ΦV10 is an Escherichia coli O157:H7-specific bacteriophage that has been used to develop luminescent reporter assays for the detection of this important foodborne pathogen. Previous work demonstrated the specificity of ΦV10 for infection of E.coli O157:H7 through interaction with the O157 antigen. In addition, modification of the lipopolysaccharide (LPS) via O-acetylation prevents ΦV10 infection in an E. coli O157:H7 expressing a phage-encoded O-acetylase gene. Through assays for phage binding, plaque formation, and lysogeny using non-O157:H7 and O157: non-H7 strains, as well as complementation of an O157:H− strain, it is demonstrated in this study that both the somatic O157 antigen and flagellar H7 antigen are required for productive infection of E. coli O157:H7 by ΦV10. Together, the results indicate that the O157 antigen is required for phage binding and that the H7 antigen is necessary to complete the infection process.

Gut dysbiosis and an inflamed bowel are growing concerns in mammals, including dogs. Probiotic supplements have been used to restore the natural microbial community and improve gastrointestinal health. Biofilm formation, antimicrobial activities, and immunological responses of probiotics are crucial to improving gut health. Thus, we tested a commercial probiotic blend (LabMAX-3), a canine kibble additive comprising Lactobacillus acidophilus, Lacticasibacillus casei, and Enterococcus faecium for their ability to inactivate common enteric pathogens; their ability to form biofilms, epithelial cell adhesion and anti-inflammatory response on Madin-Darby Canine Kidney (MDCK) cell line. Probiotic LabMAX-3 blend, or individual isolates, showed a strong inhibitory effect against Salmonella enterica, Listeria monocytogenes, enterotoxigenic Escherichia coli, and Campylobacter jejuni. LabMAX-3 formed biofilms comparable to Staphylococcus aureus. LabMAX-3 adhesion to the MDCK cell line (with or without lipopolysaccharide, LPS) pretreatment showed comparable adhesion and biofilm formation (p < 0.05) to L. casei ATCC 334 used as a control. LabMAX-3 had no cytotoxic effects on the MDCK cell line during 1 h exposure. The IL-10 and TNFα ratio of LabMAX-3, compared to the L. casei control, showed a significant increase (p < 0.05), indicating a more pronounced anti-inflammatory response. The data show that LabMAX-3, a canine kibble supplement, can improve gastrointestinal health.

In the realm of food safety, the standard practice involves collecting food product samples and sending them to a central laboratory for microbiological testing. However, this process introduces delays in obtaining the microbiological testing results and subsequently affects the timely delivery of food products to consumers. To further reduce the time-to-detection issue, we propose the development of a self-contained, battery-operated, high-sensitivity optical sensor that can be affixed to the cap of the typical food sample collection container. This device, called MPACT, offers real-time and in-transit monitoring of the contamination status of the food sample, specifically targeting E. coli O157:H7, through a bioluminescence assay. The assay exclusively targets the target pathogen and, when detected, produces minimal luminescence. As the sample is transported in the container, the number of bacterial cells multiplies, and once the luminescent signal reaches a predefined threshold, the sensor reports the results via Bluetooth. This study focuses on the design of the bottle cap sensor and examines its sensitivity by subjecting it to bioluminescence samples.

Gut dysbiosis and an inflamed bowel are growing concerns in mammals, including dogs. Probiotic supplements have been used to restore the natural microbial community and improve gastrointestinal health. Biofilm formation, antimicrobial activities, and immunological responses of probiotics are crucial to improving gut health. Thus, we tested a commercial probiotic blend (LabMAX-3), a canine kibble additive comprising Lactobacillus acidophilus, Lacticaseibacillus casei, and Enterococcus faecium for their ability to inactivate common enteric pathogens; their ability to form biofilms; epithelial cell adhesion; and their anti-inflammatory response in the Madin-Darby Canine Kidney (MDCK) cell line. Probiotic LabMAX-3 blend or individual isolates showed a strong inhibitory effect against Salmonella enterica, Listeria monocytogenes, enterotoxigenic Escherichia coli, and Campylobacter jejuni. LabMAX-3 formed biofilms comparable to Staphylococcus aureus. LabMAX-3 adhesion to the MDCK cell line (with or without lipopolysaccharide (LPS) pretreatment) showed comparable adhesion and biofilm formation (p < 0.05) to L. casei ATCC 334 used as a control. LabMAX-3 had no cytotoxic effects on the MDCK cell line during 1 h exposure. The interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) ratio of LabMAX-3, compared to the L. casei control, showed a significant increase (p < 0.05), indicating a more pronounced anti-inflammatory response. The data show that LabMAX-3, a canine kibble supplement, can improve gastrointestinal health.

The effects of air thawing (AT), water immersion thawing (WT), microwave thawing (MT) and ultrasound combined with slightly acidic electrolyzed water thawing (UST) on the myofibrillar protein (MP) properties (surface hydrophobicity, solubility, turbidity, particle size and zeta potential), protein oxidation (carbonyl content and sulfhydryl content) and structure (primary, secondary and tertiary) of frozen mutton were inves-tigated in comparison with fresh mutton (FM). The solubility and turbidity results showed that the MP properties were significantly improved in the UST treatment. UST treatment could effectively reduce the MP aggregation and enhance the stability, which was similar to the FM. In addition, UST treatment could effectively inhibit protein oxidation during thawing as well. The primary structure of MP was not damaged by the thawing methods. UST treatment could reduce the damage to MP secondary and tertiary structure during the thawing process compared to other thawing methods. Overall, the UST treatment had a positive influence in maintaining the MP properties by inhibiting protein oxidation and protecting protein structure.

Wastewater treatment is an essential process for public health and a sustainable ecosystem. Inadequate wastewater treatment can lead to the release of organic and inorganic pollutants and pathogenic bacteria into the receiving waters which could be further utilized for recreation purposes. The interaction between bacteriophage and bacteria in a wastewater treatment plant plays a major role in maintaining the treatment process. Phage therapy has been proposed as an alternative to conventional treatment methods as bacteriophages can be used on specific targets and leave useful bacteria unharmed. The bacterial species, which are responsible for bulking, foaming, and biofilm formation in a wastewater treatment plant (WWTP) have been identified and their respective phages are isolated to control their growth. Phages with lytic life cycles are preferred to lysogenic. Lytic phages can kill the specific target as they lyse the cell, infect most of the hosts, and have an immediate effect on controlling problems caused by bacteria in a WWTP. The bacteriophages such as T7, SPI1, GTE7, PhaxI, MAG1, MAG2, ϕPh_Se01, ϕPh_Se02, and Bxb1 have been investigated for the removal of bacterial biofilms from wastewater. Novel experimental setups have improved the efficiency of phage therapy in small-scale and pilot-scale experiments. Much more in-depth knowledge of the microbial community and their interaction would help promote the usage of phage therapy in large-scale wastewater treatments. This paper has covered the recent advancements in phage therapy as an effective biocontrol of pathogenic bacteria in the wastewater treatment process and has looked at certain shortcomings that have to be improved.

EDITORIAL article Front. Microbiol., 17 March 2022Sec.Food Microbiology https://doi.org/10.3389/fmicb.2022.872879

Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make phages a novel bio-recognition molecule in assay development, including biosensors for the detection of foodborne bacterial pathogens to ensure food safety. This review provides comprehensive information about the different types of phage-based biosensor platforms, such as magnetoelastic sensors, quartz crystal microbalance, and electrochemical and surface plasmon resonance for the detection of several foodborne bacterial pathogens from various representative food matrices and environmental samples.

The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15–24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2–3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15–25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibility testing , and for characterizing industrially beneficial traits. An optical scattering sensor designated BARDOT (bacterial rapid detection using optical scattering technology) that uses a red-diode laser, developed at the beginning of the 21 st century at Purdue University, some 220 years after the Petri-plate discovery can identify and study bacteria directly on the plate as a diagnostic tool akin to Raman scattering and hyperspectral imaging systems for application in clinical and food microbiology laboratories.

Overly elevated circulating non-esterified fatty acids (NEFAs) is an emerging health concern of obesity-associated energy disorders. However, methods to reduce circulating NEFAs remain elusive. The present study determined the effect of piceatannol, a naturally occurring stilbene, on adipocyte lipolysis and its underlying mechanism. Differentiated 3T3-L1 adipocytes, brown adipocytes and isolated white adipose tissue were treated with various concentrations of piceatannol for 1.5-h both in the basal and stimulated lipolysis conditions. Piceatannol significantly inhibited NEFAs and glycerol release with a concomitant reduction of ATGL, CGI-58 and PLIN1 expression in adipocytes. Using a series of inhibitor assays, piceatannol-induced degradation of these proteins was found to be mediated by upregulation of the autophagy-lysosome pathway. Moreover, we demonstrated that piceatannol is capable of stimulating autophagy in vitro. Importantly, piceatannol administration tended to lower fasting-induced serum glycerol levels in healthy mice. Furthermore, piceatannol administration lowered lipolysis, central adiposity and hyperinsulinemia in diet-induced obese mice. Our study provides profound evidence of a novel inhibitory role of piceatannol in lipolysis through autophagy-lysosome-dependent degradation of the key lipolytic proteins in adipocytes. This study offers a mechanistic foundation for investigating the potential of piceatannol-containing foods in reducing lipolysis and its associated metabolic disorders.

Food is a common vehicle for introducing Escherichia coli into humans which can result in serious disease. Efficient and rapid detection of E. coli in the multitude of food and beverage matrices available is critical to limit the health, financial, and social impacts of these infections. Compared to culture-based and molecular detection methods, bacteriophage-based detection methods perform favorably with high specificity, natural affinity, low cost, and reliable live-dead cell distinction. More recently, many rapid bacteriophage-based detection approaches have been developed coupled with advanced techniques including fluorescence, immunology, molecular biology, nanoscale science, and an array of biosensing strategies. In this review, up-to-date applications and principles of bacteriophage-based detection methods for E. coli in food are discussed, in addition to their current limitations and future prospects.

Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage ɸV10 at ambient temperature without aqueous storage solutions while limiting loss of viability. ɸV10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of ɸV10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23°C. Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for ɸV10 to form lysogens in an E . coli O157:H7 host was not negatively affected by lyophilization. Drying ɸV10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing ɸV10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.

The silicon photomultiplier (SiPM) for low light detection has many advantages when compared to existing photon counting detectors, such as high sensitivity, low cost, robustness, and compact hardware. To facilitate the use of SiPM as a portable, field deployable device, an electrical circuit was designed consisting of an amplifier, comparator, and microcontroller. In addition, a 3D printing was used to create a portable cradle for housing the SiPM. To evaluate its detection ability, a laser experiment and bioluminescent experiments, including Pseudomonas fluorescens M3A detection, E. coli O157:H7 PhiV10nluc lysogen detection, and a luminescence-based detection of E. coli O157:H7 in ground meat using the engineered luminescent-based reporter phage PhiV10nluc, were conducted. In the same experimental setting, our previously developed smartphone-based luminometer called the bioluminescent-based analyte quantitation by smartphone and a conventional photomultiplier tube-based benchtop luminometer were used to compare detection levels and applicability for supporting luminescent phage-based pathogen detection. Results showed that the SiPM provides better performance in terms of time to detection and SNR and could be used as the light detection component of the PhiV10nluc phage-based detection format.

Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillus probiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3+T cells, CD11c+ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

Multiple studies have demonstrated atmospheric cold plasma as an effective non-thermal technology for inactivating bacteria, spores, and other microbial contaminants in foods and on non-food surfaces. However, few studies have applied this technique to semi-solid food within a package. This study evaluates the efficacy and the interaction mechanism of high voltage atmospheric cold plasma (HVACP) onSalmonella enterica serovar Typhi(S.enterica) inactivation in agar gels with different compositions. HVACP was generated by a dielectric barrier discharge in air and a modified atmosphere (MA65: 65% O-2) in sealed bags. Agar gels of various densities with a pH indicator were inoculated withS.enterica(> 10(7)cfu) and exposed directly (between the electrode) or indirectly (adjacent to electrode) to 90 kV at 60 Hz for up to 1.5 h. HVACP treatment induced greater than 6 log(10)(cfu) reduction in viable bacteria (both with air and MA65) in the plasma penetrated zone exhibiting a pH change. Inactivation of bioluminescentE. coli K12-luxcells in the plasma penetrated zone confirmed that the plasma, and its generated reactive species, inactivates microbes as it penetrates into the gel. A two-minute HVACP treatment resulted in greater than 5 log(10)(cfu)S.entericareduction in applesauce. In summary, these results demonstrate that HVACP can be an effective non-thermal technology to control or even eliminate bacteria populations in semi-solid foods.

In this chapter, the toluene degradation kinetics of two Pseudomonas strains, P. putida F1 and P. putida TVA8, in liquid batch are compared. Furthermore, the TCE degradation kinetics for TVA8 are assessed. The relationship between toluene/TCE degradation and bioluminescence production in P. putida TVA8 was explored.

Luminescence based detection has been widely used in diverse science and engineering applications. The recent development of the smartphone has enabled end users to utilize this communication device as a portable detector and instruments such as a microscope, fluorimeter, colorimeter, and spectrometer. To transform the smartphone into a bioluminescence detector, our group developed an advanced signal processing algorithm and an optical chamber designed for efficient photon capture. This solution was required to overcome the typical sensitivity of the CMOS-based smartphone camera such that sub-nano to pico Watt levels of power can be measured with conventional smartphones. Preliminary experiments conducted with the bioluminescent Pseudomonas fluorescens M3A shows a detection limit of approximately 106 CFU/ml. To achieve sensitive detection while maintaining the portability, we explored using the recently developed silicon photomultiplier (SiPM), and designed a portable bioluminescence sensor which shows a 2-3 order higher sensitivity on calibration sample testing. Finally, for live sample testing, Escherichia coli O157:H7 was inoculated on a ground beef sample and subjected to luminescence phage based detection and a luminescence signal was generated from the bacteriophage infection and detected within 8-10 h after spiking.

Conventional solar disinfection (SODIS) processes rely on UVA radiation due to exclusion of shorter wavelengths by common SODIS containers. Because of this, these processes are slow and could be improved by inclusion of UVB radiation, which has been reported to be more effective for microbial inactivation than UVA on a photon basis, but is typically present at lower spectral irradiance. To examine the potential for microbial inactivation resulting from exposure to solar UVB radiation at sea-level, experiments were conducted to define the UVB/UVA action and effectiveness spectra for Salmonella typhimurium LT2, Vibrio harveyi, and Cryptosporidium parvum, which are representative of three of the most prevalent waterborne pathogens globally. For each organism, the action spectrum was similar in shape to its corresponding DNA absorption spectrum, thereby suggesting that inactivation of these organisms by UVB irradiation was largely attributable to DNA damage. Modeling and measurements of ambient solar UVB spectral irradiance were compared, indicating a trend of model over-prediction of spectral irradiance by up to 20% on cloudless days. Effectiveness spectra for organism/location pairs were calculated as the product of the action spectra and calculated spectral irradiance to identify the most effective wavelengths for inactivation. For the organisms studied, maximum predicted effectiveness appeared at wavelengths between 318 and 330 nm. At 320 nm, the simulated inactivation of C. parvum in the top 20-cm of an outdoor swimming pool (mid-latitude location in summer) after one hour of exposure was approximately 6-log10 units. These results suggest that solar UVB irradiation could yield substantial inactivation of C. parvum in outdoor recreational waters, where these protozoan parasites are responsible for a large fraction of the disease burden among swimmers.

We assessed the efficacy of bacterial inactivation using a dielectric barrier discharge in three different plasma setups: plasma gas (PG), and direct and indirect plasma activated water (PAW), where deionized water was placed either between or away from the electrodes, respectively. We used bioluminescent Escherichia coli K12 lux as a model bacteria in a biosensor format to study the inactivation kinetics and mechanism of action of produced PG and PAW. The results showed that uninterrupted application of PG decreased bioluminescence rapidly by 1 log within the first minute and 3.6-log after 10 min of treatment. Exposing the bacterial culture with a sublethal dose of PAW (1 mL) rapidly decreased the bioluminescence; however, luminescence slowly recovered after exposure. Subsequent treatment with PAW decreased the bioluminescence to a lesser extent. In addition, direct PAW induced a greater decrease in bioluminescence compared to indirect treatments for both single and multiple exposures. In contrast to the PG, PAW treatments induced a lower bactericidal effect with 0.11 to 0.22-log reduction for indirect PAW and 0.2 to 0.32-log for direct PAW. Our results also indicate that antimicrobial activity of PAW decreased slowly within 20 min of its preparation. The rapid decrease in bioluminescence followed by a partial recovery in a repeatable pattern suggests an incomplete inactivation, and that the reducing power of the cell helps them to survive. Moreover, the complete and partial oxidation of NADH solutions in vitro by PG and PAW, respectively, strongly suggest that the lux fluorophore FMNH2 and other reducing cofactors could be the target of such treatment before other cell components. This hypothesis was supported by the tendency to recover luminescence by potentially replenishing the pool of FMNH2 after plasma treatment. It is also important to consider that the reducing power of the cell (NADH, NADPH, and FMNH2) is crucial for cell viability mostly due to reducing potential for critical metabolic reactions. Therefore, in situ bioluminescence monitoring technology can potentially serve as a unique approach to elucidate the mechanism of bacteria inactivation in real time. Industrial relevance: The present study developed three dielectric barrier discharge (DBD) plasma setups to produce plasma gas and plasma activated water, which can disinfect both food products and their contact surfaces regardless of geometry. Our in situ bioluminescent technology elucidated bacterial inactivation mechanisms of plasma treatments, which may potentially suggest sufficient exposures to plasma resulting in safe food products without deteriorating their quality. The results will help food manufacturers apply new plasma based disinfection methods with appropriate treatments.