Visual Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid On-Site Detection of Escherichia coli O157: H7 in Milk Products.

(1) Background: Rapid on-site testing is an effective method for the detection of Escherichia coli O157: H7(E. coli O157: H7) in food ingredients and the environment. (2) Methods: In this study, we developed colorimetric loop-mediated isothermal amplification (LAMP) and immunochromatographic test strips (ICTs) for the rapid and visual detection of E. coli O157: H7. This study designed new specific LAMP primers for E. coli O157: H7 virulence island genes. After the LAMP amplification, the double-stranded DNA target sequence labeled with digoxin and fluorescein isothiocyanate (FITC) at both ends was bound to the anti-digoxin antibody on the gold nanoparticles. Subsequently, it was further bound to the anti-FITC antibody at the T line of the ICTs, forming a positive test result. Hydroxynaphthyl blue dye was directly added to the LAMP amplification product. A blue color indicated positive results, while a purple color indicated negative results. (3) Results: Two visualization methods showed high specificity for the target strains. The visualization tests had sensitivities of 5.7 CFU mL-1, and the detection limit of the Escherichia coli O157: H7 in artificially contaminated milk samples was 5.7 × 102 CFU mL-1, which was consistent with the results of the standard method (LAMP-electrophoresis method) used in commercial inspection. (4) Conclusions: Both methods could be useful in remote and under-resourced areas.

A colorimetric biosensor with infrared sterilization based on CuSe nanoparticles for the detection of E. coli O157:H7 in food samples.

It is critical to develop quick, accurate, and efficient sterilization for detecting Escherichia coli O157:H7 in order to prevent infections and outbreaks of foodborne illnesses. Herein, we established a colorimetric biosensor with sterilizing properties using copper selenide nanoparticles to detect E. coli O157:H7. The sample was mixed with magnetic nanoprobes and nanozyme probes to form a sandwich structure, and then the unbound nanozyme probes were collected by magnetic separation. Finally, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-hydrogen peroxide (H2O2) reporting system was added for signal amplification. The change from colorless to green can be seen with the naked eye. Under the optimal conditions, the detection range of E. coli O157:H7 was 102-106 CFU/mL, and the detection limit was 0.35 × 102 CFU/mL. The total detection time was 80 minutes, which can be successfully applied to milk and mineral water. In addition, the colorimetric sensor can kill the target bacteria by irradiating it under a 980-nm laser for 5 minutes. In conclusion, this sensor is a promising tool for rapidly detecting foodborne pathogens and promptly eliminating bacteria. IMPORTANCE: Escherichia coli O157:H7 is a major threat to public health. At present, the detection methods for E. coli O157:H7 mainly include traditional bacterial culture, immunology (enzyme-linked immune-sorbent assay) and molecular biology techniques (polymerase chain reaction). These methods have the limitations of professional operation, waste of time and energy, and high cost. Therefore, we have developed a simple, fast, bactericidal colorimetric biosensor to detect E. coli. O157:H7. The entire process was completed in 80 minutes. The method has been successfully applied to milk and mineral water samples with satisfactory results, proving that the method is an effective method for real-time detection and inactivation of bacteria.

Complete genome sequence of Escherichia coli O157:H7 phage Φ241.

We report the genome sequence of phage Φ241 infecting Escherichia coli O157:H7. Phage Φ241 was isolated from an industrial cucumber fermentation at high acidity (pH 3.7) and high salinity (5% NaCl). The phage genome consists of a 157,291 bp circular double-stranded DNA with 203 coding regions and 44.96% GC content.

Acid Adaptation Enhances Tolerance of Escherichia coli O157:H7 to High Voltage Atmospheric Cold Plasma in Raw Pineapple Juice.

Pathogens that adapt to environmental stress can develop an increased tolerance to some physical or chemical antimicrobial treatments. The main objective of this study was to determine if acid adaptation increased the tolerance of Escherichia coli O157:H7 to high voltage atmospheric cold plasma (HVACP) in raw pineapple juice. Samples (10 mL) of juice were inoculated with non-acid-adapted (NAA) or acid-adapted (AA) E. coli to obtain a viable count of ~7.00 log10 CFU/mL. The samples were exposed to HVACP (70 kV) for 1-7 min, with inoculated non-HVACP-treated juice serving as a control. Juice samples were analyzed for survivors at 0.1 h and after 24 h of refrigeration (4 °C). Samples analyzed after 24 h exhibited significant decreases in viable NAA cells with sub-lethal injury detected in both NAA and AA survivors (p < 0.05). No NAA survivor in juice exposed to HVACP for 5 or 7 min was detected after 24 h. However, the number of AA survivors was 3.33 and 3.09 log10 CFU/mL in juice treated for 5 and 7 min, respectively (p < 0.05). These results indicate that acid adaptation increases the tolerance of E. coli to HVACP in pineapple juice. The potentially higher tolerance of AA E. coli O157:H7 to HVACP should be considered in developing safe juice processing parameters for this novel non-thermal technology.

Biological Control of Escherichia coli O157:H7 in Dairy Manure-Based Compost Using Competitive Exclusion Microorganisms.

BACKGROUND: Animal manure-based compost is a valuable organic fertilizer and biological soil amendment. To ensure the microbiological safety of compost products, the effectiveness of competitive exclusion microorganisms (CE) in reducing Escherichia coli O157:H7 in dairy manure-based compost was evaluated. METHODS: A cocktail of E. coli O157:H7 strains were inoculated into dairy compost along with CE strains isolated from compost, and the reduction in E. coli O157:H7 by CE was determined in compost with 20%, 30%, and 40% moisture levels at 22 °C and 30 °C under laboratory conditions, as well as in fall, winter, and summer seasons under greenhouse settings. RESULTS: Under lab conditions, CE addition resulted in 1.1-3.36 log reductions in E. coli O157:H7 in compost, with enhanced pathogen reduction by higher moisture and lower temperature. In the greenhouse, >99% of the E. coli O157:H7 population in compost with ≥30% moisture due to cross-contamination can be effectively inactivated by CE within 2 days during colder seasons. However, it took ≥8 days to achieve the same level of reduction for heat-adapted E. coli O157:H7 cells. CONCLUSIONS: Our results demonstrated that the competitive exclusion of microorganisms can be an effective tool for controlling foodborne pathogens in compost and reducing the potential for soil and crop contamination.

Use of dishwashers fails to inactivate foodborne pathogens in home-canned model foods.

Risky home canning techniques are still performed for food preservation due to limited science-based recommendations. This study aimed to evaluate the inactivation of Shiga toxin-producing Escherichia coli O157:H7, Salmonella enterica (ser. Typhimurium, Enteritidis, and Infantis) and Listeria monocytogenes during home canning with a household dishwasher. The 450 mL of blended tomato (acidic liquid food) and potato puree (non-acidic solid food) were prepared with 1.5 % salt and 25 mL vinegar as model foods in glass jars (660 mL). The two model foods were sterilized, then inoculated with separate cocktails of each pathogen at 106-107 CFU/g. The prepared jars were placed in the bottom rack of a dishwasher and subjected to the following cycles: economic (50 °C, 122 min), express (60 °C, 54 min), and intensive (70 °C, 96 min). Temperature changes in jars were monitored by using thermocouples during heat treatment. Within the center of the jars, temperatures were measured as 45 to 53 °C in blended tomato and 44 to 52 °C in potato puree during all tested dishwasher cycles, respectively. The economic cycle treatment reduced S. enterica, E. coli O157:H7, and L. monocytogenes populations by 3.1, 4.6, and 4.2 log CFU/g in blended tomato (P ≤ 0.05), where a <1.0 log reduction was observed in potato puree (P > 0.05). All pathogens showed similar heat resistance during the express cycle treatment with a log reduction ranging from 4.2 to 5.0 log CFU/g in blended tomato and 0.6 to 0.7 log CFU/g in potato puree. Reduction in L. monocytogenes population was limited (0.6 log CFU/g) compared to E. coli O157:H7 (2.0 log CFU/g) and S. enterica (2.7 log CFU/g) in blended tomato during the intensive cycle treatment (P ≤ 0.05). Dishwasher cycles at manufacturer defined settings failed to adequately inactivate foodborne pathogens in model foods. This study indicates that home-canned vegetables may cause foodborne illnesses when dishwashers in home kitchens are used for heat processing.

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.

A Dual-mode platform for the rapid detection of Escherichia coli O157:H7 based on CRISPR/Cas12a and RPA.

Escherichia coli O157:H7 (E. coli O157:H7) is a foodborne pathogenic microorganism that is commonly found in the environment and poses a significant threat to human health, public safety, and economic stability worldwide. Thus, early detection is essential for E. coli O157:H7 control. In recent years, a series of E. coli O157:H7 detection methods have been developed, but the sensitivity and portability of the methods still need improvement. Therefore, in this study, a rapid and efficient testing platform based on the CRISPR/Cas12a cleavage reaction was constructed. Through the integration of recombinant polymerase amplification and lateral flow chromatography, we established a dual-interpretation-mode detection platform based on CRISPR/Cas12a-derived fluorescence and lateral flow chromatography for the detection of E. coli O157:H7. For the fluorescence detection method, the limits of detection (LODs) of genomic DNA and E. coli O157:H7 were 1.8 fg/µL and 2.4 CFU/mL, respectively, within 40 min. Conversely, for the lateral flow detection method, LODs of 1.8 fg/µL and 2.4 × 102 CFU/mL were achieved for genomic DNA and E. coli O157:H7, respectively, within 45 min. This detection strategy offered higher sensitivity and lower equipment requirements than industry standards. In conclusion, the established platform showed excellent specificity and strong universality. Modifying the target gene and its primers can broaden the platform's applicability to detect various other foodborne pathogens.

Contamination of Kazakhstan cheeses originating from Escherichia coli and its resistance to antimicrobial drugs.

Background and Aim: Escherichia coli, a commensal intestine bacterium of vertebrates, is widely distributed in the environment and indicates the microbiological quality of food products in relation to coliforms. In addition, virulent strains, particularly E. coli O157:H7, cause outbreaks of toxic infections caused by consuming dairy products. Because food safety studies regarding E. coli have not been conducted in Central Asia, this research aimed to study the characteristics of contamination, microbiological and genotypic properties, and resistance to antimicrobial agents of E. coli strains that contaminate various types of commercialized cheeses originating from Kazakhstan. Materials and Methods: In retail outlets, 207 samples of three types of cheese produced by 22 industrial and eight small enterprises in the central, eastern, southern, and northern regions of Kazakhstan were selected in 2020-2023. E. coli contamination was examined using standard microbiological, mass spectrometric, and molecular genetic methods. The discodiffuse European Committee on Antimicrobial Susceptibility Testing method was used to test the resistance of the identified E. coli isolates (65/207; 31.4%) to 20 antibacterial drugs. The Shiga toxin-producing E. coli (VT1 and VT2) and E. coli O157:H7 (eae) genes were investigated in all E. coli isolates using multiplex polymerase chain reaction. Results: An average of 31.4% samples of commercial Kazakhstani cheeses of various types were found to be contaminated with E. coli in almost all geographical regions of Kazakhstan, regardless of the productivity of the dairy enterprises. Soft cheeses produced by small farms (80% of samples) packaged at the retail site (100%) were the most contaminated with E. coli. The microbiological index (colony-forming unit/g) was unsatisfactory and unsuitable in 6.2% of such cheese samples. For the first time in Central Asia, the enteropathogenic strain E. coli O157:H7 was detected in 0.5% of cheese samples. E. coli isolates from cheese samples were resistant to 65% of antibacterial drugs and contained resistance genes to ß-lactams, sulfonamides, and quinolones groups. At the same time, 25% of the E. coli isolates were multi-resistant to three or more antimicrobial agents. Conclusion: The high level of contamination caused by multi-antibiotic resistant E. coli strains, including pathogenic pathogens, poses a risk to public health and highlights the need for further research on the monitoring and control of coliform enteropathogens in food products.

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.

CRISPR/Cas12a powered air-displacement enhanced evanescent wave fluorescence fiber-embedded microfluidic biochip for nucleic acid amplification-free detection of Escherichia coli O157:H7.

Simple yet ultrasensitive and contamination-free quantification of environmental pathogenic bacteria is in high demand. In this study, we present a portable clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) powered Air-displacement enhanced Evanescent wave fluorescence Fiber-embedded microfluidic Biochip (AEFB) for the high-frequency and nucleic acid amplification-free ultrasensitive detection of Escherichia coli O157:H7. The performance of AEFB was dramatically enhanced upon employing a simple air-solution displacement process. Theoretical assays demonstrated that air-solution displacement significantly enhances evanescent wave field intensity on the fiber biosensor surface and increases the V-number in tapered fiber biosensors. Consequently, light-matter interaction is strengthened, and fluorescence coupling and collection efficiency are improved, considerably enhancing sensitivity. By integrating the CRISPR biosensing mechanism, AEFB facilitated rapid, accurate, nucleic acid amplification-free detection of E.coli O157:H7 with polymerase chain reaction (PCR)-level sensitivity (176 cfu/mL). To validate its practicality, AEFB was used to detect E.coli O157:H7 in surface water and wastewater. Comparison with RT-PCR showed a strong linear relationship (R2 = 0.9871), indicating the excellent accuracy and reliability of this technology in real applications. AEFB is highly versatile and can be easily extended to detect other pathogenic bacteria, which will significantly promote the high-frequency assessment and early-warning of bacterial contamination in aquatic environments.

Carbon dots and covalent organic frameworks based FRET immunosensor for sensitive detection of Escherichia coli O157:H7.

The combination of carbon dots (CDs) with covalent organic frameworks (COFs) was used to design an innovative sensor based on fluorescence resonance energy transfer (FRET) for the detection of Escherichia coli O157:H7 (E. coli O157:H7) in food samples. Carbon dots were used as fluorescence donors, covalent organic frameworks as fluorescence acceptors. The antibody (Ab) specific to E. coli O157:H7 was used to form a CD-Ab-COF immunosensor by linking CDs and COFs. The antibody was specifically bound with E. coli O157:H7, which caused the connection between CDs and COFs to be interrupted, and the carbon dots exhibited fluorescence restoration. The sensor exhibited a linear detection range spanning from 0 to 106 CFU/mL, with the limit of detection (LOD) of 7 CFU/mL. The analytical performance of the developed immunosensor was evaluated using spiked food samples with different concentrations of E. coli O157:H7, validating the capability of assessing risks in food testing.

Conductometric immunosensor for specific Escherichia coli O157:H7 detection on chemically funcationalizaed interdigitated aptasensor.

Escherichia coli O157:H7 is a strain of Escherichia coli known for causing foodborne illness through the consumption of contaminated or raw food. To detect this pathogen, a conductometric immunosensor was developed using a conductometric sensing approach. The sensor was constructed on an interdigitated electrode and modified with a monoclonal anti-Escherichia coli O157:H7 aptamer. A total of 200 electrode pairs were fabricated and modified to bind to the target molecule replica. The binding replica, acting as the bio-recognizer, was linked to the electrode surface using 3-Aminopropyl triethoxysilane. The sensor exhibited excellent performance, detecting Escherichia coli O157:H7 in a short time frame and demonstrating a wide detection range of 1 fM to 1 nM. Concentrations of Escherichia coli O157:H7 were detected within this range, with a minimum detection limit of 1 fM. This innovative sensor offers simplicity, speed, high sensitivity, selectivity, and the potential for rapid sample processing. The potential of this proposed biosensor is particularly beneficial in applications such as drug screening, environmental monitoring, and disease diagnosis, where real-time information on biomolecular interactions is crucial for timely decision-making and where cross-reactivity or interference may compromise the accuracy of the analysis.

Survival and growth behavior of common foodborne pathogens in falafel paste under different storage temperatures.

Falafel is a popular breakfast food in the Middle East that has been recently involved in several outbreaks of foodborne illnesses. The aim of the study was to explore the growth behavior of Salmonella enterica, Escherichia coli O157:H7, Shigella sonnie, Shigella flexneri, Listeria monocytogenes and Staphylococcus aureus in falafel paste (FP) under different storage temperatures (4, 10, or 24 °C) for 14 days. FP (pH = 6.2, aw = 0.96) was inoculated with 5.0 to 6.0 log CFU/g of each of the pathogens separately. Salmonella spp. significantly declined by 1.5 log at 4 °C but grew significantly by ca. 2 and 4 log at 10 and 24 °C, respectively after 14 days. E. coli O157:H7 significantly increased (4.5 log) in FP when stored under 24 °C and survived at a level of ~105 CFU/g at 10 °C. Comparatively, Sh. sonnie and Sh. flexneri showed a better survival pattern in FP stored under 4 °C and grew (˃ 3 log) after 5 days at 10 and 24 °C. L. monocytogenes was capable of growing by 1.9 and 4.3 log after 14 d days and by 3.9 log after 3 days at 4, 10, or 24 °C, respectively. No significant decline in S. aureus counts at 4 and 10 °C occurred, however, it increased significantly to ˃ 7 log CFU/g at 24 °C. Total mesophilic count and yeast and mold count reached to spoilage levels (˃107 CFU/g) in un-inoculated FP after 1 and 3 days of storage at 24 and 10 °C, respectively. FP could support the growth of common foodborne pathogens and hence it is recommended to utilize natural antimicrobials in FP and keep the product under refrigeration (4 °C) to preclude the growth of vegetative foodborne pathogens.

Effect of Optimized UV-LED Technology on Modeling, Inactivation Kinetics and Microbiological Safety in Tomato Juice.

This research analyzed, optimized and modeled the inactivation kinetics of pathogenic bacteria (PB1: Escherichia coli O157:H7 and PB2: Listeria monocytogenes) and determined the microbiological safety of tomato juice processed by UV-LED irradiation and heat treatment. UV-LED processing conditions were optimized using response surface methodology (RSM) and were 90% power intensity, 21 min and 273-275 nm (251 mJ/cm2) with R2 > 0.96. Using the optimal conditions, levels of PB1 and PB2 resulted a log reduction of 2.89 and 2.74 CFU/mL, respectively. The Weibull model was efficient for estimating the log inactivation of PB1 and PB2 (CFU/mL). The kinetic parameter δ showed that 465.2 mJ/cm2 is needed to achieve a 90% log (CFU/mL) reduction in PB1 and 511.3 mJ/cm2 for PB2. With respect to the scale parameter p > 1, there is a descending concave curve. UV-LED-treated tomato juice had an 11.4% lower Listeria monocytogenes count than heat-treated juice on day 28 (4.0 ± 0.82 °C). Therefore, UV-LED technology could be used to inactivate Escherichia coli O157:H7 and Listeria monocytogenes, preserving tomato juice for microbiological safety, but studies are required to further improve the inactivation of these pathogens and analyze other fruit and vegetable juices.

Genomic island-encoded regulatory proteins in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important zoonotic pathogen that is a major cause of foodborne diseases in most developed and developing countries and can cause uncomplicated diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome. O islands (OIs), which are unique genomic islands in EHEC O157:H7, are composed of 177 isolated genomic features and harbour 26% of the total genes that are absent in the non-pathogenic E. coli K-12 genome. In the last twenty years, many OI-encoded proteins have been characterized, including proteins regulating virulence, motility, and acid resistance. Given the critical role of regulatory proteins in the systematic and hierarchical regulation of bacterial biological processes, this review summarizes the OI-encoded regulatory proteins in EHEC O157:H7 characterized to date, emphasizing OI-encoded regulatory proteins for bacterial virulence, motility, and acid resistance. This summary will be significant for further exploration and understanding of the virulence and pathogenesis of EHEC O157:H7.

Rapid and Ultrasensitive Colorimetric Biosensors for Onsite Detection of Escherichia coli O157:H7 in Fluids.

This study presents a breakthrough in the field of onsite bacterial detection, offering an innovative, rapid, and ultrasensitive colorimetric biosensor for the detection of Escherichia coli (E. coli) O157:H7, using chemically modified melamine foam (MF). Different from conventional platforms, such as 96-well plates and fiber-based membranes, the modified MF features a macroporous reticulated three-dimensional (3D) framework structure, allowing fast and free movement of large biomolecules and bacteria cells through the MF structure in every direction and ensuring good accessibility of entire active binding sites of the framework structure with the target bacteria, which significantly increased sensitive and volume-responsive detection of whole-cell bacteria. The biosensing platform requires less than 1.5 h to complete the quantitative detection with a sensitivity of 10 cfu/mL, discernible by the naked eye, and an enhanced sensitivity of 5 cfu/mL with the help of a smartphone. Following a short enrichment period of 1 h, the sensitivity was further amplified to 2 cfu/mL. The biosensor material is volume responsive, making the biosensing platform sensitivity increase as the volume of the sample increases, and is highly suitable for testing large-volume fluid samples. This novel material paves the way for the development of volume-flexible biosensing platforms for the record-fast, onsite, selective, and ultrasensitive detection of various pathogenic bacteria in real-world applications.

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a "noise purifier" for point-of-care testing of pathogenic bacteria in food.

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a "noise purifier" for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a "noise purifier" to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a "noise purifier" provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

Antibiofilm activity of lawsone against polymicrobial enterohemorrhagic Escherichia coli O157:H7 and Candida albicans by suppression of curli production and hyphal growth.

BACKGROUND: Most bacteria and fungi form biofilms that attach to living or abiotic surfaces. These biofilms diminish the efficacy of antimicrobial agents and contribute to chronic infections. Furthermore, multispecies biofilms composed of bacteria and fungi are often found at chronic infection sites. PURPOSE: In this study, lawsone (2­hydroxy-1,4-naphthoquinone) and its parent 1,4-naphthoquinone were studied for antimicrobial and antibiofilm activities against single-species and multispecies biofilms of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and Candida albicans. METHODS: Biofilm formation assays, biofilm eradication assays, antimicrobial assays, live cell imaging microscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), extracellular polymeric substances and indole production, cell surface hydrophilicity assay, cell motility, cell aggregation, hyphal growth, dual species biofilm formation, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and toxicity assays on plant seed germination and nematode model were utilized to investigate how lawsone affect biofilm development. RESULTS: Sub-inhibitory concentrations of lawsone (35 µg/ml) significantly inhibited single-and multispecies biofilm development. Lawsone reduced the production of curli and indole, and the swarming motility of EHEC, efficiently inhibited C. albicans cell aggregation and hyphal formation, and increased the cell surface hydrophilicity of C. albicans. Transcriptomic analysis showed that lawsone suppressed the expression of the curli-related genes csgA and csgB in EHEC, and the expression of several hypha- and biofilm-related genes (ALS3, ECE1, HWP1, and UME6) in C. albicans. In addition, lawsone up to 100 µg/ml was nontoxic to the nematode Caenorhabditis elegans and to the seed growth of Brassica rapa and Triticum aestivum. CONCLUSION: These results show that lawsone inhibits dual biofilm development and suggest that it might be useful for controlling bacterial or fungal infections and multispecies biofilms.