Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157.

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Natural approach of using nisin and its nanoform as food bio-preservatives against methicillin resistant Staphylococcus aureus and E.coli O157:H7 in yoghurt.

BACKGROUND: Natural antimicrobial agents such as nisin were used to control the growth of foodborne pathogens in dairy products. The current study aimed to examine the inhibitory effect of pure nisin and nisin nanoparticles (nisin NPs) against methicillin resistant Staphylococcus aureus (MRSA) and E.coli O157:H7 during the manufacturing and storage of yoghurt. Nisin NPs were prepared using new, natural, and safe nano-precipitation method by acetic acid. The prepared NPs were characterized using zeta-sizer and transmission electron microscopy (TEM). In addition, the cytotoxicity of nisin NPs on vero cells was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimum inhibitory concentrations (MICs) of nisin and its nanoparticles were determined using agar well-diffusion method. Further, fresh buffalo's milk was inoculated with MRSA or E.coli O157:H7 (1 × 106 CFU/ml) with the addition of either nisin or nisin NPs, and then the inoculated milk was used for yoghurt making. The organoleptic properties, pH and bacterial load of the obtained yoghurt were evaluated during storage in comparison to control group. RESULTS: The obtained results showed a strong antibacterial activity of nisin NPs (0.125 mg/mL) against MRSA and E.coli O157:H7 in comparison with control and pure nisin groups. Notably, complete eradication of MRSA and E.coli O157:H7 was observed in yoghurt formulated with nisin NPs after 24 h and 5th day of storage, respectively. The shelf life of yoghurt inoculated with nisin nanoparticles was extended than those manufactured without addition of such nanoparticles. CONCLUSIONS: Overall, the present study indicated that the addition of nisin NPs during processing of yoghurt could be a useful tool for food preservation against MRSA and E.coli O157:H7 in dairy industry.

Enhanced photodynamic inactivation against Escherichia coli O157:H7 provided by chitosan/curcumin coating and its application in food contact surfaces.

Sterilisation technologies are essential to eliminate foodborne pathogens from food contact surfaces. However, most of the current sterilisation methods involve high energy and chemical consumption. In this study, a photodynamic inactivation coating featuring excellent antibacterial activity was prepared by dispersing curcumin as a plant-based photosensitiser in a chitosan solution. The coating generated abundant reactive oxygen species (ROS) after light irradiation at 420 nm, which eradicated ≥99.999 % of Escherichia coli O157:H7. It was also found that ROS damaged the cell membrane, leading to the leakage of cell contents and cell shrinkage on the basis of chitosan. In addition, the production of ROS first excited the bacterial antioxidant defence system resulting in the increase of peroxidase (POD) and superoxide dismutase (SOD). ROS levels exceed its capacity, causing damage to the defence system and further oxidative decomposition of large molecules, such as DNA and proteins, eventually leading to the death of E. coli O157:H7. We also found the curcumin/chitosan coating could effectively remove E. coli O157:H7 biofilms by oxidative of extracellular polysaccharides and proteins. All the contributors made the chitosan/curcumin coating an efficient detergent comparable with HClO.

Shiga Toxin-Producing Escherichia coli O157:H7 Illness Outbreak Associated with Untreated, Pressurized, Municipal Irrigation Water - Utah, 2023.

During July-September 2023, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 illness among children in city A, Utah, caused 13 confirmed illnesses; seven patients were hospitalized, including two with hemolytic uremic syndrome. Local, state, and federal public health partners investigating the outbreak linked the illnesses to untreated, pressurized, municipal irrigation water (UPMIW) exposure in city A; 12 of 13 ill children reported playing in or drinking UPMIW. Clinical isolates were genetically highly related to one another and to environmental isolates from multiple locations within city A's UPMIW system. Microbial source tracking, a method to indicate possible contamination sources, identified birds and ruminants as potential sources of fecal contamination of UPMIW. Public health and city A officials issued multiple press releases regarding the outbreak reminding residents that UPMIW is not intended for drinking or recreation. Public education and UPMIW management and operations interventions, including assessing and mitigating potential contamination sources, covering UPMIW sources and reservoirs, indicating UPMIW lines and spigots with a designated color, and providing conspicuous signage to communicate risk and intended use might help prevent future UPMIW-associated illnesses.

Green-Synthesized Amino Carbons for Impedimetric Biosensing of E. coli O157:H7.

This study describes the synthesis of amino-functionalized carbon nanoparticles derived from biopolymer chitosan using green synthesis and its application toward ultrasensitive electrochemical immunosensor of highly virulent Escherichia coli O157:H7 (E. coli O157:H7). The inherent advantage of high surface-to-volume ratio and enhanced rate transfer kinetics of nanoparticles is leveraged to push the limit of detection (LOD), without compromising on the selectivity. The prepared carbon nanoparticles were systematically characterized by employing CO2-thermal programmed desorption (CO2-TPD), Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-visible), and transmission electron microscopy (TEM). The estimated limit of detection of 0.74 CFU/mL and a sensitivity of 5.7 ((ΔRct/Rct)/(CFU/mL))/cm2 in the electrochemical impedance spectroscopy (EIS) affirm the utility of the sensor. The proposed biosensor displayed remarkable selectivity against interfering species, making it well suited for real-time applications. Moreover, the chitosan-derived semiconducting amino-functionalized carbon shows excellent sensitivity in a comparative analysis compared to highly conducting amine-functionalized carbon synthesized via chemical modification, demonstrating its vast potential as an E. coli sensor.

The influence of pH on the efficacy of oxidation-reduction potential (ORP) to predict chlorine disinfection of surrogate bacteria, Escherichia coli O157:H7 and Listeria monocytogenes in oxidant demand free conditions and fresh produce wash water.

Oxidation-reduction potential (ORP) is commonly used as a rapid measurement of the antimicrobial potential of free chlorine during industrial fresh produce washing. The current study tested the hypothesis that ORP can act as a "single variable" measurement of bacterial (vegetative and endospores) inactivation effectiveness with free chlorine irrespective of the water pH value. This situation has on occasion been assumed but never confirmed nor disproven. Chlorine-dosed pH 6.5 and 8.5 phosphate buffer solutions were inoculated with Escherichia coli (E. coli), Listeria innocua (L. innocua), or Bacillus subtilis (B. subtilis) endospores. ORP, free chlorine (FC), and log reduction were monitored after 5 s (for E. coli and L. innocua) and up to 30 min (for B. subtilis spores) of disinfection. Logistic and exponential models were developed to describe how bacteria reduction varied as a function of ORP at different pH levels. Validation tests were performed in phosphate buffered pH 6.5 and 8.5 cabbage wash water periodically dosed with FC, cabbage extract and a cocktail of Escherichia coli O157:H7 (E. coli O157:H7) and Listeria monocytogenes (L. monocytogenes). The built logistic and exponential models confirmed that at equal ORP values, the inactivation of the surrogate strains was not consistent across pH 6.5 and pH 8.5, with higher reductions at higher pH. This is the opposite of the well-known free chlorine-controlled bacterial inactivation, where the antibacterial effect is higher at lower pH. The validation test results indicated that in the cabbage wash water, the relationship between disinfection efficiency and ORP was consistent with the oxidant demand free systems. The study suggests that ORP cannot serve as a reliable single variable measurement to predict bacterial disinfection in buffered systems. When using ORP to monitor and control the antibacterial effectiveness of the chlorinated wash water, it is crucial to take into account (and control) the pH.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi.

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7-1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was -0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.

Bifidobacterium longum K5 Prevents Enterohaemorrhagic Escherichia coli O157:H7 Infection in Mice through the Modulation of the Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1ß was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.

Mircrofabricating double-sided polydimethylsiloxane (PDMS) artificial phylloplane for microbial food safety research.

Leafy green surface microbiology studies often experience significant variations in results due to the heterogeneous nature of leaf surfaces. To provide a precise and controllable substitute, we microfabricated double-sided artificial leafy green phylloplanes using polydimethylsiloxane (PDMS) with a vinyl-terminated polyethylene glycol chain-based hydrophobicity modifier (PDMS-PEG) to modify PDMS hydrophobicity. We further tested the properties and applications of these artificial leaves, by examining the function of epicuticular wax, growth and survival of E. coli O157:H7 87-23 on the surface, and removal of attached E. coli cells via sanitation. The double-sided PDMS-PDMS-PEG leaves well-replicated their natural counterparts in macroscopic and microscopic structure, hydrophobicity, and E. coli O157:H7 87-23 attachment. After depositing natural epicuticular wax onto artificial leaves, the leaf surface wetting ability decreased, while E. coli O157:H7 87-23 surface retention increased. The artificial leaves supplied with lettuce lysate or bacterial growth media supported E. coli O157:H7 87-23 growth and survival similarly to those on natural leaves. In the sanitation test, the artificial lettuce leaves also displayed patterns similar to those of natural leaves regarding sanitizer efficiency. Overall, this study showcased the microfabrication and applications of double-sided PDMS-PDMS-PEG leaves as a replicable and controllable platform for future leafy green food safety studies.

The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7.

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.

Ultra-sensitive fluorescent biosensor for multiple bacteria detection based on CDs/QDs@ZIF-8 and microfluidic fluidized bed.

An ultra-sensitive fluorescent biosensor based on CDs/QDs@ZIF-8 and microfluidic fluidized bed was developed for rapid and ultra-sensitive detection of multiple target bacteria. The zeolitic imidazolate frameworks (ZIF-8) act as the carrier to encapsulate three kinds of fluorescence signal molecules from the CDs/QDs@ZIF-8 signal amplification system. Besides, three kinds of target pathogenic bacteria were automatically, continuously, and circularly captured by the magnetic nanoparticles (MNPs) in the microfluidic fluidized bed. The neutral Na2EDTA solution was the first time reported to not only dissolve the ZIF-8 frameworks from the MNPs-bacteria-CDs/QDs@ZIF-8 sandwich complexes, but also release the CDs/QDs from sandwich complexes with no loss of fluorescence signal. Due to the advantages of signal amplification and automated sample pretreatment, the proposed fluorescent biosensor can simultaneously detect Escherichia coli O157:H7, Salmonella paratyphi A, and Salmonella paratyphi B as low as 101 CFU/mL within 1.5 h, respectively. The mean recovery in spiked milk samples can reach 99.18%, verifying the applicability of this biosensor in detecting multiple bacteria in real samples.

Chip-based automated equipment for dual-mode point-of-care testing foodborne pathogens.

Foodborne pathogens have a substantial bearing on food safety and environmental health. The development of automated, portable and compact devices is essential for the on-site and rapid point-of-care testing (POCT) of bacteria. Here, this work developed a micro-automated microfluidic device for detecting bacteria, such as Escherichia coli (E. coli) O157:H7, using a seashell-like microfluidic chip (SMC) as an analysis and mixing platform. The automated device integrates a colorimetric/fluorescent system for the metabolism of copper (Cu2+) by E. coli affecting o-phenylenediamine (OPD) for concentration analysis. A smartphone was used to read the RGB data of the chip reaction reservoir to detect colorimetric and fluorescence patterns in the concentration range of 102-106 CFU mL-1. The automated device overcomes the low efficiency and tedious steps of traditional detection and enables high-precision automated detection that can be applied to POCT in the field, providing an ideal solution for broadening the application of E. coli detection.

Synthesis of silver­bismuth oxide encapsulated hydrazone functionalized chitosan (AgBi2O3/FCS) nanocomposite for electrochemical sensing of glucose, H2O2 and Escherichia coli O157:H7.

In this work, silver­bismuth oxide encapsulated 1,3,5-triazine-bis(4-methylbenzenesulfonyl)-hydrazone functionalized chitosan (SBO/FCS) nanocomposite was synthesized by a simple hydrothermal method. The amine (-NH2) group was functionalized by the addition of cyanuric acid chloride followed by 4-methylbenzenesulfonol hydrazide. The SBO/FCS has been characterized by FT-IR, X-ray diffraction, XPS, HR-SEM, HR-TEM, AFM, and thermogravimetry (TGA). Under the optimum conditions, the SBO/FCS sensor showed brilliant electrochemical accomplishment for the sensing of glucose and H2O2 by a limit of detection (LOD) of 0.057 µM and 0.006 µM. It also showed linearity for glucose 0.008-4.848 mM and for H2O2 of 0.01-6.848 mM. Similarly, the sensor exhibited a low sensitivity to glucose (32 µA mM-1 cm-2) and a good sensitivity to H2O2 (295 µA mM-1 cm-2). In addition, that the prepared electrode could be used to sense the glucose and H2O2 levels in real samples such as blood serum and HeLa cell lines. The screen printed electrode (SPE) immunosensor could sense the E. coli O157:H7 concurrently and quantitatively with a linear range of 1.0 × 101-1.0 × 109 CFU mL-1 and a LOD of 4 CFU mL-1. Likewise, the immunosensor efficiently detect spiked E. coli O157:H7 in milk, chicken, and pork samples, with recoveries ranging from 89.70 to 104.72 %, demonstrating that the immunosensor was accurate and reliable.

CobB-mediated deacetylation of the chaperone CesA regulates Escherichia coli O157:H7 virulence.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a common food-borne pathogen that can cause acute diseases. Lysine acetylation is a post-translational modification (PTM) that occurs in various prokaryotes and is regulated by CobB, the only deacetylase found in bacteria. Here, we demonstrated that CobB plays an important role in the virulence of EHEC O157:H7 and that deletion of cobB significantly decreased the intestinal colonization ability of bacteria. Using acetylation proteomic studies, we systematically identified several proteins that could be regulated by CobB in EHEC O157:H7. Among these CobB substrates, we found that acetylation at the K44 site of CesA, a chaperone for the type-III secretion system (T3SS) translocator protein EspA, weakens its binding to EspA, thereby reducing the stability of this virulence factor; this PTM ultimately attenuating the virulence of EHEC O157:H7. Furthermore, we showed that deacetylation of the K44 site, which is deacetylated by CobB, promotes the interaction between CesA and EspA, thereby increasing bacterial virulence in vitro and in animal experiments. In summary, we showed that acetylation influences the virulence of EHEC O157:H7, and uncovered the mechanism by which CobB contributes to bacterial virulence based on the regulation of CesA deacetylation.

Evaluating the Safety of Sous-Vide Cooking for Beef Products Inoculated with Single Strains of Salmonella enterica and Escherichia coli O157.

Sous-videcooking is a growing trend among retailers and consumers. Foodborne pathogens may survive the cooking if nonvalidated parameters are used or if pathogens have enhanced thermalresistance. Pathogen inactivation from sous-vide cooking was determined when introduced directly to beef products or via contaminated spices, and with or without a finishing step. Beef products (ground beef, tenderized, and nontenderized steaks) were inoculated with pathogens (Salmonella Montevideo and Escherichia coli O157:NM) in three ways: 1) directly onto the meat 2) ground black pepper incorporated into the recipe 3) ground pepper equilibrated at 30% RH (4 d) prior to incorporation. Beef samples were vacuum-packaged and submerged in a 62.5°C water bath for 120 min. Samples were sampled at 5, 10, 20, and 120 min (recommended from a partner quality study), and a duplicate was grilled to a specific internal temperature (74°C for ground beef, 57°C for steaks) and sampled. Sous-vide cooking reduced pathogen populations by >5 log CFU/g after most treatment times, but less than grilled counterparts (ca. 1-2 log CFU/g difference; p < 0.05).There were no statistically significant differences between inoculation methods, but the tenderization of steaks resulted in significantly lower reductions of pathogens from sous-vide cooking (p < 0.05). Thisresearch challenged sous-vide cooking parameters (120 min, 62.5°C). It showed sous-vide alone lowered pathogens by >4 log CFU/g after most 20-min treatments, but 120-min sous-vide treatments or grilling would be needed for >5-log reductions.Contaminated pepper led to less consistent reductions during the cooking process, yet 2-h sous-vide still achieved a 5-log reduction. Sous-vide cooking instructions must be validated as more products and recipes are marketed.

Customizable Nichrome Wire Heaters for Molecular Diagnostic Applications.

Accurate sample heating is vital for nucleic acid extraction and amplification, requiring a sophisticated thermal cycling process in nucleic acid detection. Traditional molecular detection systems with heating capability are bulky, expensive, and primarily designed for lab settings. Consequently, their use is limited where lab systems are unavailable. This study introduces a technique for performing the heating process required in molecular diagnostics applicable for point-of-care testing (POCT), by presenting a method for crafting customized heaters using freely patterned nichrome (NiCr) wire. This technique, fabricating heaters by arranging protrusions on a carbon black-polydimethylsiloxane (PDMS) cast and patterning NiCr wire, utilizes cost-effective materials and is not constrained by shape, thereby enabling customized fabrication in both two-dimensional (2D) and three-dimensional (3D). To illustrate its versatility and practicality, a 2D heater with three temperature zones was developed for a portable device capable of automatic thermocycling for polymerase chain reaction (PCR) to detect Escherichia coli (E. coli) O157:H7 pathogen DNA. Furthermore, the detection of the same pathogen was demonstrated using a customized 3D heater surrounding a microtube for loop-mediated isothermal amplification (LAMP). Successful DNA amplification using the proposed heater suggests that the heating technique introduced in this study can be effectively applied to POCT.

Comparison of Three Preharvest Sampling Strategies to Monitor Pathogens in Cattle Lairage Areas.

The objective of this study was to compare preharvest monitoring strategies by evaluating three different sampling methods in the lairage area to determine pathogen recovery for each sampling method and incoming pathogen prevalence from the cattle to inform in-plant decision making. Samples were gathered over a 5-month period, from February to June 2022, at a harvesting and processing facility located in Eastern Nebraska. Sampling methods included (i) fecal pats, (ii) boot swabs, and (iii) MicroTally swab. A total of 329 samples were collected over the study period (fecal pats: n = 105, boot swabs: n = 104, and MicroTally swabs: n = 120). Specific media combinations, an incubation temperature of 42°C, and incubation timepoints (18-24 h) were utilized for each matrix and the prevalence of Salmonella, Escherichia coli O157:H7, and six non-O157 Shiga-toxin producing E. coli (STEC) was evaluated using the BAX system Real-Time PCR assay. Overall, results from the study concluded that boot swabs were an effective sampling method for pathogen detection in the cattle lairage area. Boot swabs (97.1%) were statistically more likely to detect for Salmonella (p < 0.05) when compared to fecal pats (67.6%) and MicroTally swab (77.5%) methods. For E. coli O157:H7 and STEC - O26, O121, O45, and O103 prevalence, boot swabs were significantly better at detecting for these pathogens (p < 0.05) than MicroTally swabs (OR = 3.16 - 11.95) and a comparable sampling method to fecal pats (OR = 0.93 - 2.01, p > 0.05). Lastly, all three sampling methods detected a very low prevalence for E. coli O111 and O145; therefore, no further analysis was conducted. The boot swab sampling method was strongly favored because they require little training to implement, are inexpensive, and they do not require much sampling labor; therefore, would be a simple and effective sampling method to implement within the industry to evaluate pathogen prevalence preharvest.

Differential microbiota shift on whole romaine lettuce subjected to source or forward processing and on fresh-cut products during cold storage.

Romaine lettuce in the U.S. is primarily grown in California or Arizona and either processed near the growing regions (source processing) or transported long distance for processing in facilities serving distant markets (forward processing). Recurring outbreaks of Escherichia coli O157:H7 implicating romaine lettuce in recent years, which sometimes exhibited patterns of case clustering in Northeast and Midwest, have raised industry concerns over the potential impact of forward processing on romaine lettuce food safety and quality. In this study, freshly harvested romaine lettuce from a commercial field destined for both forward and source processing channels was tracked from farm to processing facility in two separate trials. Whole-head romaine lettuce and packaged fresh-cut products were collected from both forward and source facilities for microbiological and product quality analyses. High-throughput amplicon sequencing targeting16S rRNA gene was performed to describe shifts in lettuce microbiota. Total aerobic bacteria and coliform counts on whole-head lettuce and on fresh-cut lettuce at different storage times were significantly (p < 0.05) higher for those from the forward processing facility than those from the source processing facility. Microbiota on whole-head lettuce and on fresh-cut lettuce showed differential shifting after lettuce being subjected to source or forward processing, and after product storage. Consistent with the length of pre-processing delays between harvest and processing, the lettuce quality scores of source-processed romaine lettuce, especially at late stages of 2-week storage, was significantly higher than of forward-processed product (p < 0.05).

A Fully Integrated Handheld Electrochemical Sensing Platform for Point-of-Care Testing of Escherichia coli O157:H7.

Fully integrated devices that enable full functioning execution without or with minimum external accessories or equipment are deemed to be one of the most desirable and ultimate objectives for modern device design and construction. Escherichia coli O157:H7 (E. coli O157:H7) is often linked to outbreaks caused by contaminated water and food. However, the sensors that are currently used for point-of-care E. coli O157:H7 (E. coli O157:H7) detection are often large and cumbersome. Herein, we demonstrate the first example of a handheld and pump-free fully integrated electrochemical sensing platform with the capability to point-of-care test E. coli O157:H7 in the actual samples of E. coli O157:H7-spiked tap water and E. coli O157:H7-spiked watermelon juice. This platform was made possible by overcoming major engineering challenges in the seamless integration of a microfluidic module for pump-free liquid sample collection and transportation, a sensing module for efficient E. coli O157:H7 testing, and an electronic module for automatically converting and wirelessly transmitting signals into a single and compact electrochemical sensing platform that retains its inimitable stand-alone, handheld, pump-free, and cost-effective feature. Although our primary emphasis in this study is on detecting E. coli O157:H7, this pump-free fully integrated handheld electrochemical sensing platform may also be used to monitor other pathogens in food and water by including specific antipathogen antibodies.

Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.