Phage-based biocontrol of Porphyromonas gingivalis through indirect targeting.

Bacteriophages offer an opportunity for chemical-free, precise control of problematic bacteria, but this approach can be limited when lytic phages are difficult to obtain for the target host. In such cases, phage-based targeting of cooperating or cross-feeding bacteria (e.g., Streptococcus gordonii) can be an effective approach to control the problematic bacteria (e.g., Porphyromonas gingivalis). Using a dual-species biofilm system, phage predation of S. gordonii (108 PFU·mL-1) decreased the abundance of pathogenic P. gingivalis by >99% compared with no-treatment controls, while also inhibiting the production of cytotoxic metabolic end products (butyric and propionic acids). Phage treatment upregulated genes associated with interspecies co-adhesion (5- to 8-fold) and quorum sensing (10-fold) in residual P. gingivalis, which is conducive to increased potential to bind to S. gordonii. Counterintuitively, lower-titer phage applications (104 PFU·mL-1) increased the production of extracellular polymeric substance (EPS) by 22% and biofilm biomass by 50%. This overproduction of EPS may contribute to the phenomenon where the biofilm separated into two distinct species layers, as observed by confocal laser scanning microscopy. Although more complex mixed-culture systems should be considered to delineate the merits and limitations of this novel biocontrol approach (which would likely require the use of phage cocktails), our results offer proof of concept that indirect phage-based targeting can expand the applicability of phage-based control of pathogenic bacteria for public health protection. IMPORTANCE: Lytic phages are valuable agents for targeted elimination of bacteria in diverse applications. Nevertheless, lytic phages are difficult to isolate for some target pathogens. We offer proof of concept that this limitation may be overcome via indirect phage targeting, which involves knocking out species that interact closely with and benefit the primary problematic target bacteria. Our target (P. gingivalis) only forms a periodontal pathogenic biofilm if the pioneer colonizer (S. gordonii) offers its surface for P. gingivalis to attach. Phage predation of the co-adhesive S. gordonii significantly reduced abundance of the target pathogen by >99%, decreased the total biofilm biomass by >44%, and suppressed its production of cytotoxic metabolic byproducts. Thus, this research extends the scope of phage-based biocontrol for public health protection.

The ldh Gene Plays a Crucial Role in Mediating the Pathogen Control of Lactiplantibacillus plantarum AR113.

Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus. Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δldh, surpassed AR113Δ0273&2024, exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene's pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113ΔtagO and ΔccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

Promoting sustainable agriculture by exploiting plant growth-promoting rhizobacteria (PGPR) to improve maize and cowpea crops.

Maize and cowpea are among the staple foods most consumed by most of the African population, and are of significant importance in food security, crop diversification, biodiversity preservation, and livelihoods. In order to satisfy the growing demand for agricultural products, fertilizers and pesticides have been extensively used to increase yields and protect plants against pathogens. However, the excessive use of these chemicals has harmful consequences on the environment and also on public health. These include soil acidification, loss of biodiversity, groundwater pollution, reduced soil fertility, contamination of crops by heavy metals, etc. Therefore, essential to find alternatives to promote sustainable agriculture and ensure the food and well-being of the people. Among these alternatives, agricultural techniques that offer sustainable, environmentally friendly solutions that reduce or eliminate the excessive use of agricultural inputs are increasingly attracting the attention of researchers. One such alternative is the use of beneficial soil microorganisms such as plant growth-promoting rhizobacteria (PGPR). PGPR provides a variety of ecological services and can play an essential role as crop yield enhancers and biological control agents. They can promote root development in plants, increasing their capacity to absorb water and nutrients from the soil, increase stress tolerance, reduce disease and promote root development. Previous research has highlighted the benefits of using PGPRs to increase agricultural productivity. A thorough understanding of the mechanisms of action of PGPRs and their exploitation as biofertilizers would present a promising prospect for increasing agricultural production, particularly in maize and cowpea, and for ensuring sustainable and prosperous agriculture, while contributing to food security and reducing the impact of chemical fertilizers and pesticides on the environment. Looking ahead, PGPR research should continue to deepen our understanding of these microorganisms and their impact on crops, with a view to constantly improving sustainable agricultural practices. On the other hand, farmers and agricultural industry players need to be made aware of the benefits of PGPRs and encouraged to adopt them to promote sustainable agricultural practices.

Controlling Legionella pneumophila growth in hot water systems by reducing dissolved oxygen levels.

Legionella pneumophila, the leading cause of Legionnaires' disease in the United States, is found in lakes, ponds, and streams but poses a health risk when it grows in building water systems. The growth of L. pneumophila in hot water systems of healthcare facilities poses a significant risk to patients, staff, and visitors. Hospitals and long-term care facilities account for 76% of reported Legionnaires' disease cases with mortality rates of 25%. Controlling L. pneumophila growth in hot water systems serving healthcare and hospitality buildings is currently achieved primarily by adding oxidizing chemical disinfectants. Chemical oxidants generate disinfection byproducts and can accelerate corrosion of premise plumbing materials and equipment. Alternative control methods that do not generate hazardous disinfection byproducts or accelerate corrosion are needed. L. pneumophila is an obligate aerobe that cannot sustain cellular respiration, amplify, or remain culturable when dissolved oxygen (DO) concentrations are too low (< 0.3 mg/L). An alternative method of controlling L. pneumophila growth by reducing DO levels in a hot water model system using a gas transfer membrane contactor was evaluated. A hot water model system was constructed and inoculated with L. pneumophila at DO concentrations above 0.5 mg/L. Once the model system was colonized, DO levels were incrementally reduced. Water samples were collected each week to evaluate the effect of reducing dissolved oxygen levels when all other conditions favored Legionella amplification. At DO concentrations below 0.3 mg/L, L. pneumophila concentrations were reduced by 1-log over 7 days. Under conditions in the hot water model system, at favorable temperatures and with no residual chlorine disinfectant, L. pneumophila concentrations were reduced by 1-log, indicating growth inhibition by reducing DO levels as the sole control measure. In sections of the model system where DO levels were not lowered L. pneumophila continued to grow. Reducing dissolved oxygen levels in hot water systems of healthcare and other large buildings to control L. pneumophila could also lower the risk of supplemental chemical treatment methods currently in use.

Impacts of water activity on survival of Listeria innocua and Enterococcus faecium NRRL B-2354 in almonds during steam treatments.

Raw almonds have been associated with Salmonella outbreaks and multiple recalls related to Listeria monocytogenes contamination. While steam treatment has been approved for pasteurizing both conventional and organic whole almonds, there is limited understanding of how water activity (aw) influences the effectiveness of steam treatments in decontaminating almonds. Hence, this study aimed to assess and compare the efficacy of steam treatments against Listeria innocua and Enterococcus faecium NRRL B-2354, the known non-pathogenic surrogates, on almonds. It also sought to investigate the impact of almond's aw on bacterial resistance during steam treatments. Almond kernels were inoculated with ~8 log10 CFU/g of either E. faecium or L. innocua and equilibrated to aw 0.25 or 0.45 before being subjected to steam treatments at temperatures of 100-135 °C. Our results revealed that L. innocua exhibited lower resistance to steam compared to E. faecium, with 1.2-2.6 log10 CFU/g reductions for L. innocua and 1.0-2.0 log10 CFU/g reductions for E. faecium when the surface temperature of almonds reached 100-130 °C, depending on the aw of the almonds. The obtained DL. innocua, 100-130°C-values were 2.0-16.6 s, and DE. faecium, 100-130°C-values were 4.0-21.8 s, depending on the aw of almonds. In general, elevating steam temperatures and almond aw decreased the tolerance of L. innocua and E. faecium during steam inactivation. In addition, the z-values indicated that E. faecium on almonds was less sensitive to change in steam temperature compared to L. innocua, especially at lower aw. The zL. innocua-values were 36.6 °C and 35.7 °C, while zE. faecium-values were 48.9 °C and 42.7 °C in almonds with aw 0.25 and 0.45, respectively. Results from this study suggest that steam treatments serve as effective interventions for controlling pathogen contaminations in raw almonds.

Chickpea-Derived Modified Antimicrobial Peptides KTA and KTR Inactivate Staphylococcus aureus via Disrupting Cell Membrane and Interfering with Peptidoglycan Synthesis.

The widespread bacterial contamination caused by foodborne pathogens has continuously driven the development of advanced and potent food antimicrobial agents. In this study, two novel antimicrobial peptides (AMPs) named KTA and KTR were obtained by modifying a natural AMP, Leg2, from chickpea storage protein legumin hydrolysates. They were further predicted to be stable hydrophobic cationic AMPs of α-helical structure with no hemolytic toxicity by several online servers. Moreover, the AMPs exerted superior antibacterial activity against two representative Staphylococcus aureus strains thanks to the increased hydrophobicity and positive charge, with minimum inhibition concentration value (4.74-7.41 µM) significantly lower than that of Leg2 (>1158.70 µM). Further, this study sought to elucidate the specific antimicrobial mechanism against Gram-positive bacteria. It was found that the electrostatic interactions of the AMPs with peptidoglycan were vital for peptide activity in combating Gram-positive bacteria. Subsequently, the cell membrane of S. aureus cells was irreversibly disrupted by increasing permeability and impairing membrane components, which led to the massive release of intracellular substances and eventual cell death. Overall, this work demonstrated that KTA and KTR were active against Gram-positive bacteria via peptidoglycan targeting and membrane-disruptive mechanisms and paved the way for expanding their application potential to alleviate food contamination.

Ferrous Sulfate-Mediated Control of Phytophthora capsici Pathogenesis and Its Impact on Pepper Plant.

Phytophthora capsici, a destructive fungal pathogen, poses a severe threat to pepper (Capsicum annuum L.) crops worldwide, causing blights that can result in substantial yield losses. Traditional control methods often come with environmental concerns or entail substantial time investments. In this research, we investigate an alternative approach involving ferrous sulfate (FeSO4) application to combat P. capsici and promote pepper growth. We found that FeSO4 effectively inhibits the growth of P. capsici in a dose-dependent manner, disrupting mycelial development and diminishing pathogenicity. Importantly, FeSO4 treatment enhances the biomass and resistance of pepper plants, mitigating P. capsici-induced damage. Microbiome analysis demonstrates that FeSO4 significantly influences soil microbial communities, particularly fungi, within the pepper root. Metabolomics data reveal extensive alterations in the redox metabolic processes of P. capsici under FeSO4 treatment, leading to compromised cell membrane permeability and oxidative stress in the pathogen. Our study presents FeSO4 as a promising and cost-effective solution for controlling P. capsici in pepper cultivation while simultaneously promoting plant growth. These findings contribute to a deeper understanding of the intricate interactions between iron, pathogen control, and plant health, offering a potential tool for sustainable pepper production.

A cAMP phosphodiesterase is essential for sclerotia formation and virulence in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a plant pathogenic fungus that causes white mold or stem rot diseases. It affects mostly dicotyledonous crops, resulting in significant economic losses worldwide. Sclerotia formation is a special feature of S. sclerotiorum, allowing its survival in soil for extended periods and facilitates the spread of the pathogen. However, the detailed molecular mechanisms of how sclerotia are formed and how virulence is achieved in S. sclerotiorum are not fully understood. Here, we report the identification of a mutant that cannot form sclerotia using a forward genetics approach. Next-generation sequencing of the mutant's whole genome revealed candidate genes. Through knockout experiments, the causal gene was found to encode a cAMP phosphodiesterase (SsPDE2). From mutant phenotypic examinations, we found that SsPDE2 plays essential roles not only in sclerotia formation, but also in the regulation of oxalic acid accumulation, infection cushion functionality and virulence. Downregulation of SsSMK1 transcripts in Sspde2 mutants revealed that these morphological defects are likely caused by cAMP-dependent inhibition of MAPK signaling. Moreover, when we introduced HIGS construct targeting SsPDE2 in Nicotiana benthamiana, largely compromised virulence was observed against S. sclerotiorum. Taken together, SsPDE2 is indispensable for key biological processes of S. sclerotiorum and can potentially serve as a HIGS target to control stem rot in the field.

ESKAPEE Pathogen Biofilm Control on Surfaces with Probiotic Lactobacillaceae and Bacillus species.

Combatting the rapidly growing threat of antimicrobial resistance and reducing prevalence and transmission of ESKAPEE pathogens in healthcare settings requires innovative strategies, one of which is displacing these pathogens using beneficial microorganisms. Our review comprehensively examines the evidence of probiotic bacteria displacing ESKAPEE pathogens, with a focus on inanimate surfaces. A systematic search was conducted using the PubMed and Web of Science databases on 21 December 2021, and 143 studies were identified examining the effects of Lactobacillaceae and Bacillus spp. cells and products on the growth, colonization, and survival of ESKAPEE pathogens. While the diversity of study methods limits evidence analysis, results presented by narrative synthesis demonstrate that several species have the potential as cells or their products or supernatants to displace nosocomial infection-causing organisms in a variety of in vitro and in vivo settings. Our review aims to aid the development of new promising approaches to control pathogen biofilms in medical settings by informing researchers and policymakers about the potential of probiotics to combat nosocomial infections. More targeted studies are needed to assess safety and efficacy of different probiotic formulations, followed by large-scale studies to assess utility in infection control and medical practice.

Rapid Quantitative Method Development for Beef and Pork Lymph Nodes Using BAX® System Real Time Salmonella Assay.

The goal of this study was to develop a rapid RT-PCR enumeration method for Salmonella in pork and beef lymph nodes (LNs) utilizing BAX®-System-SalQuant® as well as to assess the performance of the methodology in comparison with existing ones. For study one: PCR curve development, pork, and beef LNs (n = 64) were trimmed, sterilized, pulverized, spiked with 0.00 to 5.00 Log CFU/LN using Salmonella Typhimurium, and then homogenized with BAX-MP media. Samples were incubated at 42 °C and tested at several time points using the BAX®-System-RT-PCR Assay for Salmonella. Cycle-Threshold values from the BAX®-System, for each Salmonella concentration were recorded and utilized for statistical analysis. For study two: Method comparison; additional pork and beef LNs (n = 52) were spiked and enumerated by (1) 3M™EB-Petrifilm™ + XLD-replica plate, (2) BAX®-System-SalQuant®, and (3) MPN. Linear-fit equations for LNs were estimated with recovery times of 6 h and a limit of quantification (LOQ) of 10 CFU/LN. Slopes and intercepts for LNs using BAX®-System-SalQuant® when compared with MPN were not significantly different (p < 0.05), while the same parameters for 3M™EB-Petrifilm™ + XLD-replica plate were significantly different (p > 0.05). The results support the capability of BAX®-System-SalQuant® to enumerate Salmonella in pork and beef LNs. This development adds support to the use of PCR-based quantification methodologies for pathogen loads in meat products.

Timing and delivery route effects of cecal microbiome transplants on Salmonella Typhimurium infections in chickens: potential for in-hatchery delivery of microbial interventions.

BACKGROUND: Exposure to microbes early in life has long-lasting effects on microbial community structure and function of the microbiome. However, in commercial poultry settings chicks are reared as a single-age cohort with no exposure to adult birds which can have profound effects on microbiota development and subsequent pathogen challenge. Microbiota manipulation is a proven and promising strategy to help reduce pathogen load and transmission within broiler flocks. However, administration of microbiota transplant products in a hatchery setting may prove challenging. Effective administration strategies are dependent on key factors, such as; the age of chicks receiving interventions and mode of delivery. This study aimed to assess these two aspects to provide supporting evidence towards microbiome manipulation strategies for use in commercial hatcheries. RESULTS: Manipulation of the microbiota between 4 and 72 h of hatch markedly reduced faecal shedding and colonisation with the foodborne pathogen Salmonella enterica serovar Typhimurium (ST4/74). Administration of transplant material via spray or gel drop delivery systems had minimal effect on the protection conferred with fewer birds in transplant groups shown to shed ST4/74 in the faeces compared to PBS-gavaged control birds. Analysis of the microbiome following transplantation demonstrated that all transplant groups had higher diversity and species richness than non-transplant groups during the first week of life and the early stages of infection with ST47/4.The relative abundance of the bacterium Faecalibacterium prausnitzii was significantly higher in CMT groups compared to PBS controls. The presence of F. prausnitzii was also shown to increase in PBS-challenged birds compared to unchallenged birds potentially indicating a role of this bacterium in limiting Salmonella infections. CONCLUSIONS: This study demonstrated that administration of microbiome transplants, using methods that would align with hatchery practices, effectively reduced colonisation and shedding of Salmonella in chickens. Age of chicks at microbiome administration had limited effect on the diversity and composition of the microbiome and conferred protection against Salmonella infections. Traditional hatchery delivery systems, such as spray or gel-drop, are sufficient to transfer donor material, alter the microbiome and confer protection against Salmonella. This study helps highlight the opportunity for use of microbiome modification methods within the hatchery.

Chryseochelins-structural characterization of novel citrate-based siderophores produced by plant protecting Chryseobacterium spp.

Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry (MS/MS) of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.

Evaluation of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to control Botryosphaeria dieback and black-foot pathogens in grapevine propagation material.

BACKGROUND: Grapevine trunk diseases (GTDs) are a complex group of diseases that lead to major economic losses in all wine-producing countries. The investigation of biocontrol agents (BCAs) capable of forestalling or at least minimizing the development of GTDs has, recently, become a priority. Nursery experiments were set up to (i) assess the biocontrol effect of Trichoderma atroviride (Ta) SC1 and Bacillus subtilis (Bs) PTA-271, alone and in simultaneous application, against Botryosphaeria dieback (BOT)- and black-foot (BF)- associated pathogens during the grapevine propagation process and (ii) evaluate the success of the BCA inoculation during the grapevine propagation process, using quantitative reverse-transcription polymerase chain reaction techniques. RESULTS: The results demonstrated a significant reduction in the percentage of potentially infected plants and the percentage of fungal isolation from wood fragments of BOT and BF pathogens in nursery material treated with Ta SC1 and Bs PTA-271, respectively. In one of the experiments, simultaneous treatments with Bs PTA-271 and Ta SC1 caused a reduction in percentages of potentially infected plants and fungal isolation, from wood fragments containing BOT and BF pathogens. CONCLUSION: These biological treatments may be relevant components of an integrated approach, using complementary management strategies to limit infection by GTD pathogens, but further research is still needed to elucidate the effectiveness of Bs PTA-271 and the benefits of simultaneous application with Ta SC1 for the control of GTD pathogens in nurseries. © 2022 Society of Chemical Industry.

Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle.

Systemic control uses the vertebrate hosts of zoonotic pathogens as "Trojan horses," killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.

Inactivation of Salmonella Enteritidis PT30 on black peppercorns in thermal treatments with controlled relative humidities.

Traditional method utilizes steam to pasteurize low-moisture ingredients like black peppercorns and almonds. Exposure to steam results in direct condensation on the product, unfavorable for a broader range of food ingredients such as dried herbs, fruits, and ground materials. Recent studies on the thermal inactivation of Salmonella in low-moisture foods suggest that the relative humidity in treatment chambers is an important factor, besides temperature, that determines the death rate of bacteria. Thus, thermal treatments with controlled high relative humidity can be an effective method to replace steam pasteurization. No condensation will occur when the products are preheated to above the dew-point temperature of the hot air in the treatment chamber, thus eliminating the need for post-treatment drying. To prove this concept, a special device was developed that preheated samples in a dry environment before exposing them to a controlled relative humidity (RH) at a high temperature. Using this device, the death rate of Salmonella Enteritidis PT30 (S. Enteritidis) in black peppercorns was determined at 80 °C and three different RH levels (60, 70, or 80 %) after the innoculated samples were heated to 78oC. The results indicate that the treatments at 80 °C and 80 % RH for 3 min, 70 % RH for 9 min, and 60 % RH for 25 min caused 5.4 ± 0.2, 6.2 ± 0.6, and 6.1 ± 1.0 log reductions, respectively. No condensation was observed on all of the treated samples. The moisture content (wet basis) of fully pasteurized (5-log reduction) black peppercorns at 60, 70, and 80 %RH reduced from 9.7 ± 0.4 % (untreated) to 8.7 ± 0.5 %, 9.2 ± 0.4 %, and 9.2 ± 0.2 %, respectively, indicating that post-drying is not required after the treatments. This study demonstrated the potential of using short-time high-RH treatments to control pathogens in low-moisture foods without the need for post-treatment drying.

Thermal death kinetics of Salmonella Enteritidis PT30 in peanut butter as influenced by water activity.

It has been a challenge in developing effective thermal pasteurization processes for foods with high-fat and low-moisture contents like peanut butter, due to a general lack of reliable data on thermal resistance of pathogens in those food matrices. Recent studies on low-moisture foods like wheat flour and almond flour suggest that temperature and water activity (at the process temperatures) are two key factors that influence thermal inactivation of bacteria. In this study, we measured high-temperature water activities of peanut butter of two moisture content (MC), 3.1% and 5.6% (dry basis), and investigated the thermal death kinetics of Salmonella enterica Enteritidis PT 30 (S. Enteritidis) in those samples at 70, 80, 90, and 100 °C. The results indicated that the water activity of peanut butter increased with increasing temperature, e.g., from 0.33 and 0.53 at 23 °C, up to 0.39 and 0.59 at 100 °C, respectively. The thermal death of S. Enteritidis in peanut butter followed the first-order kinetics. Overall, higher moisture content and a higher treatment temperature led to a smaller D-value (decimal reduction time of the survival population) of S. Enteritidis. The maximum D-value was 102.6 ± 15.2 min at MC 3.1% and 70 °C, and the minimum D-value was 0.3 min (predicted) at MC = 5.6% and 100 °C. The log D-value reduced linearly with temperature at a given aw, with Z-values equal to 15.4 °C (for MC = 3.1%) and 12.6 °C (for MC = 5.6%). Based on this study, the first-order kinetic model can be employed for developing and validating thermal pasteurization processes for peanut butter. The moisture content of peanut butter and the process temperature are two key parameters that need to be controlled for sufficient lethality.

A Novel Method to Create Efficient Phage Cocktails via Use of Phage-Resistant Bacteria.

The rapid antiphage mutation of pathogens is a big challenge often encountered in the application of phages in aquaculture, animal husbandry, and human disease prevention. A cocktail composed of phages with different infection strategies can better suppress the antiphage resistance of pathogens. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. Here, we verified that using a resistant pathogen quickly evolved under single phage infection, as the new host can easily obtain phages with different infection strategies. We randomly isolated two lytic phages (i.e., Va1 and Va2) that infect the opportunistic pathogen Vibrio alginolyticus. Whether they were used alone or in combination, the pathogen easily gained resistance. Using a mutated pathogen resistant to Va1 as a new host, a third lytic phage Va3 was isolated. These three phages have a similar infection cycle and lytic ability but quite different morphologies and genome information. Notably, phage Va3 is a jumbo phage containing a larger and more complex genome (240 kb) than Va1 and Va2. Furthermore, the 34 tRNAs and multiple genes encoding receptor binding proteins and NAD+ synthesis proteins in the Va3 genome implicated its quite different infection strategy from Va1 and Va2. Although the wild-type pathogen could still readily evolve resistance under single phage infection by Va3, when Va3 was used in combination with Va1 and Va2, pathogen resistance was strongly suppressed. This study provides a novel approach for rapid isolation of phages with different infection strategies, which will be highly beneficial when designing effective phage cocktails. IMPORTANCE The rapid antiphage mutation of pathogens is a big challenge often encountered in phage therapy. Using a cocktail composed of phages with different infection strategies can better overcome this problem. However, randomly selecting phages with different infection strategies is time-consuming and labor intensive. To address this problem, we developed a method to efficiently obtain phages with disparate infection strategies. The trick is to use the characteristics of the pathogenic bacteria that are prone to develop resistance to single phage infection to rapidly obtain the antiphage variant of the pathogen. Using this antiphage variant as the host results in other phages with different infection strategies being efficiently isolated. We also verified the reliability of this method by demonstrating the ideal phage control effects on two pathogens and thus revealed its potential importance in the development of phage therapies.

Renaissance for Phage-Based Bacterial Control.

Bacteriophages (phages) are an underutilized biological resource with vast potential for pathogen control and microbiome editing. Phage research and commercialization have increased rapidly in biomedical and agricultural industries, but adoption has been limited elsewhere. Nevertheless, converging advances in DNA sequencing, bioinformatics, microbial ecology, and synthetic biology are now poised to broaden phage applications beyond pathogen control toward the manipulation of microbial communities for defined functional improvements. Enhancements in sequencing combined with network analysis make it now feasible to identify and disrupt microbial associations to elicit desirable shifts in community structure or function, indirectly modulate species abundance, and target hub or keystone species to achieve broad functional shifts. Sequencing and bioinformatic advancements are also facilitating the use of temperate phages for safe gene delivery applications. Finally, integration of synthetic biology stands to create novel phage chassis and modular genetic components. While some fundamental, regulatory, and commercialization barriers to widespread phage use remain, many major challenges that have impeded the field now have workable solutions. Thus, a new dawn for phage-based (chemical-free) precise biocontrol and microbiome editing is on the horizon to enhance, suppress, or modulate microbial activities important for public health, food security, and more sustainable energy production and water reuse.

Monoglyceride reduces viability of porcine epidemic diarrhoea virus in feed and prevents disease transmission to post-weaned piglets.

Outbreaks of African swine fever virus (ASFv) and porcine epidemic diarrhoea virus (PEDv) have revealed the susceptibility of livestock to disease transmitted through feed. Several viruses, including PEDv, survive in feed and may introduce disease that causes significant morbidity and mortality. In 2013, PEDv, which causes severe diarrhoea and vomiting, reached North America after spreading for decades across Eurasia. The global exchange of ingredients has created demand for products that prevent disease transmission from feed. Formaldehyde-based products are highly effective at inactivating enveloped viruses when applied at 3.25 kg/t. Alternative products to formaldehyde, including carboxylic acids, essential oils and medium chain fatty acids (MCFAs), have exhibited mixed efficacy against PEDv and require application rates higher than formaldehyde. Amphiphilic molecules like MCFAs disrupt the bilayer-lipid membranes that protect viral nucleic acids through the formation of micelles. Monoglycerides form micelles at lower concentrations than MCFAs, which suggests they may be more potent against enveloped viruses. The potential efficacy of monoglycerides against enveloped viruses in feed led to the development and examination of an experimental monoglyceride blend. The proprietary monoglyceride blend significantly (p < .0001) reduced PEDv viability in vitro after application to feed at 1.5, 2.5 and 3.5 kg/t. The monoglyceride was tested in a natural feeding behaviour challenge model in piglets. The feed was contaminated with ice-blocks containing viable PEDv, and the piglets were exposed to PEDv through the feed bin for 20 days. At the end of the 20-day challenge period, all pigs were rectally swabbed and tested for PEDv by qPCR. In the untreated control group 54.8% of the piglets tested positive for PEDv, whereas none of the MCFA-treated feed (10 kg/t inclusion) transmitted PEDv. Strikingly, the monoglyceride-treated groups (1.5, 2.5 and 3.5 kg/t) all exhibited 100% protection from PEDv. These data support the use of this proprietary monoglyceride blend in mitigation and prevention of viral disease transmission to piglets from contaminated feed.

Highly sensitive time-resolved fluoroimmunoassay for the quantitative onsite detection of Alternaria longipes in tobacco.

AIMS: Alternaria longipes is a causal agent of brown spot of tobacco, which remains a serious threat to tobacco production. Herein, we established a detection method for A. longipes in tobacco samples based on the principle of time-resolved fluoroimmunoassay, in order to fulfil the requirement of rapid, sensitive and accurate detection in situ. METHODS AND RESULTS: A monoclonal antibody against A. longipes was generated, and its purity and titration were assessed using western blot and ELISA. The size of europium (III) nanospheres was measured to confirm successful antibody conjugation. The method described here can detect A. longipes protein lysates as low as 0.78 ng ml-1 , with recovery rates ranging from 85.96% to 99.67% in spiked tobacco. The specificity was also confirmed using a panel of microorganisms. CONCLUSIONS: The fluorescent strips allow rapid and sensitive onsite detection of A. longipes in tobacco samples, with high accuracy, specificity, and repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel detection method provides convenience of using crude samples without complex procedures, and therefore allows rapid onsite detection by end users and quick responses towards A. longipes, which is critical for disease control and elimination of phytopathogens.