Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are foodborne pathogens that cause hemolytic uremic syndrome and fatal infant diarrhea, respectively, but the characterization of these bacteria from imported food in China are unknown. A total of 1577 food samples from various countries during 2015–2021 were screened for STEC and EPEC, and the obtained isolates were tested for antimicrobial resistance and whole genome sequencing analysis was performed. The prevalence of STEC and EPEC was 1.01% (16/1577) and 0.51% (8/1577), respectively. Antimicrobial resistances to tetracycline (8%), chloramphenicol (8%), ampicillin (4%), ceftazidime (4%), cefotaxime (4%), and trimethoprim-sulfamethoxazole (4%) were observed. The antimicrobial resistance phenotypes corresponded with genotypes for most strains, and some resistance genes were related to mobile genetic elements. All 16 STEC isolates were eae negative, two solely contained stx1 (stx1a or stx1c), 12 merely carried stx2 (stx2a, stx2d, or stx2e), and two had both stx1 and stx2 (stx1c + stx2b, stx1a + stx2a + stx2c). Although they were eae negative, several STEC isolates carried other adherence factors, such as iha (5/16), sab (1/16), and lpfA (8/16), and belonged to serotypes (O130:H11, O8:H19, and O100:H30) or STs (ST297, ST360), which have caused human infections. All the eight EPEC isolates were atypical EPEC; six serotypes and seven STs were found, and clinically relevant EPEC serotypes O26:H11, O103:H2, and O145:H28 were identified. Two STEC/ETEC (enterotoxigenic E. coli) hybrids and one EPEC/ETEC hybrid were observed, since they harbored sta1 and/or stb. The results revealed that food can act as a reservoir of STEC/EPEC with pathogenic potential, and had the potential ability to transfer antibiotic resistance and virulence genes.

Postbiotics possess various functional activities, closely linked to their source bacterial strains and preparation methods. Therefore, the functional activities of postbiotics need to be evaluated through in vitro and in vivo methods. This study aims to prepare a postbiotic and explore its antihemolytic, anti-inflammatory, antioxidant, and antibacterial activities. Specifically, a postbiotic preparation named PostbioP-6 was prepared by intercepting 1–5 kDa of Lacticaseibacillus paracasei Postbiotic-P6 fermentation broth. The results demonstrate that PostbioP-6 exhibited notable biological activities across multiple assays. It showed significant antihemolytic activity, with a 4.9–48.1% inhibition rate at 10–50% concentrations. Anti-inflammatory effects were observed both in vitro, where 8–40% PostbioP-6 was comparable to 259.1–645.4 μg/mL diclofenac sodium, and in vivo, where 3.5 and 4.0 μL/mL PostbioP-6 significantly reduced neutrophil counts in inflamed zebrafish (p < 0.05). Antioxidant properties were evident through increased reducing power (OD₇₀₀ increased from 0.279 to 2.322 at 1.25–12.5% concentrations), DPPH radical scavenging activity (38.9–92.4% scavenging rate at 2.5–50% concentrations), and hydroxyl radical scavenging activity (4.66–10.38% scavenging rate at 0.5–4% concentrations). Additionally, PostbioP-6 demonstrated antimicrobial activity against two Gram-positive bacteria, eight Gram-negative bacteria, and one fungus. Furthermore, PostbioP-6 significantly inhibited the increase in peroxide value and malondialdehyde content in cookies, highlighting its potential application in food preservation. In conclusion, we prepared a novel postbiotic, termed PostbioP-6, which proved to have prominent anti-hemolytic, anti-inflammatory, antioxidant, and broad-spectrum antimicrobial activities. The multifunctional properties of PostbioP-6 position it as a potentially effective functional food supplement or preservative. In the future, further research is necessary to elucidate the precise mechanisms of action, identify the active components, and validate its biological activities in animal models or clinical trials.

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. 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. 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. 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. The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. 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.

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.

Listeria monocytogenes is an important foodborne pathogen with worldwide prevalence. Understanding the variability in the potential pathogenicity among strains of different subtypes is crucial for risk assessment. In this study, the growth, survival, and virulence characteristics of 16 L. monocytogenes strains isolated from imported meat in China (2018–2020) were investigated. The maximum specific growth rate (μ_max) and lag phase (λ) were evaluated using the time-to-detection (TTD) method and the Baranyi model at different temperatures (25, 30, and 37 °C). Survival characteristics were determined by D-values and population reduction after exposure to heat (60, 62.5, and 65 °C) and acid (HCl, pH = 2.5, 3.5, and 4.5). The potential virulence was evaluated via adhesion and invasion to Caco-2 cells, motility, and lethality to Galleria mellonella. The potential pathogenicity was compared among strains of different lineages and subtypes. The results indicate that the lineage I strains exhibited a higher growth rate than the lineage II strains at three growth temperatures, particularly serotype 4b within lineage I. At all temperatures tested, serotypes 1/2a and 1/2b consistently demonstrated higher heat resistance than the other subtypes. No significant differences in the log reduction were observed between the lineage I and lineage II strains at pH 2.5, 3.5, and 4.5. However, the serotype 1/2c strains exhibited significantly low acid resistance at pH 2.5. In terms of virulence, the lineage I strains outperformed the lineage II strains. The invasion rate to Caco-2 cells and lethality to G. mellonella exhibited by the serotype 4b strains were higher than those observed in the other serotypes. This study provides meaningful insights into the growth, survival, and virulence of L. monocytogenes, offering valuable information for understanding the correlation between the pathogenicity and subtypes of L. monocytogenes.