Metal-Phenolic Network Directed Coating of Single Probiotic Cell Followed by Photoinitiated Thiol-Ene Click Fortification to Enhance Oral Therapy.

Probiotics-based oral therapy has become a promising way to prevent and treat various diseases, while the application of probiotics is primarily restricted by loss of viability due to adverse conditions in the gastrointestinal (GI) tract during oral delivery. Layer-by-layer (LbL) single-cell encapsulation approaches are widely employed to improve the bioavailability of probiotics. However, they are generally time- and labor-intensive owing to multistep operation. Herein, a simple yet efficient LbL technique is developed to coat a model probiotic named Escherichia coli Nissle 1917 (EcN) through polyphenol-Ca2+ network directed allyl-modified gelatin (GelAGE) adsorption followed by cross-linking of GelAGE via photoinitiated thiol-ene click reaction to protect EcN from harsh microenvironments of GI tract. LbL single-cell encapsulation can be performed within 1 h through simple operation. It is revealed that coated EcN exhibits significantly improved viability against acidic gastric fluid and bile salts, and enhanced colonization in the intestinal tract without loss of proliferation capabilities. Furthermore, oral therapy of coated EcN remarkably relieves the pathological symptoms associated with colitis in mice including down-regulating inflammation, repairing epithelial barriers, scavenging reactive oxygen species (ROS), and restoring the homeostasis of gut microbiota. This simplified LbL coating strategy has great potential for various probiotics-mediated biomedical and nutraceutical applications.

Development of Recombinase Aided Amplification (RAA)-Exo-Probe Assay for the Rapid Detection of Shiga Toxin-Producing Escherichia coli.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. OBJECTIVE: Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. METHODS: Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. RESULTS: These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. CONCLUSIONS: Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. HIGHLIGHTS: As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

Sensitive and Rapid Detection of Escherichia coli O157:H7 From Beef Samples Based on Recombinase Aided Amplification Assisted CRISPR/Cas12a System.

BACKGROUND: Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming. OBJECTIVE: In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced. METHODS: First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA's Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. RESULTS: The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10-4 ng/µL, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples. CONCLUSIONS: The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. HIGHLIGHTS: The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.

Biofilm Formation of Listeria monocytogenes and Pseudomonas aeruginosa in a Simulated Chicken Processing Environment.

This study aims to investigate the mono- and dual-species biofilm formation of Listeria monocytogenes and Pseudomonas aeruginosa incubated in different culture mediums, inoculum ratios, and incubation time. The planktonic cell population and motility were examined to understand the correlation with biofilm formation. The results showed that chicken juice significantly inhibited the biofilm formation of L. monocytogenes (p < 0.05). Pseudomonas aeruginosa was the dominant bacteria in the dual-species biofilm formation in the trypticase soy broth medium. The dynamic changes in biofilm formation were not consistent with the different culture conditions. The growth of planktonic L. monocytogenes and P. aeruginosa in the suspension was inconsistent with their growth in the biofilms. There was no significant correlation between motility and biofilm formation of L. monocytogenes and P. aeruginosa. Moreover, scanning electron microscopy (SEM) results revealed that the biofilm structure of L. monocytogenes was loose. At the same time, P. aeruginosa formed a relatively dense network in mono-species biofilms in an initial adhesion stage (24 h). SEM results also showed that P. aeruginosa was dominant in the dual-species biofilms. Overall, these results could provide a theoretical reference for preventing and controlling the biofilm formation of L. monocytogenes and P. aeruginosa in the food processing environment in the future.

Development of Recombinase-Aided Amplification (RAA)-Exo-Probe and RAA-CRISPR/Cas12a Assays for Rapid Detection of Campylobacter jejuni in Food Samples.

Campylobacter jejuni is the major cause of campylobacteriosis, one of the most common foodborne illnesses worldwide. Here, we report the development of RAA-exo-probe and RAA-CRIPSR/Cas12a assays for the detection of C. jejuni in food samples. The two assays were found to be highly specific to C. jejuni and highly sensitive, as they were one log more sensitive compared to the traditional culture method, with detection thresholds of 9 and 5 copies per reaction, respectively. These assays successfully detected C. jejuni in spiked chicken samples and natural meat samples (chicken, beef, mutton, etc.) and were overall less dependent on expensive equipment, only requiring a fluorescent reader. Their ease of use compared to other nucleic acid amplification-based methods indicates that these assays could be adapted for the rapid, routine surveillance of C. jejuni contamination in food samples, particularly for work done in the field or poorly equipped labs.

Inactivation and Subsequent Growth Kinetics of Listeria monocytogenes After Various Mild Bactericidal Treatments.

This study was carried out to investigate the effects of mild heat, lactic acid, benzalkonium chloride and nisin treatments on the inactivation, sublethal injury, and subsequent growth of Listeria monocytogenes. Results showed that the Bigelow model successfully described the thermal inactivation kinetics, while the Log-linear model with tail consistently offered the most accurate fit to LA, BC, and nisin inactivation curves of cells. Differential plating indicated that percentage of sublethal injury for nisin treated cells was significantly higher than that for the other three treatments. Compared to non-treated cells, significant extension of lag time was observed for all treated cells. The longer exposures to heat treatment contributed to the extended lag time of the survivors. While for LA, BC and nisin treated cells, the longest lag time was not observed at the most severe treatment conditions. The correlation analysis of sublethal injury percentage on the duration of lag time revealed that only heat treatment showed the significant correlation. Overall, the lag time analysis could evaluate a wide range of bacterial injury. Lag time of treated cells was significantly influenced by stress treatments and temperatures of recovery, however, there were not any significant changes in the maximum specific growth rate between treated and non-treated cells under isothermal recovery conditions. The information generated from this study is valuable for utilizing intervention strategies in the elimination or growth inhibition of L. monocytogenes.

Antibiotic Resistance Patterns of Staphylococcus aureus Isolates from Retail Foods in Mainland China: A Meta-Analysis.

Foodborne Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is increasingly threatening human health. Pooled prevalence rates of S. aureus contamination have been extensively studied in retail food products in mainland China, but data regarding antibiotic resistance rates of S. aureus remain scattered. This study was designed to collect researches published between 2007 and 2017 in mainland China and to evaluate the antibiotic resistance of S. aureus from retail foods using a meta-analytic approach. We systematically searched the China National Knowledge Infrastructure (CNKI) and Web of Science databases to identify peer-reviewed literature. A number of multilevel random-effects models were fitted to estimate mean occurrence rates of antibiotic-resistant S. aureus, and subgroup analyses were performed to compare antibiotic resistance rates of S. aureus throughout the years and among the methods to determine the antimicrobial susceptibility. Among the considered antibiotics, S. aureus showed the highest resistance rate to penicillin G (87%, 95% confidence interval [CI] 83-90%), followed by ampicillin (72%, 95% CI 62-81%) and erythromycin (41%, 95% CI 36-46%). MRSA showed the highest resistance rate to ampicillin (98%, 95% CI 89-100%), followed by oxacillin (97%, 95% CI 80-100%) and penicillin G (96%, 95% CI 89-99%). Multidrug resistance (MDR) of S. aureus was most frequently observed to three antibiotics (17%, 95% CI 12-22%), and MRSA showed the highest resistance rate to four antibiotics (24%, 95% CI 5-67%). Subgroup analyses results proved that sources of heterogeneity among studies were neither publication year nor detection method. In conclusion, the meta-analysis showed that ß-lactam antibiotics resistance of S. aureus and MRSA strains isolated from retail foods remained the most serious, and MDR of S. aureus and MRSA were also observed. Therefore, it is important to monitor the antibiotic resistance of S. aureus and MRSA in food chain, and food safety measures should be taken to reduce the transmission of this bacterium from foods to human beings.