Isolation and characterization of phage ISTP3 for bio-control application against drug-resistant Salmonella.

Salmonella including drug-resistant strains are major foodborne pathogens causing serious illness and pose a great threat to the prevention and control for food safety. Phages can naturally defect the bacterium, is considered as a new and promising biological antimicrobial agent in the post-antibiotic era. A poultry facility in Wuhan, China provided wastewater samples from which a collection of 29 phages were isolated and purified. A broad host spectrum phage ISTP3, which capable of infecting all tested Salmonella, including drug-resistant Salmonella enterica, were examined. Additionally, the effectiveness of this phage ISTP3 in reducing drug-resistant S. enterica was assessed in diverse food samples. Transmission electron microscopy (TEM) and whole genome sequencing demonstrated that ISTP3 was found to belong to family Ackermannviridae. The one-step growth experiment and assays of stability demonstrated that ISTP3 exhibited short periods of inactivity before replicating, produced a significant number of viral progeny during infection, and remained high stable under varying pH and temperature conditions. We evaluated the efficacy of phage ISTP3 against drug-resistant Salmonella on chicken breast and lettuce samples at different temperatures. When applying phage ISTP3 in food matrices, the drug resistant Salmonella count significantly reduced at 4°C and 25°C at an MOI of 100 or 1,000 within a timescale of 12 h. Overall, the results, such as broad host ranges, strictly lytic lifestyles, absence of lysogenic related genes, toxin genes, or virulence genes in the genome, demonstrate that the application of phage ISTP3 as a biocontrol agent has promising potential for preventing and controlling drug-resistant S. typhimurium in the context of food safety, processing, and production.

The power of phages: revolutionizing cancer treatment.

Cancer is a devastating disease with a high global mortality rate and is projected to increase further in the coming years. Current treatment options, such as chemotherapy and radiation therapy, have limitations including side effects, variable effectiveness, high costs, and limited availability. There is a growing need for alternative treatments that can target cancer cells specifically with fewer side effects. Phages, that infect bacteria but not eukaryotic cells, have emerged as promising cancer therapeutics due to their unique properties, including specificity and ease of genetic modification. Engineered phages can transform cancer treatment by targeting cancer cells while sparing healthy ones. Phages exhibit versatility as nanocarriers, capable of delivering therapeutic agents like gene therapy, immunotherapy, and vaccines. Phages are extensively used in vaccine development, with filamentous, tailed, and icosahedral phages explored for different antigen expression possibilities. Engineered filamentous phages bring benefits such as built in adjuvant properties, cost-effectiveness, versatility in multivalent formulations, feasibility of oral administration, and stability. Phage-based vaccines stimulate the innate immune system by engaging pattern recognition receptors on antigen-presenting cells, enhancing phage peptide antigen presentation to B-cells and T-cells. This review presents recent phage therapy advances and challenges in cancer therapy, exploring its versatile tools and vaccine potential.

Combine thermal processing with polyvalent phage LPEK22 to prevent the Escherichia coli and Salmonella enterica contamination in food.

Thermal processing is the most frequently used method to destruct bacteria in food processing. However, insufficient thermal processing may lead to the outbreak of foodborne illness. This study combined thermal processing with thermostable phage to prevent food contamination. The thermostable phages were screened which can retain activity at 70 °C for 1 h. Among them, the polyvalent phage LPEK22 was obtained to lyse Escherichia coli and Salmonella enterica, especially several multi-drug resistant bacteria. In milk (liquid food matrix), LPEK22 significantly reduced the E. coli by 5.00 ± 0.18 log10 CFU/mL and S. enterica by 4.20 ± 0.23 log10 CFU/mL after thermal processing at 63 °C for 30 min. For beef sausage (solid food matrix), LPEK22 significantly reduced the E. coli by 2.34 ± 0.17 log10 CFU/cm2 and S. enterica by 1.54 ± 0.13 log10 CFU/cm2 after thermal processing at 66 °C for 90 s. Genome analysis revealed that LPEK22 was a novel phage with a unique tail spike protein belonging to the family of Ackermannviridae. LPEK22 did not contain lysogenic, drug-resistant, and virulent genes that may compromise the safety of food application. These results determined that LPEK22, a novel polyvalent Ackermannviridae phage, could combine with thermal processing to prevent drug-resistant E. coli and S. enterica both in vitro and in foods.

Isolation, Identification and Genetic Characterization of Antibiotic Resistant Escherichia coli from Frozen Chicken Meat Obtained from Supermarkets at Dhaka City in Bangladesh.

Antimicrobials have been used to improve animal welfare, food security, and food safety that promote the emergence, selection, and dissemination of antimicrobial-resistant (AMR) bacteria. In this study, 50 E. coli were isolated from frozen chicken meat samples in Dhaka city. Antibiotic sensitivity patterns were assessed through the disk diffusion method and finally screened for the presence of antimicrobial resistance genes (ARG) using the polymerase chain reaction (PCR). Among the 160 samples, the prevalence of E. coli was observed in fifty samples (31.25%). All of these isolates were found resistant to at least one antimicrobial agent, and 52.0% of the isolates were resistant against 4-7 different antimicrobials. High resistance was shown to tetracycline (66.0%), followed by resistance to erythromycin (42.0%), ampicillin and streptomycin (38.0%), and sulfonamide (28.0%). In addition, the most prevalent ARGs were tet(A) (66.0%), ereA (64.0%), tet(B) (60.0%), aadA1 and sulI (56.0%), blaCITM (48.0%) and blaSHV (40.0%). About 90.0% of isolates were multidrug resistant. This study reveals for the first time the current situation of E. coli AMR in broilers, which is helpful for the clinical control of disease as well as for the development of policies and guidelines to reduce AMR in broilers production in Bangladesh.

Application of a novel phage vB_SalS-LPSTLL for the biological control of Salmonella in foods.

Salmonella is one of the most common foodborne pathogens around the world. Phages are envisioned as a new strategy to control foodborne pathogenic bacteria and food safety. A Salmonella specific lytic phage vB_SalS-LPSTLL (LPSTLL) was selected for food applications on the basis of lytic range, lytic efficiency, functional stability and characteristics. Phage LPSTLL was able to lyse 11 Salmonella serotypes, which represents the broadest range reported Salmonella phages, and was able to suppress the growth of Salmonella enterica in liquid culture over nine hours. LPSTLL exhibited rapid reproductive activity with a short latent period and a large burst size in one-step growth experiment. LPSTLL remained active over a pH range of 3.0 to 12.0, and at incubation temperatures up to 60 °C for 60 min, indicating wide applicability for food processing and storage. Significant reductions of viable Salmonella were observed in diverse foods (milk, apple juice, chicken and lettuce) with reductions up to 2.8 log CFU/mL recorded for milk. Sensory evaluation indicated that treatment with phage LPSTLL did not alter the visual or tactile quality of food matrices. Genome analysis of LPSTLL indicated the absence of any virulence or antimicrobial resistance genes. Genomic comparisons suggest phage LPSTLL constitutes a novel member of a new genus, the LPSTLLvirus with the potential for Salmonella biocontrol in the food industry.

Construction and Characterization of an Aeromonas hydrophila Multi-Gene Deletion Strain and Evaluation of Its Potential as a Live-Attenuated Vaccine in Grass Carp.

Aeromonas hydrophila is an important pathogen that causes motile Aeromonas septicemia (MAS) in the aquaculture industry. Aerolysin, hemolysin, serine protease and enterotoxins are considered to be the major virulence factors of A. hydrophila. In this study, we constructed a five-gene (aerA, hly, ahp, alt and ast) deletion mutant strain (named Aeromonas hydrophila five-gene deletion strain, AHFGDS) to observe the biological characteristics and detect its potential as a live-attenuated vaccine candidate. AHFGDS displayed highly attenuated and showed increased susceptibility to fish blood and skin mucus killing, while the wild-type strain ZYAH72 was highly virulent. In zebrafish (Danio rerio), AHFGDS showed a 240-fold higher 50% lethal dose (LD50) than that of the wild-type strain. Immunization with AHFGDS by intracelomic injection or immersion routes both provided grass carp (Ctenopharyngodon idella) significant protection against the challenge of the strain ZYAH72 or J-1 and protected the fish organs from serious injury. Further agglutinating antibody titer test supported that AHFGDS could elicit a host-adaptive immune response. These results suggested the potential of AHFGDS to serve as a live-attenuated vaccine to control A. hydrophila infection in aquaculture.

Characterization of Salmonella Phage LPST153 That Effectively Targets Most Prevalent Salmonella Serovars.

Foodborne diseases represent a major risk to public health worldwide. In this study, LPST153, a novel Salmonella lytic phage with halo (indicative of potential depolymerase activity) was isolated by employing Salmonella enterica serovar Typhimurium ATCC 13311 as the host and had excellent lytic potential against Salmonella. LPST153 is effectively able to lyse most prevalent tested serotypes of Salmonella, including S. Typhimurium, S. Enteritidis, S. Pullorum and S. Gallinarum. Morphological analysis revealed that phage LPST153 belongs to Podoviridae family and Caudovirales order and could completely prevent host bacterial growth within 9 h at multiplicity of infection (MOI) of 0.1, 1, 10 and 100. LPST153 had a latent period of 10 min and a burst size of 113 ± 8 PFU/cell. Characterization of the phage LPST153 revealed that it would be active and stable in some harsh environments or in different conditions of food processing and storage. After genome sequencing and phylogenetic analysis, it is confirmed that LPST153 is a new member of the Teseptimavirus genus of Autographivirinae subfamily. Further application experiments showed that this phage has potential in controlling Salmonella in milk and sausage. LPST153 was also able to inhibit the formation of biofilms and it had the ability to reduce and kill bacteria from inside, including existing biofilms. Therefore, the phage LPST153 could be used as a potential antibacterial agent for Salmonella control in the food industry.

Application of a Broad Range Lytic Phage LPST94 for Biological Control of Salmonella in Foods.

Salmonella, one of the most common food-borne pathogens, is a significant public health and economic burden worldwide. Lytic phages are viable alternatives to conventional technologies for pathogen biocontrol in food products. In this study, 40 Salmonella phages were isolated from environmentally sourced water samples. We characterized the lytic range against Salmonella and among all isolates, phage LPST94 showed the broadest lytic spectrum and the highest lytic activity. Electron microscopy and genome sequencing indicated that LPST94 belongs to the Ackermannviridae family. Further studies showed this phage is robust, tolerating a wide range of pH (4-12) and temperature (30-60 °C) over 60 min. The efficacy of phage LPST94 as a biological control agent was evaluated in various food products (milk, apple juice, chicken breast, and lettuce) inoculated with non-typhoidal Salmonella species at different temperatures. Interestingly, the anti-Salmonella efficacy of phage LPST94 was greater at 4 °C than 25 °C, although the efficacy varied between different food models. Adding phage LPST94 to Salmonella inoculated milk decreased the Salmonella count by 3 log10 CFU/mL at 4 °C and 0.84 to 2.56 log10 CFU/mL at 25 °C using an MOI of 1000 and 10000, respectively. In apple juice, chicken breast, and lettuce, the Salmonella count was decreased by 3 log10 CFU/mL at both 4 °C and 25 °C after applying phage LPST94 at an MOI of 1000 and 10,000, within a timescale of 48 h. The findings demonstrated that phage LPST94 is a promising candidate for biological control agents against pathogenic Salmonella and has the potential to be applied across different food matrices.

Application of a Phage Cocktail for Control of Salmonella in Foods and Reducing Biofilms.

Salmonella contamination in foods and their formation of biofilms in food processing facility are the primary bacterial cause of a significant number of foodborne outbreaks and infections. Broad lytic phages are promising alternatives to conventional technologies for pathogen biocontrol in food matrices and reducing biofilms. In this study, 42 Salmonella phages were isolated from environmentally-sourced water samples. We characterized the host range and lytic capacity of phages LPSTLL, LPST94 and LPST153 against Salmonella spp., and all showed a wide host range and broad lytic activity. Electron microscopy analysis indicated that LPSTLL, LPST94, and LPST153 belonged to the family of Siphoviridae, Ackermannviridae and Podoviridae, respectively. We established a phage cocktail containing three phages (LPSTLL, LPST94 and LPST153) that had broad spectrum to lyse diverse Salmonella serovars. A significant decrease was observed in Salmonella with a viable count of 3 log10 CFU in milk and chicken breast at either 25 °C or 4 °C. It was found that treatment with phage cocktail was able to significantly reduced biofilm on a 96-well microplate (44-63%) and on a stainless steel surface (5.23 to 6.42 log10). These findings demonstrated that the phage cocktail described in this study can be potentially used as a biological control agent against Salmonella in food products and also has the effect to reduce Salmonella formed biofilms.

The two-component regulatory system CpxA/R is required for the pathogenesis of Aeromonas hydrophila.

Aeromonas hydrophila is a causative pathogen of many infectious diseases in fish and human. The two-component regulatory system enables bacteria to respond to a wide range of stimuli, growth conditions and environments. The two-component system CpxA/R is prevalent in microorganisms and involved in the pathogenesis of a number of pathogens. In this study, we generated a ΔcpxA/R mutant of A. hydrophila ZYAH72 to explore the biological functions mediated by the CpxA/R in this organism. Compared with the wild type strain, the knockout mutant strain ΔcpxA/R exhibited a significant impairment in adherence to human epithelial cells Caco-2 and resistance against host killing in fish blood. However, the mutant strain and the wild type strain showed no difference in the cytotoxicity assay, which revealed that the absence of CpxA/R did not influence the cytopathic effect of this pathogen in vitro. Furthermore, the virulence of ΔcpxA/R was attenuated in zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idella) infections, with reduced mortality or delayed death time. These findings suggest that CpxA/R is required for the virulence of A. hydrophila.