Lysis Ability Research Articles

Antioxidant, membrane stabilizing and thrombolytic potentials of the leaves of erythrina fusca lour

Erythrina fusca (Family: Fabaceae) is famous for many traditional healing. In the present study, the leaves of E. fusca were collected, dried, powdered and then extracted with methanol. It was further partitioned into n-hexane, dichloromethane and aqueous soluble fractions. All the extracts were subjected to antioxidant, membrane stabilizing and thrombolytic assays. In case of free radical scavenging assay, the aqueous soluble fraction showed highest scavenging activity. In ferric reducing anti-oxidant power assay, the dicholoromethane extract showed highest reduction of ferric ion in a dose dependent manner. In total antioxidant assay, the methanol extract and dichloromethane extract showed stronger capacity as antioxidant. During the determination of total phenolic and flavonoid contents, the methanol extract and dichloromethane soluble fraction showed higher contents of phenolics and flavonoids, respectively. The erythrocyte membrane stabilizing assay revealed the capacities of methanol extract and its aqueous fraction to stabilize the RBC membrane in both hypotonic solution- and heat- induced hemolytic conditions. However, the clot lysis ability of the E. fusca leaf extracts was not so prominent. Bangladesh J. Bot. 53(3): 439-445, 2024 (September)

Development and application of a bacteriophage cocktail for Shigella flexneri biofilm inhibition on the stainless steel surface

Food contamination and biofilm formation by Shigella in food processing facilities are major causes of acute gastrointestinal infection and mortality in humans. Bacteriophages (phages) are promising alternatives to antibiotics in controlling plankton and biofilms in food matrices. This study isolated two novel phages, S2_01 and S2_02, with lytic activity against various Shigella spp. From sewage samples. Transmission electron microscopy revealed that phages S2_01 and S2_02 belonged to the Caudovirales order. On characterizing their lytic ability, phage S2_01 initially exhibited relatively weak antibacterial activity, while phage S2_02 initially displayed rapid antibacterial activity after phage application. A combination of these phages in a 1:9 ratio was selected, as it has been suggested to elicit the most rapid and sustained lysis ability for up to 24 h. It demonstrated lytic activity against various foodborne pathogens, including six Shigella spp. The phage cocktail exhibited biofilm inhibition and disruption abilities of approximately 79.29% and 42.55%, respectively, after 24 h in a 96-well microplate. In addition, inhibition (up to 23.42%) and disruption (up to 19.89%) abilities were also observed on stainless steel surfaces, and plankton growth was also significantly suppressed. Therefore, the phage cocktail formulated in this study displays great potential as a biological control agent in improving food safety against biofilms and plankton.

Characterization of a novel lytic bacteriophage VPMCC14 which efficiently controls Vibrio harveyi in Penaeus monodon culture.

Vibrio harveyi causes luminous vibriosis diseases in shrimp, which lead to shrimp mortalities. Considering the emergence of antibiotic-resistant bacteria, a Vibrio-infecting bacteriophage, VPMCC14, was characterized, and its lysis ability was evaluated on a laboratory scale. VPMCC14 was shown to infect V. harveyi S5A and V. harveyi ATCC 14126. VPMCC14 also exhibited a latent period of 30 min, with a burst size of 38 PFU/cell on its propagation strain. The bacteriophage was stable at a wide range of pHs (3-9), temperatures (0-45°C), and salinities (up to 40 ppt). VPMCC14 exhibited strict virulence properties as the bacteriophage entirely lysed V. harveyi S5A in liquid culture inhibition after 5 h and 4 h at very low MOIs such as MOI 0.1 and MOI 1, respectively. VPMCC14 could control V. harveyi infection in aquariums at MOI 1 and decrease the mortality of Penaeus monodon challenged by V. harveyi. VPMCC14 genome was 134,472 bp long with a 34.5 G+C% content, and 240 open reading frames. A unique characteristic of VPMCC14 was the presence of the HicB family antitoxin-coding open reading frame. Comparative genomic analyses suggested that VPMCC14 could be a representative of a new genus in the Caudoviricetes class. This novel bacteriophage, VPMCC14, could be applied as a biocontrol agent for controlling V. harveyi infection.

Developing “Off-the-Shelf” CLL1 CAR-DNT Therapeutics for the R/R Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is one of the most common and highly heterogeneous hematological malignancy. The prognosis is poor especially for the elderly patients. For Relapsed/Refractory(R/R) AML patients, the one-year overall survival rate is merely 20%. CAR-T cells have shown tremendous therapeutic benefits for ALL, DLBCL and MM. One of the limitations of current CAR-T therapies is the use of autologous T cells. This can be limiting due to the cost, complicated manufacturing process, rapid disease progression, and manufacturing failures etc. Therefore, there is a need to develop the ready-to-use allogeneic cell product. Double negative T (DNT) cells are the ideal candidate for AML cell therapy as they naturally kill AML cells, and have been successfully administered to AML patients with therapeutic benefit without causing GvHD. Relying on their own natural immune activated receptors, DNT cells could lyse a wide range of tumors independent of TAA expression, which have more advantages compared to the conventional CAR-T cells. In our study, we developed the allogeneic product of CAR-DNT cells targeting C-type lectin-like molecule-1 (CLL1), which is a promising target due to its over-expression in primary AML cells and leukemia stem cells, while absence on hematopoietic stem/progenitor cells. Methods: We developed a humanized single chain variable fragment (scFv) with specific recognition of human CLL1 antigen by screening from 62 murine-derived CLL1 antibodies. Then, we designed a second-generation CLL1 CAR structure containing 4-1BB-CD3ζ and transduced it into the DNT cells by lentivirus. To clarify the GvH and HvG reactions of DNT cells, we performed the mixed lymphocyte reaction experiments in vitro and compared DNT cells with TCR and B2M double knockout T(DKO) cells and TCR knockout T(TKO) cells. We evaluated the anti-tumor function of CLL1 CAR-DNT cells through killing experiments by different effector-to-target ratios and multiple stimulations with AML cell lines (THP1 and HL60). Furthermore, we speculated that the CAR structure plays a critical role in both tumoricidal function and persistence of CAR-DNT cells. We designed nearly 20 enhanced CAR structures based on the signaling pathway of DNT cells. These newly designed structures have been tested in vitro and will be confirmed in vivo. Results: The humanized anti-CLL1 scFv candidate H1-3Q2 demonstrated high specificity toward human CLL1 antigen at the Membrane Protein Array. H1-3Q2 based CAR-T cells showed good tumor suppression in vivo. However, due to the heterogenous expression of CLL1 in AML, CLL1 CAR-T cells may not be able to eliminate AML cancer cells completely. While DNT cells could naturally lyse tumor cells through the receptors like NKG2D and DNAM-1, providing them with lots of advantages. Moreover, DNT cells have the potential to be used as allogenic cell therapy product. Co-culturing allogenic PBMCs or umbilical cord blood with DNT cells for up to 72 hours resulted in little or no killing reaction, indicated a low risk of GvHD. Conversely, the PBMCs from three different donors were used to attack allogenic DNT cells or DKO/TKO T cells in vitro. The killing effect on DKO/TKO T cells gradually increased over time, while no effect on DNT cells was observed even after 192 hours. This suggested that DNT cells are not easily cleared by host immune cells. DNT cells could efficiently lyse various hematological and solid tumor cell lines, particularly showing high sensitivity towards AML cells. The introduction of the CAR moiety further enhanced the specificity of DNT cells. The in vitro killing results demonstrated that CLL1 CAR-DNT cells exhibited stronger and faster AML resistance compared with CAR-T cells, and even eliminated tumor cells that have lost TAA. However, CLL1 CAR-DNT cells showed limited proliferation in the multiple-stimulation assays. To address this, we designed enhanced CAR structures. Screening by multiple-challenge assays, the fifth-generation CAR exhibited outstanding proliferation and superior tumor lysis ability in vitro. Conclusions: Our findings demonstrate that CAR-DNT product is an ideal candidate for allogeneic adoptive immunotherapy and exhibits superior tumoricidal function. This study underscores the potential of the “off-the-shelf” CLL1 CAR-DNT cell product for treating R/R AML.

Oral phage therapy with microencapsulated phage A221 against Escherichia coli infections in weaned piglets

BackgroundEscherichia coli (E. coli) is a common pathogen that often causes diarrhea in piglets. Since bacteria are becoming more and more resistant to antibiotics, phages have become a promising alternative therapy. However, the therapy of oral phage often fails to achieve the desired effect. A novel phage named A221 was isolated by using E. coli GXXW-1103 as host strain, characterized by electron microscopy, genomic sequencing and analyzed by measuring lysis ability in vitro.ResultsPhage A221 was identified as a member of Ackermannviridae, Aglimvirinae, Agtrevirus with 153297 bp genome and effectively inhibited bacterial growth in vitro for 16 h. This study was conducted to evaluate the therapeutic effect of oral microencapsulated phage A221 on E. coli GXXW-1103 infections in weaned piglets. The protective effect of phage was evaluated by body weight analysis, bacterial load and histopathological changes. The results showed that with the treatment of phage A221, the body weight of piglets increased, the percentage of Enterobacteriaceae in duodenum decreased to 0.64%, the lesions in cecum and duodenum were alleviated, and the bacterial load in the jejunal lymph nodes, cecum and spleen were also significantly different with infected group (P < 0.001).ConclusionsThe results showed that phage A221 significantly increased the daily weight gain of piglets, reduced the bacterial load of tissues and the intestinal lesions, achieved the same therapeutic effect as antibiotic Florfenicol. Taken together, oral microencapsulated phage A221 has a good therapeutic effect on bacterial diarrhea of weaned piglets, which provides guidance for the clinical application of phage therapy in the future.

Effects of Hypobaric Hypoxia on Coagulation in Healthy Subjects Exposed to 3,500 m Altitude.

Kammerer, Tobias, Anna Walzl, Thomas Müller, Philipp Groene, Giulia Roveri, Rachel Turner, Johanna Roche, Hannes Gatterer, Christoph Siebenmann, and Simon T. Schäfer. Effects of hypobaric hypoxia on coagulation in healthy subjects exposed to 3,500 m altitude. High Alt Med Biol. 24:94-103, 2023. Background: Hypoxia is discussed as a trigger for prothrombotic changes both in intensive care and high altitude medicine. This research study aimed to evaluate the effect of isolated hypobaric hypoxia (HH) on coagulation in females in a highly standardized setting. Methods: Twelve healthy female subjects were studied under HH (equivalent to 3,500 m) and normoxia (NX) during two 4-day sojourns, in a strictly controlled crossover design. Nutrition, fluid intake, hormonal status (i.e., menstrual cycle variation), and physical stress were standardized. Functional coagulation and blood lysis were measured by viscoelastometry and compared between HH and NX. In addition, plasma-based coagulation tests (PBCTs), namely prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII coagulation activity (FVIII:C), von Willebrand factor antigen (vWF:Ag), and von Willebrand factor ristocetin cofactor activity (vWF:RCo) were measured. Results: Neither for Viscoelastic Haemostatic Assays nor for PBCTs significant changes were found for HH compared with NX (all p > 0.05). Specifically, the lysis ability, as well as clotting time, clot formation, clot amplitude, and maximum clot firmness unchanged were similar between HH and NX. This also applied to all other variables. Conclusion: We demonstrate that moderate HH per se has no influence on blood coagulation in healthy females.

Sex-specific innate immunity and ageing in long-lived fresh water turtles (Kinosternon flavescens: Kinosternidae)

BackgroundThe progressive deregulation of the immune system with age, termed immunosenescence, has been well studied in mammalian systems, but studies of immune function in long-lived, wild, non-mammalian populations are scarce. In this study we leverage a 38-year mark-recapture study to quantify the relationships among age, sex, survival, reproductive output and the innate immune system in a long-lived reptile, yellow mud turtles (Kinosternon flavescens; Testudines; Kinosternidae).MethodsWe estimated rates of survival and age-specific mortality by sex based on mark-recapture data for 1530 adult females and 860 adult males over 38 years of captures. We analyzed bactericidal competence (BC), and two immune responses to foreign red blood cells - natural antibody-mediated haemagglutination (NAbs), and complement-mediated haemolysis ability (Lys) - in 200 adults (102 females; 98 males) that ranged from 7 to 58 years of age captured in May 2018 during their emergence from brumation, and for which reproductive output and long-term mark-recapture data were available.ResultsWe found that females are smaller and live longer than males in this population, but the rate of accelerating mortality across adulthood is the same for both sexes. In contrast, males exhibited higher innate immunity than females for all three immune variables we measured. All immune responses also varied inversely with age, indicating immunosenescence. For females that reproduced in the preceding reproductive season, egg mass (and therefore total clutch mass) increased with age,. In addition to immunosenescence of bactericidal competence, females that produced smaller clutches also had lower bactericidal competence.ConclusionsContrary to the general vertebrate pattern of lower immune responses in males than females (possibly reflecting the suppressive effects of androgens), we found higher levels of all three immune variables in males. In addition, contrary to previous work that found no evidence of immunosenescence in painted turtles or red-eared slider turtles, we found a decrease in bactericidal competence, lysis ability, and natural antibodies with age in yellow mud turtles.

Salmonella Phage CKT1 Effectively Controls the Vertical Transmission of Salmonella Pullorum in Adult Broiler Breeders

Phage therapy is widely being reconsidered as an alternative to antibiotics for the treatment of multidrug-resistant bacterial infections, including salmonellosis caused by Salmonella. As facultative intracellular parasites, Salmonella could spread by vertical transmission and pose a great threat to both human and animal health; however, whether phage treatment might provide an optional strategy for controlling bacterial vertical infection remains unknown. Herein, we explored the effect of phage therapy on controlling the vertical transmission of Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum), a poultry pathogen that causes economic losses worldwide due to high mortality and morbidity. A Salmonella phage CKT1 with lysis ability against several S. enterica serovars was isolated and showed that it could inhibit the proliferation of S. Pullorum in vitro efficiently. We then evaluated the effect of phage CKT1 on controlling the vertical transmission of S. Pullorum in an adult broiler breeder model. The results demonstrated that phage CKT1 significantly alleviated hepatic injury and decreased bacterial load in the liver, spleen, heart, ovary, and oviduct of hens, implying that phage CKT1 played an active role in the elimination of Salmonella colonization in adult chickens. Additionally, phage CKT1 enabled a reduction in the Salmonella-specific IgG level in the serum of infected chickens. More importantly, the decrease in the S. Pullorum load on eggshells and in liquid whole eggs revealed that phage CKT1 effectively controlled the vertical transmission of S. Pullorum from hens to laid eggs, indicating the potential ability of phages to control bacterial vertical transmission.

A Highly Effective Bacteriophage-1252 to Control Multiple Serovars of Salmonella enterica.

Salmonella enterica (S. enterica) is the most common foodborne pathogen worldwide, leading to massive economic loss and a significant burden on the healthcare system. The primary source of S. enterica remains contaminated or undercooked poultry products. Considering the number of foodborne illnesses with multiple antibiotic resistant S. enterica, new controlling approaches are necessary. Bacteriophage (phage) therapies have emerged as a promising alternative to controlling bacterial pathogens. However, the limitation on the lysis ability of most phages is their species-specificity to the bacterium. S. enterica has various serovars, and several major serovars are involved in gastrointestinal diseases in the USA. In this study, Salmonella bacteriophage-1252 (phage-1252) was isolated and found to have the highest lytic activity against multiple serovars of S. enterica, including Typhimurium, Enteritidis, Newport, Heidelberg, Kentucky, and Gallinarum. Whole-genome sequencing analysis revealed phage-1252 is a novel phage strain that belongs to the genus Duplodnaviria in the Myoviridae family, and consists of a 244,421 bp dsDNA, with a G + C content of 48.51%. Its plaque diameters are approximately 2.5 mm to 0.5 mm on the agar plate. It inhibited Salmonella Enteritidis growth after 6 h. The growth curve showed that the latent and rise periods were approximately 40 min and 30 min, respectively. The burst size was estimated to be 56 PFU/cell. It can stabilize and maintain original activity between 4 °C and 55 °C for 1 h. These results indicate that phage-1252 is a promising candidate for controlling multiple S. enterica serovars in food production.

Optimization of Crude Protease Production from Bacillus thuringiensis HSFI-12 and Thrombolytic Activity Its Enzyme Dialysate

Cardiovascular disease (CVD) is among the leading causes of death in the world caused by thrombosis. Thrombosis is the formation of excessive blood clots on the walls of blood vessels called thrombus. This could lead to fatal blockage of the heart muscle or brain. Thrombosis can be treated using enzyme-type drugs such as thrombolytic proteases. There have been many studies of bacteria producing thrombolytic enzymes isolated from fermented foodstuffs. However, commercial production of bacterial enzymes to fulfill the need of thrombolytic agents is still limited, causing high price of CVD therapy. Bacillus sp. HSFI-12 (Holothuria scabra Fermented Intestine-12) isolated from the fermented intestine of sea cucumber Holothuria scabra had been reported to have a competitive thrombolytic activity to the commercial Nattokinase. This study aimed to determine bacterial optimum incubation time based on enzyme activity after bacterium molecular identification was done. It also aimed to obtain the dialysate of bacterial crude protease and then determine the thrombolytic activity of the obtained dialysate. Thrombolytic activity was tested on 4 different types of blood (A, B, AB and O). Results of molecular identification showed that strain HSFI-12 shared similarity level of 99.80 %, with Bacillus thuringiensis. Activity of crude protease from the incubated cultures peaked at 48 h of incubation with activity of 191.5 U/mL The activity of concentrated protease after precipitation and dialysis process (dialysate) was 2-times higher by 355,7 U/mL. The percentage of lysis of blood clots produced by crude blood groups A, B, AB and O showed a range of values ​​of 66.423 - 67.656 % while those that produce dialysate are 77.564 - 78.861 %. As conclusion, the best (optimized) incubation time to produce crude enzyme from B. thuringiensis HSFI-12 with the highest activity was 48 h. The process of concentrating the crude thrombolytic protease of B. thuringiensis HSFI-12 increases both activity and thrombolytic ability of the bacterial enzyme. HIGHLIGHTS The most optimum incubation time for protease production from Bacillus thuringiensis is 48 h Partial purification on bacterial crude protease through resulted in 86 % increased activity Partial purification on bacterial crude protease resulted in 17 % increased blood clot lysis ability Clot lysis ability of both crude and dialysate proteases are higher than that of Nattokinase (NK) GRAPHICAL ABSTRACT

Oral Administration of a Phage Cocktail to Reduce Salmonella Colonization in Broiler Gastrointestinal Tract-A Pilot Study.

Salmonella contamination in poultry meat products can lead to serious foodborne illness and economic loss from product recalls. It is crucial to control Salmonella contamination in poultry from farm to fork. Bacteriophages (phages) are viruses of bacteria that offer several advantages, especially their specificity to target bacteria. In our study, three Salmonella phages (vB_SenS_KP001, vB_SenS_KP005, and vB_SenS_WP110) recovered from a broiler farm and wastewater treatment stations showed high lysis ability ranging from 85.7 to 96.4% on over 56 serovars of Salmonella derived from several sources, including livestock and a broiler farm environment. A three-phage cocktail reduced S. Enteritidis and S. Typhimurium, in vitro by 3.9 ± 0.0 and 3.9 ± 0.2 log units at a multiplicity of infection (MOI) of 103 and 3.8 ± 0.4 and 4.1 ± 0.2 log units at MOI of 104 after 6 h post-phage treatment. A developed phage cocktail did not cause phage resistance in Salmonella during phage treatments for three passages. Phages could survive under simulated chicken gastrointestinal conditions in the presence of gastric acid for 2 h (100.0 ± 0.0% survivability), bile salt for 1 h (98.1 ± 1.0% survivability), and intestinal fluid for 4 h (100 ± 0.0% survivability). Each phage was in the phage cocktail at a concentration of up to 9.0 log PFU/mL. These did not cause any cytotoxicity to human fibroblast cells or Caco-2 cells as indicated by the percent of cell viability, which remained nearly 100% as compared with the control during 72 h of co-culture. The phage cocktail was given to broilers raised in commercial conditions at a 9 log PFU/dose for five doses, while naturally occurring Salmonella cells colonized in the gastrointestinal tract of broilers were significantly reduced as suggested by a considerably lower Salmonella prevalence from over 70 to 0% prevalence after four days of phage treatment. Our findings suggest that a phage cocktail is an effective biocontrol agent to reduce Salmonella present in the guts of broilers, which can be applied to improve food safety in broiler production.

Isolation and characterization of a Vibrio owensii phage phi50-12

Vibrio owensii is a widely distributed marine vibrio species that causes acute hepatopancreatic necrosis in the larvae of Panulirus ornatus and Penaeus vannamei, and is also associated with Montipora white syndrome in corals. We characterized V. owensii GRA50-12 as a potent pathogen using phenotypic, biochemical, and zebrafish models. A virulent phage, vB_VowP_phi50-12 (phi50-12), belonging to the N4-like Podoviridae, was isolated from the same habitat as that of V. owensii GRA50-12 and characterized. This phage possesses a unique sequence with no similar hits in the public databases and has a short latent time (30 min), a large burst size (106 PFU/infected cell), and a wide range of pH and temperature stabilities. Moreover, phi50-12 also demonstrated a strong lysis ability against V. owensii GRA50-12. SDS-PAGE revealed at least nine structural proteins, four of which were confirmed using LC–MS/MS analysis. The size of the phi50-12 genome was 68,059 bp, with 38.5% G + C content. A total of 101 ORFs were annotated, with 17 ORFs having closely related counterparts in the N4-like vibrio phage. Genomic sequencing confirmed the absence of antibiotic resistance genes or virulence factors. Comparative studies have shown that phi50-12 has a unique genomic arrangement, except for the well-conserved core regions of the N4-like phages. Phylogenetic analysis demonstrated that it belonged to a group of smaller genomes of N4-like vibrio phages. The therapeutic effect in the zebrafish model suggests that phi50-12 could be a potential candidate for application in the treatment of V. owensii infection or as a biocontrol agent. However, further research must be carried out to confirm the efficacy of phage50-12.

Evaluation of the Thrombolytic and Antioxidant Activity of Leaf Extracts of Plumbago zeylanica L.

Abstract: Background: Plumbago zeylanica L. is one of the extremely accessible conventionally used herbal plants with various biological activities. However, actions of P. zeylanica L on blood clotting and other complications of blood were indisposed therapeutically studied. Therefore, the scope of the current exploration is to screen the thrombolytic, antioxidant, and cytotoxic effects of leaf extracts. Materials and Methods: Thrombolytic activity (in vitro) was assessed with clot lysis and thrombin inhibitory ability. Further, thrombolytic activity (in vivo) was evaluated by a thrombotic tail (carrageenan-induced) animal model. DPPH and nitric oxide (free radical) scavenging methods were employed to check the in vitro antioxidant property. Further cytotoxicity and acute oral toxicity were assessed for plant extract. Results: The quantitative analysis elicits the presence of the magnificent amount of the total phenolic content (96.8±7.92 mg GAE/g) and total flavonoid content (63.52±4.54 mg QE/g) on the dry weight basis. The maximum clot lysis (96.83%±0.657) of methanolic leaf extract was detected in in vitro model at 800 μg/mL in 72 hr. A strong thrombin inhibition (94.63±2.12%) effect was observed for methanol leaf extract at 2 mg/mL. In in vivo studies a significant (p< 0.001) clot lysis was achieved at the tested dose (100, 200 and 300 mg/kg). DPPH radical and nitric oxide scavenging activity showed the IC50 value of 25.47±0.51 and 56.32±0.85, respectively. The methanolic extract was found safer up to the highest lethal dose of 2000 mg/kg. Conclusion: These findings suggested that the plant leaves are comprised of significant thrombolytic properties. It could be a promising source for the existence of antioxidant and thrombolytic agents. Keywords: Plumbago zeylanica L., Thrombolytic activity, Anti-oxidant activity, Cytotoxicity, Streptokinase, DPPH radical etc.

Broad lytic spectrum of novel Salmonella phages on ciprofloxacin-resistant Salmonella contaminated in the broiler production chain.

Background and Aim:Ciprofloxacin (CIP) is recommended for salmonellosis treatment as the drug of choice; however, overuse of this drug can cause drug resistance issues and failure to treat diseases. Phage therapy is an alternative approach for combatting CIP-resistant infection. This study aimed to estimate the prevalence of CIP-resistant Salmonella isolated from the broiler production chain and evaluated the lytic ability of novel Salmonella phages isolated from water samples.Materials and Methods:Samples were obtained from the broiler production chain and used for Salmonella isolation. serovar and CIP resistance of each isolate were characterized through latex agglutination and agar disk diffusion test, respectively. Water samples from different sources were acquired for phage isolation. The lytic activity of novel-isolated phages was also examined.Results:In this study, 51 Salmonella isolates were recovered from the broiler production chain (two commercial farms, one free-range farm, two slaughterhouses, and three stalls from the wet market). Kentucky was the major serovar characterized (16), followed by Typhimurium (9), Agona (5), Corvalis (5), Schwarzengrund (5), Singapore (3), Weltevreden (3), Mbandaka (2), Give (2), and Albany (1). The serovars that exhibited CIP resistance were 14/16 isolates of serovar Kentucky (87.5%) and one isolate of serovar Give (50%), whereas eight other serovars were susceptible to this drug. Overall, the prevalence of CIP-resistant Salmonella recovered from the sources included in this study was 29.4%. This study identified 11 Salmonella phages isolated from wastewater samples derived from broiler farms, wastewater treatment stations, and natural reservoirs. Our phages showed the total percentage of lysis ability ranging from 33.3% to 93.3% against CIP-resistant isolates. However, only one bacterial isolate, namely 210SL, recovered from the food contact surface of a wet market stall and was resistant to all phages.Conclusion:Diverse serovars of Salmonella were recovered in the broiler production chain in this study, while the isolates presenting CIP-resistant Salmonella were as high as 29.4%. Overall, Salmonella phages showed high lysis ability against these CIP-resistant Salmonella isolates, suggesting the potential application of phage-based treatments or biocontrol in the broiler production chain.

Phage amplification-based technologies for simultaneous quantification of viable Salmonella in foodstuff and rapid antibiotic susceptibility testing

Salmonella, especially drug-resistant Salmonella poses a serious threat to food safety and human health. Herein, we proposed a rapid, accurate and sensitive phage amplification-based analysis (PAA) based on an isolated Salmonella phage T156 with broad host range and potent lysis ability for the quantification of viable Salmonella, as well as rapid antibiotic susceptibility testing. This assay has been successful applied to quantify viable Salmonella in food matrices such as milk and lettuce, with a detection limit of 1 colony forming units (CFU) /mL and high specificity for only detect live bacteria not dead bacteria. When combined with real-time PCR (qPCR), PAA-qPCR further reduced the detection time from 6.5 h to 3.5 h, with a detection limit of 10 CFU/mL and without complicate DNA extraction or purification process. Moreover, this assay could also specifically detect drug-resistant Salmonella, shortening the detection time from 24 h to 6.5 h. The results were consistent with that of the conventional paper disc diffusion method (DDM). These PAA methods were evaluated and applied for Salmonella quantification, as well as the detection of drug-resistant Salmonella, providing a promising platform to trace the foodborne pathogen in the complex food matrix, as well as the detection of drug-resistant Salmonella.

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Combined effects of Salmonella phage cocktail and organic acid for controlling Salmonella Enteritidis in chicken meat

Salmonella contaminated in poultry meat remains an important food safety issue as this pathogen leads to a serious foodborne illness worldwide. A number of poultry meat products have often been recalled or rejected due to Salmonella contamination leading to high economic losses each year. In poultry meat processing, various approaches with a strong bacteriostatic effect on Salmonella have been introduced including application of bacteriophages or organic acids. This study aimed to investigate the combined effects of a Salmonella phage cocktail at lower multiplicity of infection (MOI) and organic acid applied to artificially contaminated chicken meat under storage conditions. Newly isolated Salmonella phages (n = 32) showed diverse lysis profiles exhibiting lysis ability ranging from 27.7 to 87.8% on 29 serovars of Salmonella. Two selected phages, vB_SenM_P7 and vB_SenP_P32 presenting the highest lysis ability were used in a phage cocktail formulation. Acetic acid, butyric acid, propionic acid and lactic acid included in this study showed the inhibitory effect on Salmonella from 0.09 to 0.25% for the minimum inhibitory concentration (MIC) and more than 0.5% for the minimum bactericidal concentration (MBC). A phage cocktail showed highest % survival in propionic acid which was combined with the phage treatment. Use of this phage-acid combination not only could completely kill Salmonella in-vitro but could also lower Salmonella number on artificially contaminated chicken meat at 4 °C during a 3-day storage by 1.4 log units while a reduction of 0.4 and 1.2 log units was observed in treatment with a phage cocktail only and propionic acid only, respectively. Overall, findings suggest a combined treatment using phage and organic acid as an effective alternative for controlling Salmonella in the poultry meat industry to improve food safety.

Potential In vitro and In vivo Bioactivities of Schleichera oleosa (Lour.) Oken: A Traditionally Important Medicinal Plant of Bangladesh

People in Bangladeshi village area have long practice to take plant-based products for their basic health care. Schleichera oleosa (Lour.) Oken (Family: Sapindaceae) is an important folk medicine in Bangladesh, India that has been used to cure a wide variety of human ailments. Here, the crude methanol extract of S. oleosa leaf (MESOL) and its various solvent (Hexane, dichloromethane, ethyl acetate, aqueous) fractions were evaluated to determine the level of biological activities by both In vitro and in vivo approaches. The crude methanol extract along with its different solvent fractions was investigated for antioxidant activity by measuring total phenolic content and DPPH radical scavenging assay. Cytotoxic activity was performed by brine shrimp lethality bioassay method. The blood clot lysis ability was screened using aspirin as standard. In vitro anti-inflammatory test was performed by RBC membrane stabilizing activity. Beside In vitro analysis, tail immersion procedure and formalin-induced writhing test were carried out to evaluate the analgesic activity of the plant extract in mice. In addition, the anti-diarrheal activity was determined by castor oil-induced diarrheal model in mice. The ethyl acetate fraction of S. oleosa showed prominent antioxidant activity by scavenging DPPH radical with an IC50 value of 9.46 μg/ml, possibly due to its highest phenol content (103.23 mg of GAE/g of plant extract). The crude methanol extract revealed significant cytotoxicity towards brine shrimp with an LC50 value of 16.79 μg/ml. The dichloromethane fraction showed moderate blood clot lysis ability (28.93% clot lysis) while the crude methanol extract of S. oleosa leaf produced the highest 74.62% inhibition of hemolysis that was induced by hypotonic solution. During in vivo assay, the crude methanol extract of S. oleosa leaf produced significant (p&lt;0.05) and dose-dependent pain response and anti-diarrheal effect in mice. The present study revealed that Schleichera oleosa possesses significant pharmacological activities. However, additional studies are compulsory to discover the mechanism of action of this plant extract.

Hybrid Vesicles Based on Autologous Tumor Cell Membrane and Bacterial Outer Membrane To Enhance Innate Immune Response and Personalized Tumor Immunotherapy.

Tumor heterogeneity, often leading to metastasis, limits the development of tumor therapy. Personalized therapy is promising to address tumor heterogeneity. Here, a vesicle system was designed to enhance innate immune response and amplify personalized immunotherapy. Briefly, the bacterial outer membrane vesicle (OMV) was hybridized with the cell membrane originated from the tumor (mT) to form new functional vesicles (mTOMV). In vitro experiments revealed that the mTOMV strengthened the activation of innate immune cells and increased the specific lysis ability of T cells in homogeneous tumors. In vivo experiments showed that the mTOMV effectively accumulated in inguinal lymph nodes, then inhibited lung metastasis. Besides, the mTOMV evoked adaptive immune response in homologous tumor rather than the heterogeneous tumor, reversibly demonstrating the effects of personalized immunotherapy. The functions to inhibit tumor growth and metastasis accompanying good biocompatibility and simple preparation procedure of mTOMV provide their great potential for clinical applications.

Encapsulation and Algicidal Properties of Fermentation Products From Vibrio brasiliensis H115

Algicidal bacteria offer an eco-friendly and promising approach for controlling harmful algae blooms (HABs). In this study, repeated batch fermentation of immobilized algicidal bacterium Vibrio brasiliensis H115 was preformed to enhance the productivity of the algicidal compounds. The highest algicidal efficiency of the fermentation products against Akashiwo sanguinea (100%) was achieved when the fermentation time was decreased from 24 to 14 h. The cell-free fermentation broth was then spray-dried and floating microcapsules were prepared from the dried powder. The optimum preparation conditions for floating microcapsules were: sodium alginate (SA), 3%; CaCO3: SA (mass ratio), 3:4; CaCl2, 3%; citric acid, 4%; ethylcellulose, 2%; crosslinking time, 30 min. Under the optimal conditions, the floating microcapsules displayed efficient A. sanguinea cell lysis ability and the algicidal efficiency increased from 10.62% (4 h) to 100% (24 h). These results suggest that the floating microcapsules could potentially be practically used for controlling the outbreaks of A. sanguinea.