.

Integral LOCA experiments to study FFRD behavior of high burnup nuclear fuels

Integral LOCA experiments to study FFRD behavior of high burnup nuclear fuels

Isolation and Characterization of Two Novel Lytic Bacteriophages against Salmonella typhimurium and Their Biocontrol Potential in Food Products.

Foodborne pathogens, such as Salmonella, are major factors that pose significant threats to global food safety and public health. Salmonella typhimurium is a prominent serotype contributing to non-typhoidal salmonellosis, which is a prevalent foodborne illness affecting humans and animals. Bacteriophages are considered one of the most environmentally friendly biocontrol agents, particularly in the food industry, owing to their high specificity and high safety. However, the emergency of phage-resistant mutants limits the biocontrol effect of phage treatment, leading to the requirement for a high diversity of lytic phages. Therefore, the study isolated and characterized two novel lytic Salmonella bacteriophages (SPYS_1 and SPYS_2) targeting S. typhimurium ATCC14028 and evaluated their effectiveness in reducing the contamination rates for milk and chicken tenders. Morphological and genomic analyses indicated that Salmonella phages SPYS_1 and SPYS_2 are novel species classified under the genus Skatevirus and the genus Berlinvirus, respectively. Both phages exhibited high stability across a broad range of thermal and pH conditions. The one-step growth curve result suggested that both phages had a short adsorption time and a large burst size in a single lytic cycle. The phage SPYS_1 demonstrated a noteworthy inhibition effect on the growth of S. typhimurium ATCC14028 in milk, resulting in a ~2-log reduction within the 2 to 4 h range. Overall, both phages have shown significant potential for application in food safety in the future.

Characterization and genome analysis of a broad host range lytic phage vB_SenS_TUMS_E19 against Salmonella enterica and its efficiency evaluation in the liquid egg.

Salmonella enterica serovars are zoonotic bacterial that cause foodborne enteritis. Due to bacteria's antibiotic resistance, using bacteriophages for biocontrol and treatment is a new therapeutic approach. In this study, we isolated, characterized, and analyzed the genome of vB_SenS_TUMS_E19 (E19), a broad host range Salmonella bacteriophage, and evaluated the influence of E19 on liquid eggs infected with Salmonella enterica serovar Enteritidis. Transmission electron microscopy showed that the isolated bacteriophage had a siphovirus morphotype. E19 showed rapid adsorption (92% in 5min), a short latent period (18min), a large burst size (156 PFU per cell), and a broad host range against different Salmonella enterica serovars. Whole-genome sequencing analysis indicated that the isolated phage had a 42 813bp long genome with 49.8% G+C content. Neither tRNA genes nor those associated with antibiotic resistance, virulence factors, or lysogenic formation were detected in the genome. The efficacy of E19 was evaluated in liquid eggs inoculated with S. Enteritidis at 4 and 25 °C, and results showed that it could effectively eradicate S. Enteritidis in just 30min and prevented its growth up to 72h. Our findings indicate that E19 can be an alternative to a preservative to control Salmonella in food samples and help prevent and treat salmonellosis.

Physiochemical characterization of a potential Klebsiella phage MKP-1 and analysis of its application in reducing biofilm formation.

The common intestinal pathogen Klebsiella pneumoniae (K. pneumoniae) is one of the leading causes of fatal superbug infections that can resist the effects of commonly prescribed medicines. The uncontrolled use or misuse of antibiotics has increased the prevalence of drug-resistant K. pneumoniae strains in the environment. In the quest to search for alternative therapeutics for treating these drug-resistant infections, bacteriophages (bacterial viruses) emerged as potential candidates for in phage therapy against Klebsiella. The effective formulation of phage therapy against drug-resistant Klebsiella infections demands thorough characterization and screening of many bacteriophages. To contribute effectively to the formulation of successful phage therapy against superbug infections by K. pneumoniae, this study includes the isolation and characterization of a novel lytic bacteriophage MKP-1 to consider its potential to be used as therapeutics in treating drug-resistant Klebsiella infections. Morphologically, having a capsid attached to a long non-contractile tail, it was found to be a siphovirus that belongs to the class Caudoviricetes and showed infectivity against different strains of the target host bacterium. Comparatively, this double-stranded DNA phage has a large burst size and is quite stable in various physiological conditions. More interestingly, it has the potential to degrade the tough biofilms formed by K. pneumoniae (Klebsiella pneumoniae subsp. pneumoniae (Schroeter) Trevisan [ATCC 15380]) significantly. Thus, the following study would contribute effectively to considering phage MKP-1 as a potential candidate for phage therapy against Klebsiella infection.

Isolation and Characterization of the Lytic Enterococcus faecium Phage φEc-ZZ2

Infections with vancomycin-resistant Enterococcus (VRE) have been increasingly reported, especially in immunocompromised patients. Bacteriophages (phages) are often considered as potential therapeutic candidates in treating infectious diseases. To search for therapeutic phages as antimicrobial agents for the treatment of Enterococcus faecium infections, eight E. faecium phages were isolated from the environments. One of these, phage φEc-ZZ2, which was capable of eliciting efficient lysis of E. faecium, was selected for analysis. Transmission electron microscopy revealed its resemblance to members of the family Siphoviridae, with a tail 200 nm long and an icosahedral head 45 nm in diameter. Phage φEc-ZZ2 exhibited rapid adsorption (> 99% adsorbed in 10 min), a short latency period of 20 min and a large burst size of 180 PFU/cell. Host range determination shows that φEc-ZZ2 has a relatively broader host range (specificity for 54.5% of tested E. faecium strains). Furthermore, when φEc-ZZ2 alone was incubated at different pH, the phage was stable over a wide pH range (5 to 9). These characteristics of φEc-ZZ2 will provide useful information for further research on the interaction between Enterococcus phages and their hosts. Moreover, in consideration of its pH stability and broader host range, φEc-ZZ2 may be a good candidate as a therapeutic/disinfectant agent to control nosocomial infections caused by E. faecium.

Proliferation in malaria parasites: How resource limitation can prevent evolution of greater virulence.

For parasites, robust proliferation within hosts is crucial for establishing the infection and creating opportunities for onward transmission. While faster proliferation enhances transmission rates, it is often assumed to curtail transmission duration by killing the host (virulence), a trade-off constraining parasite evolution. Yet in many diseases, including malaria, the preponderance of infections with mild or absent symptoms suggests that host mortality is not a sufficient constraint, raising the question of what restrains evolution toward faster proliferation. In malaria infections, the maximum rate of proliferation is determined by the burst size, the number of daughter parasites produced per infected red blood cell. Larger burst sizes should expand the pool of infected red blood cells that can be used to produce the specialized transmission forms needed to infect mosquitoes. We use a within-host model parameterized for rodent malaria parasites (Plasmodium chabaudi) to project the transmission consequences of burst size, focusing on initial acute infection where resource limitation and risk of host mortality are greatest. We find that resource limitation restricts evolution toward higher burst sizes below the level predicted by host mortality alone. Our results suggest resource limitation could represent a more general constraint than virulence-transmission trade-offs, preventing evolution towards faster proliferation.

A Novel and Effective Therapeutic Method for Treating Aeromonas schubertii Infection in Channa maculata.

Aeromonas schubertii is a pathogen that severely affects aquatic animals, including the snakehead, Channa maculata. Lytic bacteriophages have been recognized as effective alternatives to antibiotics for controlling bacterial infections. However, there have been no reports of A. schubertii phages as far as we know. In this study, a lytic bacteriophage SD04, which could effectively infect A. schubertii, was isolated from pond water cultured with diseased snakehead. The SD04 phage formed small, round plaques on Petri dishes. Electron microscopy revealed a hexagonal head and a contractile tail. Based on its morphology, it may belong to the Myoviridae family. Two major protein bands with molecular weights of 50 and 38 kilodaltons were observed after the phage was subjected to SDS-PAGE. The phage showed a large average burst size, high specificity, and a broad host range. When stored at 4 °C, phage SD04 had high stability over 12 months and showed almost no variation within the first six months. All fish were healthy after both intraperitoneal injection and immersion administration of SD04, indicating the safety of the phage. After treatment with SD04, Channa maculata in both phage therapy groups and prevention groups showed high survival rates (i.e., 83.3 ± 3.3% and 100 ± 1.3%, respectively). Phage therapy inhibits bacterial growth in the liver, the target organ of the infected Channa maculat. The experimental results indicate the potential use of phage SD04 for preventing A. schubertii infection in Channa maculata.

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 (5min) 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.

Potential application of phage vB_EfKS5 to control Enterococcus faecalis and its biofilm in food

Contaminated food with antibiotic-resistant Enterococcus spp. could be the vehicle for transmitting Enterococcus to humans and accordingly cause a public health problem. The accumulation of biogenic amines produced by Enterococcus faecalis (E. faecalis) in food may have cytological effects. Bacteriophages (phage in short) are natural antimicrobial agents and can be used alone or in combination with other food preservatives to reduce food microbial contaminants. The aim of this study was to isolate a novel phage against E. faecalis and determine its host range to evaluate its potential application. Bacteriophage, vB_EfKS5, with a broad host range, was isolated to control the growth of E. faecalis. The vB_EfKS5 genome is 59,246 bp in length and has a GC content of 39.7%. The computational analysis of phage vB_EfKS5 genome confirmed that it does not contain any lysogenic, toxic, or virulent genes. Phage vB_EfKS5 exhibited lytic activity against most E. faecalis isolates with different multiplicities of infections and it infected 75.5% (22/29) of E. faecalis isolates and 42.3% (3/7) of E. faecium isolates. It was also able to destroy the biofilm formed by E. faecalis with different MOIs. Phage vB_EfKS5 alone or in combination with nisin could control the growth of E. faecalis in broth and milk. Based on its high productivity, stability, short latent period, and large burst size, phage vB_EfKS5 has a high potential for applications both in food and medical applications.

Characterization of a Vibrio parahaemolyticus-targeting lytic bacteriophage SSJ01 and its application in artificial seawater.

Vibrio parahaemolyticus is a major foodborne pathogen causing serious illnesses. In this study, a new lytic bacteriophage SSJ01 that infects V. parahaemolyticus was isolated and characterized. It had a short non-contractile tail and belonged to the Caudoviricetes class. It rapidly adsorbed onto host cells, exhibited a short latent period, and has a large burst size. It showed lytic activities under a broad range of temperature (-18 to 60°C), pH (5 to 11), and salinity (0 to 6%). It contained 35 open reading frames with a G + C content of 49.16% without toxic or lysogen-forming genes. The MOI of 105 phage-treated group in vitro reduced the target cells up to 3.49-log CFU/mL at 6°C and 3.47-log CFU/mL at 25°C, respectively. In aquatic environments (6 and 25°C), bactericidal activities showed a significant decrease within 2h. Therefore, the bacteriophage SSJ01 has potential as a biocontrol agent to control V. parahaemolyticus in marine culture.

Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.